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LIN, Chîî-fa, 1940- THE CASEIN STABILIZING FUNCTION OF SULFATED HYDROCOLLOIDS.

The Ohio State University, Ph.D., 1971 Food Technology

5 University Microfilms, A XEROX Company, Ann Arbor, Michigan |

THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED THE CASEIN STABILIZING FUNCTION OF SULFATED HYDROCOLLOIDS

A Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of the Ohio State University

Chii-fa Lin, B. Sc., M. Sc.,

The Ohio State University

1971

Approved by

Adviser Department of Foocf Science a Nutritio PLEASE NOTE;

Some pages have small and indistinct print. Filmed as received.

UNIVERSITY MICROFILMS. This investigation was supported in part by Public Health

Service Research Grant No. FD-00117 from the Food and Drug Adminis tra tio n , Washington, D.C. and by a donation from Ross Laboratories,

Columbus, Ohio. Dedicated

to my wife, Kathy,

and my daughter, Judy, for th e ir help, patience and encouragement. ACKNOWLEDGEMENTS

My gratitude and sincere appreciation are expressed to

Dr. P. M. T. Hansen, Associate Professor of the Department of Food

Science and Nutrition, for his able guidance in this research and in assisting with the preparation of the manuscript and to Mrs. J ill Hansen for her help and encouragement offered to me and rny family during my graduate study. Without either of them, this work could not have been accomplished. Special thanks are due to Dr. I . Kristoffersen and Dr. W. J. Harper for their advice and keen personal interest in this study. November 25, 1940 ...... Born - Hsinchu, Taiwan, China

1963 ...... B. Sc., National Taiwan University Taipei, Taiwan, China

1966-1968 ...... Graduate Research Assistant, Department of Dairy Technology, The Ohio State University Columbus, Ohio

1968 ...... M. Sc., The Ohio State University, Columbus, Ohio

Graduate Research Associate, Department of Dairy Technology, The Ohio State University Columbus, Ohio

Graduate Research Associate, Department of Food Science & N utrition, The Ohio State University Columbus, Ohio TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS...... iv

VITA ...... V

LIST OF TABLES...... ix

LIST OF FIGURES...... x

INTRODUCTION ...... 1

LITERATURE REVIEW ...... 3

A. Interaction between K-casein and calcium sensitive c a s e in s ...... 3 B. Interaction between sulfated polysaccharides and proteins ...... 9

1. Chondroitin sulfate ...... 11 2. H eparin ...... 12 3. Carrageenan ...... 17

SCOPE OF INVESTIGATION...... 21

EXPERIMENTAL PROCEDURE...... 22

A. Protein sa m p le s ...... 22 B. Fractionation of carrageenans from Chondrus crispus . . 22

C. Polysaccharide samples ...... 25

D. Stabilization t e s t ...... 28

E. Determination of protein concentration ...... 29

F. Polysaccharide film s for infrared study ...... 29

G. Butter solution ...... 29

H. Sedimentation velocity ...... 30

v i TABLE OF CONTENTS co n tin u e d

Page

I. Viscosity measurement ...... 30

J. Density measurement ...... 30

K. Sedimentation equilibrium ...... 30

L. Mi H i pore electrophoresis ...... 31

M. Polyacrylamide gel electrophoresis ...... 31

RESULTS...... 32

A. Isolation and properties of carrageenan fractions ...... 32

1. F ra c tio n a tio n ...... 32 2. Electrophoretic pattern ...... 34 3. Monomer/polymer formation ...... 34 4. Infrared sp e ctra ...... 39 5. Stabilizing ability ...... 42

B. Factors affecting the s ta b ility of carrageenan/ag-casein s y s te m ...... 49

1. pH e f f e c t ...... 52 2. Heating time and temperature effects ...... 55 3. Polymer size e f f e c t ...... 60 (a) V is c o s it y ...... 61 (b) Molecular weight ...... 64 4. Concentration effects ...... 66 (a) Hydrocolloid concentration ...... 69 (b) Protein concentration ...... 69 5. Sedimentation velocity ...... 74 C. Interaction between synthetically sulfatedpolysaccharides and ag-casein ...... 74

1. Comparison of chemically derived polysaccharide s u lfa te s ...... 74 2. pH e f f e c t ...... • • • • ^0 3. Heat e f f e c t ...... 83 4. Concentration e f f e c t ...... 83

DISCUSSION...... 96 TABLE OF CONTENTS c o n tin u e d

Page

REFERENCES...... HI

APPENDIX...... H 8 LIST OF TABLES

Table Page

1. RECOVERY AND PROPERTIES OF FRACTIONATED CARRAGEENANS FROM CHONDRUS CRISPUS ...... 33

2. VISCOSITY AND MOLECULAR WEIGHT OF CARRAGEENAN FRACTIONS OF VARYING STABILIZING A B IL IT Y...... 65

3. SOLUBILITY OF FREEZE-DRIED STABILIZED COMPLEX FROM ALBG/as-CASEIN SYSTEM ...... 93

4. COMPARISON OF THE STABILIZING PROPERTIES OF KNOWN STABILIZERS OF as-CASEIN ...... 104

5. AN EXAMPLE OF THE COMPUTER OUT-PUT ...... 121

6. AN EXAMPLE OF THE IN-PUT DATA CARDS...... 125 LIST OF FIGURES

Fi gure Page

1. FAG-ELECTROPHORETIC PATTERNS OF as"CASEIN AND K-CASEIN...... 23

2. FLOW DIAGRAM OF THE PROCEDURE FOR THE FRACTIONATION OF CARRAGEENAN EXTRACT FROM CHONDRUS CRISPUS ...... 26

3. FLOW DIAGRAM OF THE PROCEDURE FOR THE FRACTIONATION OF k-CARRAGEENAN...... 27

4. ELECTROPHORETIC PATTERNS OF CARRAGEENAN FRACTIONS FROM CHONDRUS CRISPUS (CELLULOSE ACETATE) ...... 35

5. ELECTROPHORETIC PATTERNS OF ks“ AND Ki-CARRAGEENAN AT DIFFERENT CONCENTRATION (CELLULOSE ACETATE) ...... 3 7

6 . ELECTROPHORETIC PATTERNS OF k- AND x-CARRAGEENANS AT DIFFERENT CONCENTRATIONS (CELLULOSE ACETATE) ...... 4 0

7. INFRARED SPECTRA OF CARRAGEENAN FRACTIONS FROM CHONDRUS CRISPUS...... 43

8 . STABILIZATION OF as-CASEIN BY CARRAGEENAN FRACTIONS FROM CHONDRUS CRISPUS (0.15% p ro te in , pH 7.2 and 0.01 M CaCl2) ...... 50

9. EFFECT OF pH ON THE STABILIZING ABILITY OF K-CASEIN, k-carrageenan and X-CARRAGEENAN ...... 5 3

10. EFFECT OF HEATING TIME AT 85 C ON THE STABILIZING ABILITY OF K-CASEIN, K-CARRAGEENAN AND X-CARRAGEENAN . . 55

11. EFFECT OF TEMPERATURE OF THE STABILIZING ABILITY OF k- casein, k- carrageenan and X-CARRAGEENAN ...... 58

12. RELATIONSHIP BETWEEN VISCOSITY AND STABILIZING ABILITY . 62

13. RELATIONSHIP BETWEEN MOLECULAR WEIGHT OR CARRAGEENANS AND ITS STABILIZING A B IL IT Y...... 57

14. STABILIZATION OF Og-CASEIN BY K-CARRAGEENAN AT THREE DIFFERENT PROTEIN CONCENTRATIONS ...... 70 LIST OF FIGURES continued

Figure Page

15. EFFECT OF ag-CASEIN CONCENTRATION ON THE STABILIZING ABILITY OF K-CARRAGEENAN AND K-CASEIN ...... 72

16. SEDIMENTATION VELOCITY PAHERNS OF K-CARRAGEENAN, ag- CASEIN AND MIXTURE OF TWO IN THE ABSENCE OF CALCIUM IONS ...... , 75

17. STABILIZATION OF as-CASEIN BY SELECTED CHEMICALLY SUL FATED POLYSACCHARIDES ...... 77

18. EFFECT OF pH ON THE STABILIZING ABILITY OF SLBG.... 81

19. EFFECT OF HEATING TIME AT 85 C ON THE STABILIZING ABILITY OF SLBG AND SGG...... 84

20. EFFECT OF TEMPERATURE ON THE STABILIZING ABILITY OF SLBG...... 86

21. STABILIZATION OF as-CASEIN BY SLBG AT THREE DIFFERENT PROTEIN CONCENTRATIONS ...... 88

22. EFFECT OF as-CASEIN CONCENTRATION ON THE STABILIZING ABILITY OF SLBG...... 91

23. EFFECT OF DILUTION OF THE STABILITY OF THE SLBG/ag- CASEIN COMPLEX ...... 94 INTRODUCTION

The physical s ta b ility of flu id type foods is a determining factor in judging the quality as well as the s h e lf-life o f the products. In s ta b ility of the products is often caused by the incom p a tib ility of the components o f the flu id or by the unwarranted interaction between components. Some of the means to improve the s ta b ility of the flu id type foods include heat treatment, mechanical treatment and the use o f additives. Heat treatment, such as fore warming, is used in evaporated milk processessing to enhance the s ta b ility o f the products. Mechanical treatment by homogenization is applied to flu id foods of the emulsion type to prevent fa t separation. However, i t is not always possible to create a fu lly stable product by manipulation of processing steps and in such instances i t may be necessary to resort to the use of sta b ilizin g agents. The use of such additives is known as a beneficial practice in the manufacturing o f evaporated m ilk, chocolate m ilk, ice cream and other formulated flu id type beverages.

Most of the stabilizers used in the food industry are derived either from plants or marine algae. With the seasonal variation, age difference and d iffe re n t local environments of these plant

species, it is not surprising to find that the stabilizer extracts

perform d iffe re n tly from time to time. Moreover, in a product

1 2 consisting of proteins, lipids, carbohydrates and electrolytes, the in te ra c tio n between various components needs to be well balanced so that a stable system can be achieved. A slight inconsistency in performance of the stabilizer may offset the delicate balance of the system resulting in an unstable product.

The physical function of stabilizers has been recognized to depend in part upon their ability to build the necessary body characteristics of the product by increasing the viscosity of the f lu id and to prevent separation by in te ra c tin g w ith components existing in the different phases. Although the addition of stabi lizers is widely accepted in the food industry, no uniform guideline

is available pertaining to their proper use. In fact, the appli

cation of certain stabilizers in a particular product relies to a

large extent upon a rb itra ry t r ia ls . Thus, problems such as fa t

separation, syneresis, component sedimentation and gelation s till

occur very frequently in fluid type foods.

Therefore, the underlying purpose of this study has been to

explore methods for evaluation of the performance of sulfated

stabilizers, to search for products with uniformity of performance,

and to increase the basic knowledge concerning the in te ra c tio n

between these polysaccharides and proteins. LITERATURE REVIEW

Macromol ecu!ar interactions occur in all biological systems where proteins, lipids, carbohydrates and electrolytes exist together. The interaction between various components has been a

subject for extensive review by many investigators; for example,

Waugh (71) has treated protein-protein interaction with emphasis

on association, aggregation and dénaturation. Bettelheim-Jevons

(6) has reviewed complex formation between proteins and aminopoly

saccharides and mucopolysaccharides. Polysaccharide-lipoprotein

interactions have been described in detail with reference to

molecular complex formation between some chemically sul fated poly

saccharides and lipoproteins in a paper by Cornwell and Kruger (18).

The effect of cations on protein stability has been dealt with in

several reviews (28,45,52,61). The particular bindings and effects

of calcium on casein and serum proteins have been reviewed by

Greenberg (25). The present review deals with the specific protein- protein interactions in casein which lead to the formation of stable

micelles and the specific protein-polysaccharide interactions which

occur in complex biological systems.

A. Interaction between K-casein and calc i um sensitive caseins.

Casein, the acid-precipi table fraction of milk proteins, was

long considered to be a homogeneous substance. In 1939, Mellander

3 (44) observed the resolution of acid casein into three peaks by moving boundary electrophoresis at pH 8.6. He designated these

three peaks as a, 8, and y in order of decreasing mobility. Waugh and Von Hippel (72) later showed the a-casein actually contained

two fractions which subsequently were named K-casein and og-casein.

K-casein was found to be soluble in the presence of calcium ions, whereas otg-casein was strongly sensitive to calcium.

The distinction between sensitivity and insensitivity to

calcium refers to the behavior of casein fractions in the presence

of 0.01 M calcium chloride in the neutral pH range. Calcium-sensi

tive caseins tend to aggregate and precipitate in this ionic environ

ment, whereas insensitive caseins either remain soluble or form

stable, non-precipi table, colloidal aggregates.

The discovery by Z ittle (83, 88) that K-casein possesses the

a b ility to stabilize og-casein and 3-casein against calcium precipi

tation aroused much interest among milk protein chemists and has

been the basis fo r much of the accumulated knowledge of casein micelle

reactions. Several factors affecting the stabilizin g a b ility of

K-casein have been elucidated. These factors include heat treatment,

the presence of salts (particularly sodium chloride and calcium

chloride), pH and modification of side chain.amino acids on either .

K-casein or otg-casein.

Early work by Zittle (83) demonstrated that various prepara

tions of K-casein behaved differently towards heat treatment.

Some preparations were heat-stable and some were heat-labile, that 5 is, they lost their stabilizing ability after heating. Alais e t.a l.-

(1) isolated a caseino-glycopeptide by heating K-casein solutions at 120'C for 20 minutes. The amino acid composition of the heat- released caseino-glycopeptide was found to be nearly the same as that of the caseino-glycopeptide obtained by the action of rehnin.

Since the glycopeptide is the hydrophilic portion of the molecule, a release of this moeity would definitely be expected to impair the s ta b iliz in g a b ilit y o f K-casein. However, on the basis o f experiments involving less severe heat treatments (100"c for 5 minutes), Z ittle (83) has excluded the possibility of any release of glycopeptide as result of moderate heating. He interpreted his observation of the heat-labile K-casein as being a result of either the chemical manipulation during the preparation of K-casein or more likely the result of proteolysis by coprecipitated enzymes.

(70) In recent experiments, Z ittle (85, 86) reported that none of the K-casein prepared by the u re a -s u lfu ric acid method (87) were h e a t-la b ile . These preparations o f K-casein were, however, found to be h e a t-la b ile in the presence o f 0.06M sodium chloride or 0.01 M of calcium chloride solution. Z ittle concluded that the effect o f sodium chloride might be due to the reduction o f e le c tro s ta tic

repulsion between the charged K-casein by providing an io n ic

envelope by which K-casein could be brought closer for aggregation

during heating. In the case of the divalent cation, calcium, the

e ffe c t was probably to enhance the association between K-casein

molecules, therefore, the cross-bonding between K-casein could be 6 brought about more extensively by heat. When the salt concentration was increased, a solubilization effect was observed. At 0.15 M calcium chloride, no precipitation of K-casein occurred. The author speculated that in this case a direct effect of the salt on the heat induced aggregation process and to some extent a salting-in effect might have been responsible for this observed solubilization.

Experiments (85, 86) on pH effects revealed that at the pH range tested (6.2 to 7.9) in sodium chloride/imidazole buffer, the heat-

1ability was somewhat greater at the lower pH values. The explana tion advanced by Z ittle was that K-casein molecules carried less charge at lower pH, thus, greater interaction of the heated molecules could occur. The addition of reducing compounds like mercaptoethanol alone to the K-casein did not affect the heat-1 ability of K-casein.

However, when mercaptoethanol was introduced in the presence of salts,

the combined effect was greater than that of salts alone. I t is of

interest to notice that the addition of ag-casein in 1 : 1 weight

ratio will prevent heat-1 ability of K-casein in mercaptoethanol and

salts solution. The protective action of ag-casein presumably is

a specific one, since other proteins like a-lactalbumin show negative

result in preventing the heat-1 ability of K-casein.

Some of the early research in the area of K-casein/ag-casein

interaction focused on the hydrophilic carbohydrate portion of the

K-casein molecule. Thompson and Pepper (65) studied the effect of

neuraminidase (NANAse) on K-casein and reported that up to two thirds

of the NANA (sialic acid) could be removed without damage to the 7 stabilizing ability. They fe lt that the carbohydrate remaining a fte r NANA removal, phosphate and possibly d is u lfid e groups c o n tri buted to the ability to stabilize ag-casein against calcium precipi tation. In order to elucidate the role of phosphate in this inter actio n , Pepper and Thompson (49) dephosphorylated both K-casein and ag-casein with phosphatase. Dephosphorylation of K-casein did not change significantly its stabilizing power, but dephosphorylation of ag-casein impaired drastically its ability to be stabilized by K-casein. MacKinlay and Wake (43) studied the ro le o f d is u lfid e groups by modification of K-casein into S-sulfo derivatives and showed that the SO^-K-casein were s t i l l able to s ta b iliz e the ag-casein against calcium precipitation.

Since n eith er phosphates nor d is u lfid e groups ;. proved to be related to the stabilizing ability of K-casein, the attention was turned to the functional groups o f amino acids. Woychik (80) blocked the free amino groups o f K-casein w ith t r i f 1uoroace tylation.

Following this treatment, K-casein was not able to stabilize ag-casein in the presence of calcium ions, but this ability was fully restored a fte r removal o f the blocking groups. H ill and Craker (33) modified lysine to the extent of 2-3 lysine residues per molecule of K-casein and reported that at this level of substitution, k "asein retained its ability to stabilize ag- and 3-caseins against calcium precipita tion. Blocking of lysine residues on ag- and 3-caseins did not change th e ir a b ility to be s ta b iliz e d by K-casein. S im ilar re su lts were observed for the modification of arginine side chains. Hill (32) reported that 8 there was little effect on the stabilizing ability of K-casein when .

1.5 arginine residues were m odified per mole o f K-casein, and the modified ag-casein was fu lly stabilized by K-casein too. Although

H ill's observations have demonstrated that neither lysine nor arginine are involved in the stabilization of ag-casein at the specified level of modification, this does not totally rule out the possible participation of these two amino acids side chains in the k - and ag-casein interaction. The possibility that a greater extent of modification is needed in order to impair the stabilizing a b ility of K-casein has been observed in the case of tyrosine

residues. Woychik and Wondolowski (82) reported th a t n itra tio n o f

up to four tyrosines in K-casein was without effect, but that

additional nitration progressively decreased the stabilizing ability

of the protein, and the ability was totally impaired after all eight

tyrosines were nitrated. Nitration of only four of the twelve

tyrosines in ag-casein le ft this protein unable to be stabilized

against calcium precipitation. Z ittle (84) reported when all

histidines and 70% of the trypotophan were photooxidized, K-casein

lo s t it s s ta b iliz in g a b ility completely. Woychik and Wondolowski

(81) coupled para-K-casein with blood serum albumin (BSA) which

resulted in the formation o f soluble para-K-BSA deriva tives. The

complex derivatives possessed the a b ility to s ta b iliz e ag-casein

suggesting that the interaction between ag-casein and K-casein is

a property of the para-K-casein molecule.

In summary, studies on the interaction between calcium sensitive caseins and K-casein fay various investigators have indicated that free amino groups (80), tyrosine (82), h is tid in e (84) and tryptophan

(84) in K-casein molecule are involved in this interaction. The site of association would fae located in the para-K-casein rather than in the glycomacropeptide (81). Results also rule out the p a rtic ip a tio n o f phosphate (49), d is u lfid e (43, 79) and to some ex tent sialic acid (65) of the K-casein molecule. Although arginine

(32) and lysine (33) have faeen reported to fae insignificant in re spect to the stabilizing ability, further research is needed in order to completely rule out any participation. In the case of ag-casein, evidence has suggested th a t a t le a s t phosphate (49) and tyrosine (82) are involved in K-casein/ag-casein interaction.

B. Interaction between sulfated polysaccharides and proteins.

Polysaccharides are universally present in all living organisms.

They are most abundant in higher orders of land plants and in sea weeds where they maintain the structural integrity of cell walls.

In animals, they are important components of cartilage, of body

fluids, of skin, and of mucosa. Polysaccharides are polymers of sugar units and can fae divided

into two general classes (73) according to the type of their repeat

ing units. CLASSIFICATION DESCRIPTION EXAMPLE

Homoglyean Containing only one Cellulose type of sugar u n it. Dextran

Heteroglycan Containing two or more Carrageenans kinds o f sugar units. Locust bean gum

The physical characteristics of polysaccharides are to a large extent due to structural arrangements and a classification is possible on this basis;

CLASSIFICATION DESCRIPTION EXAMPLE Linear Sugar units arranged Cellulose in straight chains. Carrageenans

Branched Sugar units arranged Guar gum in branched form. Locust bean gum

From the point of view of potential protein interaction, it is important to recognize differences in polysaccharides due to any variation in functional groups such as in the following scheme:

CLASSIFICATION DESCRIPTION EXAMPLE

Neutral Carrying no ionic group. Locust bean gum

Cationic Carrying positive charges. Cationic guar

Anionic Carrying anionic groups Carrageenans such as sulfate. heparin n 1. Chondroitin sulfate.

The strongly acidic sulfated polysaccharides which are the subject of interest in the present study occupy a prominent place among polysaccharides carrying negative charges. The major naturally occurring sulfated polysaccharides are chondroitin sulfate, heparin and carrageenan. I t should be recognized, however, that chondroitin sulfate and carrageenan each represents groups o f polysaccharides varying in some detail with respect to structure but, generally with sim ilar physiological functions.

The different chondroitin sulfates are structurally related in the following manner:

TERM REPEATING UNITS OCCURRENCE

Chondroitin sulfate A D-glucuronic acid, Cartilage (Chondroitin 4-sulfate) 2-Acetamido-2-deoxy-4- Animal bone 0-sulpho-D-gal actose

Chondroitin sulfate B L-iduronic acid, Pig skin (B-heparin, or 2-Acetami do-2-deoxy-4- Lung Dermatan sulfate) 0-sulpho-D-galactose

Chondroitin sulfate C D-glucuronic acid, Cartilage (Chondroitin 6-sulfate) 2-Acetamido-2-deoxy-6- Animal bone 0-sulpho-D-galactose Umbilical cord

Chondroitin sulfate D ** See footnote Cartilage

Infrared spectrum of chondroitin sulfate D is identical to that of chondroitin sulfate C, but chemical analysis shows higher sulfate content than that of chondroitin sulfate C. Structure of this compound is not yet completely established (11). 12

Chondroitin sulfate along with the protein, collagen, is a major constituent of cartilage, the structural building blocks of tissue. The basic unit of this complex consists of a protein core, approximately 4,000 A in length, to which are attached by covalent linkages about 60 chains of chondroitin sulfate of molecular weight

50,000 and with a length approximately 1,000 A (11). Anderson,

Hoffman and Meyer (3) reported that the hydroxyl group of serine is involved in linking the polysaccharide. Roden (5) has indicated that the D-galactose of chondroitin sulfate probably participates in this protein-polysaccharide linkage.

2. Heparin.

Heparin, the widely distributed mucopolysaccharide found in high concentration in liver, lung and spleen, possesses important biological properties, notably as a blood anticoagulant. Since its

discovery some fifty years ago, heparin has received considerable

attention, but despite intensive investigations, the basis of its

anticoagulant activity has not been fully established. The backbone structure of heparin resembles that.of chondroitin

sulfate. I t contains equimolar amounts of D-glucuronic acid and

2-amino-2-deoxy-D-glucose residues. I t is distinguished from

chondroitin sulfate by having the amino-sugars N-sulfated rather

than N-acetylated and by its high 0-sulfate content. The linkage

in heparin has bean reported by Uolfrom, Vercellotti and Horton (77)

as a, a, -D-(l-4)-hexuronic acid linkage to 2-amino-2-deoxy-D-glucose.

Wolfrom, Wang and Honda (78) have also reported that besides N-sulfate

there are two additional sulfate groups per tetrasaccharide unit. 13

These are located on the C-6 of 2-amino-2-deoxy-D-glucose residues. •

The D-glucuronic acid residues are not sulfated. The term heparin generally refers to the sodium salt of heparinic acid.

The blood anticoagulant activity of heparin is an exceedingly

important physiological function of this polysaccharide. To fully

appreciate this function, i t is necessary to consider some of the

reactions involved in the blood clotting mechanism. The primary

blood clotting material is fibrinogen which is present in the blood

plasma. Fibrinogen, upon the action of the enzyme, thrombin, yields

fibrin and fibrinopeptide. Following this event, fibrin undergoes

polymerization and a coagulation of the blood is the end result.

I t is obvious that thrombin cannot be present as such in circulating

blood, however a precursor of thrombin, prothrombin, is present.

The change of prothrombin to thrombin requires the presence of calcium

ions and of the substance, thromboplastin. There are two sources of

thromboplastin in the body, that is (a) the tissues and (b) the plasma

its e lf. When the tissue is injured, thromboplastin is promptly

liberated, particulary from the blood vessels. The released thrombo

plastin will induce the subsequent steps leading to the formation of fibrin and blood will be clotted in few minutes to prevent excessive

bleeding (5).

The coagulation of blood is an example of gel formation and

resembles in many ways the coagulation of milk by rennin, even to the

extent of the onset of syneresis. A comparison between rennin action

on casein and thrombin action on fibronogen is given in the following: SUBSTRATE/ENZYME BONDS CLEAVED PRODUCTS COAGULATION

K-casein/renm'n Phe-Met Para-casein, Ca-linkages Glycoinacropeptide

Fibrinogen/thrombin Arg-Gly Fibrin Polymerization Fibrinopeptides

In addition, i t should be noticed that both of the released peptides contain sialic acid and hexosamine, and a positive relationship exists between the s ia lic acid content and the substrate s ta b ility .

The role of heparin in blood anti coagulation presumably is to in terfere with the production o f thromboplastin and to in h ib it the throm bin action on fibrinogen. Early reports by Howell (34) disclosed that heparin did not possess any antithrombin a c tiv ity per se. However, when heparin is added to blood, i t generates the anti thrombin a c tiv ity by complex!ng with a thermolabile constituent of the plasma. Porter, et. al. (51) have confirmed that heparin has no anti thrombin a c tiv ity at levels less than 0.5 unit/m l. However, in the presence of plasma protein fractions containing a cofactor much smaller quantities exhi bited strong a c tiv ity . As to the nature of the cofactor, they were able to show that i t had a molecular weight sim ilar to albumin and an is o le ctric point sim ilar to y-globulin. From this evidence, they speculated that possibly ag-globulin of plasma would be the cofactor.

Thus, they postulated that thrombin is inactivated by the blocking of active enzyme sites with a suitably oriented heparin-cofactor complex

(50). Thrombin a c tiv ity may be released from the inactive complex by the addition o f a heparin antagonist such as protamine. 15

Altliough i t has been demonstrated that heparin can participate in the inhibition of thrombin, other investigators (7, 20, 57) have accumulated evidence for yet another reaction of heparin by which this polysaccharide in low concentration prevents the formation of blood thromboplastin, a substance needed in conversion of thrombin from its precursors. Thus, Douglas (20) and Shanbe/gs et. al. (57) have concluded that the possible main action of heparin is to inter fere with Idle production of thromboplastin rather than to inhibit

the thrombin action of fibrinogen.

Heparin contains a high amount of ester sulfate which confers

to strong polyelectrolytic character to this molecule. Stivala and

co-worker (63) have demonstrated that desulfation of heparin greatly

impairs its anticoagulant activity. Yet, Wolfrom et. al. (76) report

ed that sulfation of a highly active heparin resulted in a decrease

in activity of more than 50% rather than an increase. Meyer, Piroue

and Odier (46) reported that dextran sulfate having a sulfate con

tent equal or higher than heparin exhibited anticoagulant activity

considerably below that of heparin. Thus, i t is evident that the

degree of sulfation per se is not a controlling factor in the bio

logical activity of heparin, and i t may be worthwhile speculating i f perhaps the location of sulfate may be more important than the

total sulfate content. Doane and Whistler (19) tested the anticoagu

lant activity of sulfated amylose, sulfated amylopectin and sulfated

guaranand reported that sulfation did increase the activity especially

in the branched polysaccharide, amylopectin.. However, even here the

activity was low in comparison with heparin. 16

Since none of the polysaccharides with 0-sulfate groups approach ed the anticoagulant a c tiv ity of heparin, Doane and Whistler (19)

postulated that N-sulfate groups in heparin are more critica lly

associated with anticoagulant a c tiv ity than 0-sulfates. Velluz and

co-workers (67) have reported that heparin from which N-sulfate has

been removed is completely inactive as an anticoagulant. Walton

et. al. (68) have also obtained data which supports this hypothesis.

However, Warner and Coleman (69) have demonstrated that selective

N-sulfation of chitosan resulted in a product which exhibited no

a c tiv ity . On the contrary, when chitosan was sulfated on both N-

and 0- (75%) positions, it showed considerable anticoagulant activity.

Stivala and Liberti (62) have reported a positive relationship

between binding capacity of heparin and its biological a c tiv ity .

They indicated that besides sulfate groups, carboxyl groups may also

contribute to the anticoagulant a c tiv ity .

Laurent (38) has reported that anticoagulant a c tiv ity increases

with increasing molecular weight fo r fractions of single preparations

of heparin. Lasker and Stivala (37) have also observed a near-linear

relationship between biological activity and molecular weight of

heparin. A sim ilar result has also been obtained by Walton and

co-workers (68). In a detailed study, Liberti and Stivala (40)

have observed that the anticoagulant a c tiv ity of heparin increases

with molecular weight up to about 10,000. Beyond that the increase

in molecular weight shows no appreciable difference in a c tiv ity .

Under acidic conditions, heparin rapidly loses its anticoagulant 17 a c tiv ity . Jensen, Snell man and Sylven (35) have reported that heparin retains only 44% (and in some preparation 28%) of its original activity when treated with warm acetic acid. Chemical analysis indicates no sig nifican t change in sulfur content of the acid treated heparin. Molecular weight study (35) as revealed by diffusion constant shows a s lig h t decrease from 17,000 to 16,600 afte r inactivation. However, sedimentation velocity experiment indicates an increase in $20 value from 2.07 to 2.7. This evidence suggests that inactivation of heparin in acidic condition is not associated with any depolymerization but rather a structural re arrangement has occurred during Inactivation.

In summary, the actual mechanism of heparin's action may involve the in it ia l formation of a heparin/cofactor comples which in h ib its specific enzyme active sites on thrombin or i t may involve in addition an interference with the production of thromboplastin.

However, the principal contributing factors in the anticoagulant a c tiv ity of heparin are (a) location of sulfate groups; (b) degree of dissociation of sulfate and carboxyl groups and (c) the molecular shape and size.

3. Carraqeenan.

To quotate from the Encyclopedia o f Chemical Technology:

The use of carrageenan, an increasingly important seaweed hydrocolloid, can be traced back at least two centuries. Long before the polysaccharide its e lf became commercially available, housewives along the coasts of Ireland and France gathered the sun-bleached fronds of Chondrus crispus and cooked them with milk to make a pudding, called blancmange. 18

In 1871, Bourgade (10) f ir s t developed an alcoholic precipitation process to extract carrageenans from the seaweeds. But i t was not u n til 1937 that carrageenan was produced commercially. Since then, many patents (9, 64, 75) have been granted to various investigators fo r the use of carrageenan in pudding, chocolate m ilk, ice cream and other food products. Moirano recently patented (47) a process in which calcium sensitive carrageenan was used in dessert gels to suppress the syneresis e ffect caused by locust bean gum and K-carrageenan. Although many useful applications o f carrageenan have been developed during the past three decades, very few reports in the lite ra tu re give reasons which account fo r the action of carrageenan.

Statements lik e "Much of the value of carrageenan lie s in its a b ility to react with protein in milk" (16) are a ll too common in the tech nical lite ra tu re . Even as late as 1969, a textbook (22) presumed to be authorative, resort to a superficial speculation about the involvement of 3,6-anhydrogalactose and sulfate groups without a shade of documentation. In fact, there has been an emerging aware ness that carrageenan polysaccharides possess unique properties due to th e ir chemical constitution and considerable volume of data on the functional properties has been accumulated.

Hansen (29) observed that carrageenan when added to milk does not exist in free dispersion, but interacts with the micellar pro

teins. The interaction was shown to be dependent upon the presence of calcium ions. Subsequently, i t was reported (30) that carrageenan

possesses the a b ility to stabilize ag-casein against calcium precipi 19 tation, an effect sim ilar to that achieved by the action of ic-casein.

The interacted product of carrageenan and ag-casein was shown to be more tolerant to the influence o f heat as well as calcium ions.

By using polyacrylamide gel electrophoresis, Grindrod and

Nickerson (25) were able to show that carrageenan completed with

K-casein preventing i t from entering PAG. The same author found that carrageenan interacted with bovine serum albumin and altered the migration pattern on PAG. Hidalgo and Hansen (31) studies the interaction of carrageenan and several other anionic polysaccharides with whey proteins. The interactions were s tric tly dependent upon pH, ionic strength, and the relative proportions of the colloids.

On this basis, the authors concluded that the interactions were of a general type, occurring between soluble proteins and the poly electrolytes, involving only ionic attractions, presumably through long-range electrical forces acting between parallel double layers.

In contrast, the interaction between as-casein and K-carrageenan is a specific interaction which depends on other factors than simple e lectrica l attractions. The structural requirements o f carrageenans have been partially elucidated. At least three factors are contribut ing to the action of carrageenan; that is , the location of ester sulfate groups in the pyranose units, polymer size and the backbone structure of the polymer. Sulfate groups located on C-6 were found to be detrimental to the micelle forming a b ility , whereas those on

C-2 and C-4 were effective. Recently, Chakraborty and Hansen (12) have studied numerous combination of. carrageenan and ag-casein 20 complexes in the electron microscope. As a result of this study, they have postulated a micro-gel lik e system fo r the stable carrageenan/og-casein complex. In this system, carrageenan forms a stable three-dimensional network in which Og-casein are interspersed. De ta ils of the binding between og-casein and carrageenan are as yet not known. The concept that i t is possible to form a three-dimen sional network from the linear strands of carrageenan has been ad vanced by Rees (53). SCOPE OF INVESTIGATION

This study was undertaken to investigate the stabilizatio n of

calcium sensitive caseins by sulfated hydrocolloids. The specific

objectives were:

1. To increase the basic knowledge concerning the stabilizatio n of

Ug-casein by carrageenan against calcium precipitation.

2. To search fo r hydrocolloids with better performance in s ta b ili

zation of ag-casein and to elucidate th e ir mode of action.

3. To employ equilibrium ultracentrifugation for the determination

o f molecular weights o f the hydrocolloids and to compile a computer

program for the analysis o f the data. EXPERIMENTAL PROCEDURE

A. Protein samples.

The proteins used in this study were as-casein and K-casein.

These were obtained by standard fractionation procedures of milk obtained from Ayrshire cows at the University Dairy Farm. The purity of the preparations was checked by polyacrylamide gel electo- phoresis. The preparations were stored in small portions as their

frozen solutions at -15 "C. 1. as-casein (Variant s).- — Prepared according to the procedure by

Z ittle and Custer (87). The electrophoretic pattern is shown in

Figure 1.

2. K-casein — Prepared by extraction of whole casein with urea-

su lfu ric acid, supplemented with ethanol precipitation according to

the method o f Z ittle r and Custer (87). The electrophoretic pattern

is shown in Figure 1. B. Fractionation o f carrageenans from Chondrus crispus.

A supply of dried seaweeds of Chondrus crispus was obtained

through the courtesy of Mr. J. Claude Wallace, Manager of the

Marine Plants Experimental Station, from the coast of Miminegash,

Prince Edward Island, Canada. Samples had been previously washed,

picked and mechanical dried. Extraction o f carrageenan in the

laboratory was started by digesting 20 grams of dried seaweeds in

1900 ml d is tille d water fo r 2 hours at 90 ± 2°C with repeated 22 FIGURE 1. PAG-ELECTROPHORETIC PATTERNS OF a^-CASEIN AND K-CASEIN. 24

fî 25 agitation. The thick slurry was centrifuged at 3,000 RPM in a

International Centrifuge Model SBV for 20 minutes. The supernatant was collected and the sediment was re-digested and centrifuged as

before. A ll supernatants were combined and then slowly mixed with

twice the volume o f isopropyl alcohol under vigorous agitation. A

fibrous product appeared which could be easily removed manually.

This carrageenan extract was washed twice in 500 ml ethanol and

twice in 300 ml ethyl ether. The crude extract was firs t air-dried

and then dried in vaccum overnight at room temperature (54, 60),

The procedure given in Figure 2 was used to separate k- and

X-components from the crude extract. Fractionation of and K-j-

carrageenan was performed according to the procedure (Figure 3)

developed in our laboratory (41).

C. Polysaccharide samples.

Five carrageenan fractions obtained from the fractionation of

dry seaweed were used in th is study. These were: (a) crude extract,

(b) potassium sensitive fraction, (c) potassium insensitive fraction,

(d) potassium/calcium sensitive fractio n, and (e) potassium sentitiv e /

calcium insensitive fraction. In addition, sulfated locust bean gum

and sulfate guar gum prepared by Kelco Company of California were

also used. These polysaccharides reportedly had an average of one.

sulfate per hexose unit and was preferentially sulfated on C-6

position (4). Sul fated starch (D. S. 0.92) and alkali modified

sulfated locust bean gum were obtained from Marine Colloids, Inc. (27). Dissolve 7 grams of unfractionated carrageenan in 2100^ml d is tille d water.

Add 700 ml KCl solution containing 41.7 grams KCl.

Centrifuge at 3,000 RPM fo r 30 minutes in a International Centrifuge Model SBV.

Supernatant I» Precipitate the polysaccharide with 2 volumes of isopropyl alcohol. 1

Wash twice in 300 ml ethyl alcohol. i I Wash twice in 300 ml ethyl ether. I I Air-dried then dry under vaccum overnight. i 1 K-carrageenan x-carrageenan

FIGURE 2. FLOW DIAGRAM OF THE PROCEDURE FOR THE FRACTIONATION OF CARRAGEENAN EXTRACT FROM CHONDRUS CRISPUS. 0.2% K-carrageenan

Add equal volume of 0.04 M CaCl2

Centrifuge at 35,000 G fo r 30 minutes at 3-5 “C.

Supernatant

Disperse into d is tille d water, to make concentration approximately 0 . 2%

Dialysis against 0.1 M NaCl ■; 3-5 "C fo r’ ■ 1 day. ■

Dialysis against distilled water at 3-5"C fo r 1 day.

15 ,000 GCentrifuge 15,000 GCentrifuge fo r 30 minutes at room temperature.!

Sediment, Supernatant i f any, discard. I' Freeze dry Freeze dry

Ks ■carrageenan -carrageenan

FIGURE 3. FLOW DIAGRAM OF THE PROCEDURE FOR THE FRACTIONATION OF K-CARRAGEENAN. 28 D. Stabilization test.

The experimental design for the stabilizatio n test is illu s tr a t ed in the following table;

Stock solution:

A : 0.5 O;;-casein in water, pH 7.3.

B : 0.05% (or 0.1%) hydrocolloid in water, pH 7.0 -

C : 0.1 M CaClg solution.

be No. A B H2O C Ratio of hydrocol ag-casein

ml. ml. ml. ml.

1 3.0 0 6.0 1.0 . 0

2 3.0 1.0 5.0 1.0 1/30

3 3.0 2.0 4.0 1.0 1/15

4 3.0 3.0 3.0 1.0 1/10

5 3.0 4.0 2.0 1.0 1/7.5

6 3.0 5.0 1.0 1.0 1/6

7 3.0 6.0 0 1.0 1/5

8 3.0 0 7.0 0 Control

9 0 3.0 7.0 0 Control

10 0 0 9.0 1.0 Control

The test was performed by f ir s t mixing the protein and hydro-

colloid solutions with the required amount o f water. The calcium

solution was injected la s t by a 1 ml. syringe. Following the

calcium addition, the tubes were incubated at 30°C fo r 15 minutes 29 followed by centrifugation at 3,000 G for 5 minutes and decanting of the supernatant into a dry test tube. One drop of 20% NaOH was added to clarify the decanted supernatant. The protein concentration was measured spectrophotometrically at 280 mu.

E. Determination of protein concentration.

Protein concentration was determined spectrophotometrically on a Hitachi Perkin-Elmer Model 139 Spectrophotometer equipped with a

digital readout. Extinction coefficients for ag-casein was deter mined experimentally by comparing the absorbance at 280 mu for

different dilutions of the protein. The protein content of the

casein preparations were determined by Micro-Kjeldahl analysis.

A linear relationship was observed between the UV-absorbance at

280 mu and the Kjeldahl values for this protein. The extinction

coefficients was calculated from the slope of this line. An

extinction coefficient of 1.22 for 0.1% solution was used for estima

tion of K-casein concentration (87).

F. Polysaccharide films for infrared study.

Infrared studies were applied to determine the location of ester

sulfate in carrageenans. For this purpose, dry films were prepared

according to the procedure described by Lin and Hansen (42).

G. Buffer solution. Two different buffer solutions were used in this study.

1. Cacodyl ate buffer: This buffer was used in the sedimentation

velocity study and had the follov/ing,composition; 0.15 M NaCl, 0.05 30 M cacodylic acid and the pH was adjusted to 7.0 by NaOH (48).

2. Malonate buffer: This buffer was used in the Mi H i pore electro phoresis for differentiating carrageenan samples. It has the follow ing composition: 0.1 M malonic acid, 10% DMSO (v/v), 15% alcohol

(v/v), 0.02 M CaClg and the final pH was adjusted to 3.0 by Ca(0H)2 powder.

H. Sedimentation velocity.

In order to study the interaction between ag-casein and k - carrageenan in the absence of calcium ions, sedimentation velocity experiments were carried out at 20 in a Beckman Model E Analytical

Ultracentrifuge using an AN-D rotor at 44,000 RPM for 2 hours for ag-casein, K-carrageenan and mixtures of two. All samples were

prepared in cacodyl ate buffer. Measurement of the peak movement was

accomplished by using a Nikon Profile Projector.

I. Vi scosi ty-measurement.

The viscosity measurements used for the calculation of $20 were

conducted in cacodyl ate buffer at 30"c by using a Cannon-Fenske

Viscometer type 50 E 452.

0. Density measurement.

The density measurements were performed at 20"c by using a pycnometer. All samples were prepared in cacodyl ate buffer.

K. Sedimentation equilibrium.

Simultaneous measurements of partial specific volume and mol ecu- 31 lar weight of polysaccharide samples were accomplished by sedimentation equilibrium experiments in H2O and D2O solutions according to the method developed by Edelstein and Schachman (21). A multi-channel

Yphantis cell was used in the experiment. All calculations were completed by using the computer program tabulated in Appendix.

L. Mil 11 pore electrophoresis.

Mi 11ipore electrophoresis was conducted in malonate buffer by a modification of a method previously used for hydrocolloids (13).

Prior to the application of samples, cellulose acetate strips were soaked in malonate buffer for 10 - 20 minutes. Sample applicators were allowed 1 minute contact time on the strips. Then, the entire

Millipore electrophoresis unit was incubated at 40“c for 30 minutes.

Electrophoresis was conducted at 100 volts for 15 minutes at 40"c.

The strips were then stained in 1% Alcin Blue in 3% acetic acid solution (pH 3.0) for 15 minutes followed by a washing with 5% acetic acid solution 5 - 6 times.

M. Polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis was accomplished by using

7% gel in a EC 470 Vertical Gel Electrophoresis Cell. Tris-EDTA-

borate buffer (pH 8.3) was used and both samples and gel contained

4.5 M urea and 0.3% mercaptoethanol. Detail procedures were refered to the Technical Bulletin 128 by E-C Apparatus Corporation. RESULTS

A. Isolation and properties of carrageenan fractions.

The primary objective o f this research was to seek further understanding o f the mechanism of interaction between sulfated .poly saccharides and milk proteins. The underlying concept has been that the protection of calcium sensitive caseins against the precipitating influence by calcium ions is a key element fo r maintaining physical s ta b ility o f the caseinate system. Therefore, the experimental plan was specifically directed towards obtaining information about (a) the properties and the performance of components in the carrageenan complex and of the chemically sulfated locust bean gum, and (b) using this information fo r delineating the mode of action of the poly saccharides with calcium sensitive caseins.

1. Fractionation.

A fractionation of carrageenan from Chondrus crispus was achieved by f ir s t using Smith's (60) potassium method following by a treatment with calcium chloride at low temperature to separate the potassium sensitive material into calcium sensitive and calcium insensitive fractions. Table 1 gives the recovery and sta b ilizin g a b ility of these fractions. The recovery of crude carrageenan extract from 20 grams of dry seaweed was 8,7, 9.1 and 9.4 grams for three independent trials. Fractionation of 7 grams of crude extract yielded 2.15-2.68

32 TABLE 1. RECOVERY AND PROPERTIES OF FRACTIONATED CARRAGEENANS FROM CHONDRUS CRISPUS.

SENSITIVITY as-CASEIN ^ FRACTIONS RECOVERY TO CATION STABILIZED K+ Ca++ U) Crude extract 43.5-47.0 79.8

X-carrageenan 30.7-38.3 - 37.0 K-carrageenan 58.6-61.3 + ± 98.4

Kj-carrageenan 45.0 + 76.1

* Experiment conditions: protein = 0.15% carrageenan = 0.03% pH 7.2 and 0.01 M CaCl 2 . 34 grams o f A-carrageenan and 4.10-4.29 grams of K-carrageenan. Fraction a tion o f 2.5 grams of K-carrageenan yielded 1.11 grams o f K^-carragee nan and 0.95 grams of Kg-carrageenan.

2. Electrophoretic patterns.

Millipore electrophoresis for all five carrageenans obtained from the fractionation of Chondrus crispus is given in Figure 4.

The unfractionated material showed a somewhat diffuse pattern with three separate zones clearly in evidence. Although the subfractions were s till characterized by m ultiplicity of zones, the intensity of the individual zones varied in a definite manner so that K-carrageenan resembled the fa s t moving band and A-carrageenan the d iffu se band in the crude extract. The pattern for K^-carrageenan was sim ilar to that of K-carrageenan except the intensity of the stationary zone was more prominent. In the case of Kf-carrageenan,

the pattern was complex; the d iffu s e pattern resembled somewhat the

crude extract but displayed at least 4 separated bands. Overall,

the electrophoretic results suggested that the fractionation scheme

did achieve separation of fractions which were distinctly different,

although the separation are not yet pure.

3. Monomer/polymer formation.

Figure 5 shows a series of electrophoretic patterns of Kg- and

Kj-carrageenan in the concentration range of 0.05% to 0.5%. It is

evident that at 0.5% concentration K$-carrageenan gives a very intense

stationary zone together with a very faint fast moving band. With ELECTROPHORETIC PATTERNS OF CARRAGEENAN FRACTIONS FROM CHONDRUS CRISPUS. (CELLULOSE ACETATE)

(Note the diffuse zone in X-carrageenan which is characteristic for this fraction.) CRUDE EXTRACT K X . FIGURE 5. ELECTROPHORETIC PATTERNS OF Kg- AND -CARRAGEENAN AT DIFFERENT CONCENTRATION. (CELLULOSE ÂCETATE)

(Note the prominent stationary zone increasing with concentration.) Ks K; i^S '^1 n-S 0.05% 0 . 1% 0.2% 0.3% 0. 4 % 0.5% 39 decreasing concentration the intensity of the stationary zone also decreased, while the relative intensity of the fast moving band increased. At low concentration (0.05%) only the fast moving band was observed without any trace of a stationary zone. Similar observations were obtained for the other x-carrageenan varieties with no appreciable change in the X-carrageenan as shown in Figure

6. These electrophoresis results suggest that the stationary zone may be an aggregated form of the polysaccharide and not a separate component. At high concentration, these polysaccharides apparently polymerize in such a way as to preclude electrophoretic migration either due to a loss of net electrical charge or due to massive gelation. When the concentration of the polysaccharide is low, monomerization is favored, thus, all the material moves out from

the stationary zone. This concentration dependent behavior of

carrageenan may well be of importance in considering its physical

functions.

4. Infrared spectra.

In order to determine the location of the ester sulfate groups

in the pyranose rings of these five fractions, infrared spectra were obtained and the results are presented in Figure 7. K-carrageenan

(Figure 7-A) exhibited a strong 850 peak indicating the presence of

C-4 sulfate (8) and a small 805 peak showing C-2 sulfate on 3,6-

anhydrogalactose. The intense 930 peak reflected the presence of

3,6-anhydrogalactose which typically is weak or absent in the case

of X-carrageenan (Figure 7-B). For X-carrageenan, the board 800-850 ELECTROPHORETIC PATTERNS OF k- AND X-CARRAGEENANS AT DIFFERENT CONCENTRATIONS. (CELLULOSE ACETATE)

(Note again the characteristic diffuse zone for X-carrageenan and also the intensity of the stationary zone which is concentration dependent.) K. X. K X. K X.

0.05% 0.1% 0.3% 0. 5 °/< 42 peak represented the combination of three d ifferent types of sulfates.

In general, the spectra observed fo r k - and A-carrageenan were in good agreement with other studies (8, 42).

Although the crude extract was a combination of k - and A-carragee nan the spectrum (Figure 7-C) resembled fa r more K-carrageenan than an expected intermediate between k - and A-carrageenan. The sub-fractions of K-carrageenan, Kg- and K^-carrageenan (Figure 7-D,E), resembled th e ir parent material except the 805 peak was absent in K-j-carrageenan and the 850 and 930 peaks were re la tive ly less intense in Kg-carragee nan. In comparison with e a rlie r findings from fractionation of carra geenan (41), K-j-carrageenan showed good agreement with previous re su lts, however, the Kg-carrageenan of the present study did not show the resemblance to i-carrageenan which was previously noticed.

Thus, the results have shown that fractionation may lead to va ri

able results. A possible cause may be the differences in raw material due to geographical, seasonal, and age variations noticed by Black et.

a l. (8). A point o f interest is the apparent effect of K-carrageenan

on n u llify in g the IR characteristic o f A-carrageenan in the crude ex

tra c t which after a ll contained more than one-third of the A-component.

We speculate that interactions between A- and K-component may lead to

conformational changes which may affect not only the IR peaks (15) but

may have a pronounced effect on the sta b ilizin g property of such

mixtures.

5. Stabilizing a b ility .

Curves illu s tra tin g the stabilizatio n of as-casein by these five INFRARED SPECTRA OF CARRAGEENAN FRACTIONS FROM CHONDRUS CRISPUS.

Spectrum A ; K-carrageenan B : A-carrageenan C : crude extract D : Kg-carrageenan E : Ki-carrageenan 6.0 7.0 microns 9 0 10.0 (2.0 14.0 0.0 0.0

I 0

.2 0

30 .3 0

,50 .5 0

70 .7 0

1600 1400 1200 1000 8 0 0 FREQUENCY (cm“ l) 7.0 MICRONS 90 |Q.o 12.0 14.0 0.0 0.0

I 0

2 0 .2 0

30 .3 0

50 .5 0

70 . 7 0

1600 1400 1200 1000 8 0 0 FREQUENCY (cm“ ‘ ) 6.0 7.0 m i c r o n s 9 0 10.0 12.0 14.0 0.0 0.0

I 0

. 2 0 .2 0

3 0 .3 0

.50 .5 0

,70 .70

0 0 1600 14001200 1000 8 0 0 FREQUENCY (cm”*) 6.0 7.0 microns 90 10.0 12.0 14.0 0.0 0.0

I 0

.2 0 .2 0

3 0 .3 0

.50 .5 0

70 .70

00 1600 1400 1200 1000 8 0 0 FREQUENCY {cm”*) 6.0 7.0 microns 9 0 10.0 12.0 0.0 0.0

I 0

o .2 0 .2 0

30 .3 0

.50 .5 0

70 .70

CO 1600 1400 1200 1000 80 0 FREQUENCY (cm"**) 49 fractions are given in Figure 8 . The highly effective K-carrageenan completely stabilized ag-casein at the hydrocolloid/protein ra tio of

0.25, whereas the re la tive ly ineffective X-carrageenan gave only 60% stabilizatio n of otg-casein at the highest ra tio (0.33) tested. Stabi liza tio n by the crude extract was intermediate to k- and X-carrageenan.

The sub-fractions of K-carrageenan, Kg- and k^-carrageenan, exhibited no appreciable difference in th e ir performance when compared to one another. However, when compared with K-carrageenan, these fractions were slightly less effective, and the difference became more prominent at the low concentration of carrageenan tested. One possible reason may be that fractionation may have altered the properties of the polysaccharides. When alii mixture of Kg- and

K^-carrageenan was subjected to the stabilizatio n te s t, no improve ment in the sta b ilizin g a b ility was observed suggesting that such alteration would be an irreversible one.

According to these results, the s ta b ilizin g a b ility of K-carra geenan does not reside in a specific component of the complex. Rather, since K-carrageenan was better than either of the sub-fractions, i t supports the concept that its s ta bilizing function may relate to a tertiary structure of the polysaccharide.

B. Factors affecting the s ta b ility o f carrageenan/as-casein system.

Since studies on the isolation and properties of carrageenan

fractions revealed that the physical functions of these polysaccharides might well relate to th e ir te rtia ry structure and that exhaustive

purification might destroy the stabilizing ability, further investi- FIGURE 8 . STABILIZATION OF «s-CASEIN BY CARRAGEENAN FRACTIONS FROM CHONDRUS CRISPUS. (0.15% protein, pH 7.2 and 0.01 M CaClg) 100 :-CARRAGEENAN

lixture ^ Kg & <1

Crude extract

•Kj-Fraction

X-Carrageenan

0 0.1 0.2 0.3

RATIO OF CARRAGEENAN/as-CASEIN (w/w) 52 gâtions were directed towards the exploration of specific factors which would affect the stability of the interacted product between protein and the k - and X-carrageenan fractions, and a comparison of the results with the native protective colloid, K-casein.

1 . pH effect.

The effect of pH on the stabilizing ability of k- and X-carrageenans and K-casein is given in Figure 9. At the pH range of 5-9,

K-carrageenan exhibited a 100% stabilizing ability at the ratio tested. Below pH 5, the stabilizing ability of K-carrageenan sharply decreased, and at pH 3 or below, it was no longer effective.

In the case of X-carrageenan, the stabilizing ability was lost gradually as the pH was decreased. Below pH 4, it s performance was close to that of K-carrageenan. For the natural protective colloid, K-casein, a complete precipitation was observed at the

pH range o f 3.5 - 5.0. When the pH was e ith e r above or below th is

range, stability was rapidly regained.

The change in stabilizing ability as a function of pH may be

related to: (a) an aggregation of ag-casein in which the protective

action by the hydrocolloid is excluded, or (b) an alteration in

the conformation of the polysaccharide to a less effective type.

Since K-casein, k - and X-carrageenan all gave drastically different

response to the pH change, i t is u n lik e ly th a t an a lte ra tio n o f

«s-casein is determining the change in s t a b ilit y o f the complex.

Therefore, i t is lik e ly th a t the pH -effect was caused by an a lte ra tio n

of the structure of the protective colloid. FIGURE 9. EFFECT OF pH ON THE STABILIZING ABILITY OF K-CASEIN, k - carrageenan and a - carrageenan . •

(Protein concentration 0.15%, hydrocolloid 0.0375% and 0.01 M CaCl 2 ) STABILITY OF os-CASEIN (%) 55

2. Heating time and temperature effects.

The effect of heating time at constant temperature on the stabi lizing ability of K-casein, k - and A-carrageenan is shown in Figure

10. K-casein showed a d ra stic decrease in the f i r s t ten minutes o f heating, after that the decrease was more slow but s till progressive; at the end of one hour heating at 85"C, 60% of its stabilizing ability was lost. This observation was consistent with the K-casein study by

Z itt le (83). For k - and A-carrageenan at the higher ratio (0.25) of hydrocolloid/protein tested, no significant change in stability was found. However, heat e ffects were observed fo r these hydrocolloids at the lower hydrocolloid/protein ratio ( 0 . 1 ) where the complex lo s t about 35% of its stability as a result of heat treatment.

The effect of temperature at constant heating time on the stabi lizing ability of these protective colloids is shown in Figure 11.

For K-carrageenan, the response was a s tra ig h t, negative lin e a r re la tionship for both concentrations. At lOO'c, 30% of the protection was lost. This type of relationship indicates that secondary forces, probably hydrogen bonding, would be im portant fo r the s ta b iliz in g

reaction. The curve for A-carrageenan also exhibited a temperature

dependence, but i t was not lin e a r and it s performance reached maximum

at 60**C. However, the difference was only approximately 10%.

The curves for K-casein showed a distinct peak at 20*^0 indicating

a lower stabilizing ability for this native protective colloid at o “c

and none a t 100“C. This observation was in agreement with the report

(36) th a t the association between K-casein and ag-casein is not FIGURE 10. EFFECT OF HEATING TIME AT 85'C ON THE STABILIZING ABILITY OF K-CASEIN, K-CARRAGEENAN AND X-CARRAGEENAN.

(Protein concentration 0.15%, pH 7.2 and 0.01 M CaCl 2 . Note the values in parentheses denote ra tio o f hydrocol 101 d/protein.) 100

K"Carrageenan (0.25)

K-Carrageenan ( 0 . 1 )

A-Carrageenan ( 0 .2 b)

40 K-Casein (0,25)

20 A-Carrageenan (0.1)

HEATING TIME (MIN. AT 85*0) EFFECT OF TEMPERATURE ON THE STABILIZING ABILITY OF k- casein, K-CARRAGEENAN AND X-CARRAGEENAN.

(Experimental conditions were same as given in Fiaure 10 except heating time was fixed fo r 15 minutes.) 100

Carrageenan ( 0 . 1 2 )

60 K-Carrageenan (0.10)

K-Casein (0.1)

20 X-Carrageenan (0.1)

0 20 40 60 80 100

TEMPERATURE (°C) 60 favored at high or low temperatures.

The e ffe c ts on s t a b ilit y o f the complexed casein system as a result of heat treatment might be due to heat induced changes either in the « 5 -casein or in the protective colloids. According to the findings by Kenkare (36), ag-casein is extremely heat stable whereas

K-casein is not. Therefore, the observed effects are more likely a result of heat-induced changes in the protective colloid, whether this be K-casein or one of the carrageenans.

3. Polymer size effect.

Hydrocolloids are characterized by their extreme viscosity- building properties and many of their uses have been correlated with this property. Therefore, the possibility must not be over looked that the stabilization of calcium sensitive proteins by hydrocol!oids may be related to an increased solvent viscosity whereby the rate of aggregation is retarded. Restriction of flow

is an important physical function of viscosity. According to Stoke's

law, ^ = 6 rniY, where is friction factor, n is the viscosity of the

solution and Y is the radius of the particle in solution (59), i t is

apparent that the friction factor, (|), for a particle increases as the

viscosity of the solution increases. Consequently, the rate of com

ponent separation or aggregation, will be strongly retarded by the

incorporation of viscosity building additives. The viscosity of

hydrocolloid solutions is a consequence of molecular size and confor

mation. The close relationship between viscosity, molecular size and 61 configuration of the polymer can be expressed by the equation pro posed by Mark and Houwink, (59) [q ] = KM^^, where [q ] is the in tr in s ic viscosity, M is the molecular weight, K is a constant, and a another constant which depends on the configuration of the polymer. This equation demonstrates the im portant combined e ffe c ts o f the molecular weight and configuration on the viscosity.

(a). Viscosity.

Previous findings (41) from an acid degradation study of carragee nans have revealed that for K-carrageenan the stabilizing ability de creased as the degradation time was increased. Further e ffo rts were made to secure a number o f K-carrageenan samples from Chondrus crispus differing in viscosity and the stabilization curves for these samples are given in Figure 12. The re su lts again demonstrated th a t a re la tio n sh ip existed between the s t a b ilit y of the complex and the carrageenan v is c o s ity . However, the re su lts did not provide any clue as to whether the dependency resulted from a co n trib u tio n o f v is c o s ity to the solventsystem or from va ria tio n s in the polymer

structure or size of the hydrocolloid. It is important to make

th is d is tin c tio n , because i t was previously pointed out th a t some

degree of stabilization may be achieved through restriction of flow

due to viscosity. If, however, viscous effects may be discounted

the s ta b iliz a tio n by carrageenans must occur by a mechanism in which

not only the functional groups are im portant but where polymer

size and conformation exert a controlling influence. Further studies

were, therefore, made on the molecular weight relationship to stabi- 62

FIGURE 12. RELATIONSHIP BETWEEN VISCOSITY AND STABILIZING ABILITY.

(Protein concentration 0.15%, K-carrageenan 0.03%, pH 7.2 and 0.01 M CaCle- The v is c o s ity values are for a 1.5% solution at 75 C.) 100

0 1 10 100 1000 VISCOSITY (CPS) 6 4 lizing ability of the protective colloids.

(b). Molecular weight.

Since molecular weight measurements by equilibrium ultracentri fugation are generally independent of molecular shapes and viscosity effects, this method was selected for analysis of the polysaccharides in this study. Carrageenans are polyanions and their charge groups tend to influence the molecular weight determination. In order to minimize the charge effect, 1 M sodium chloride solution was used as the dispersing solvent throughout the study. According to Van Hoi de

(66) under this high ionic strength condition, the charge effect is no longer significant and any correction for this effect is not needed. The results of the molecular weight studies are presented in

Table 2 together with viscosity data. After equilibrium was achieved

(generally after 2 days), all of the plots of logarithmic fringe number vs r^ were examined for linearity. In none of the cases were these preparations homogeneous as revealed by the curvature of the logarithmic plot. Because of the heterogeneity, i t was not deemed justifiable to attempt the simultaneous determination of the partial specific volume in these experiments and all calculations were performed with the value 0.50 as reported by Cook et. al. (17).

According to this table, there is no relationship between stabilizing effectiveness and viscosity and, therefore, viscous effects may be discounted in the stabilization mechanism. Although the molecular weights were variable the results in the table indicate that stab ili- TABLE 2. VISCOSITY AND MOLECULAR WEIGHT OF CARRA6 EENAN FRACTIONS OF VARYING STABILIZING ABILITY

Stabilizing strength in descending order ninh* Molecular weight (Mw)

C. crispus (k fraction) 1 1 .6 107,000

G. stel lata (unfractionated) 14.4 300,000

E. spinosum (i fraction) 7.2 2 0 0 ,0 0 0

G. pistil lata (k fraction) 10.5 350,000

C. crispus (X fraction) 14.4 95,000

G. pistil lata (X fraction) 2 0 .6 300,000

P. rotundus (unfractionated) 5.1 600,000

* Inherent viscosity values were taken from data reported by Black et. al. ( 8 ). 66 zation was not favored by the higher molecular weights, since

Spearman's rank correlation coefficient was -0.39. The relationship between stabilization of ag-casein by carrageenans of different molecular weights was examined further and the results are shown in

Figure 13. The plot revealed that maximum stabilization occurred in

the molecular weight range of 100,000 - 300,000. When the molecular weight of hydrocolloids was either higher or lower than this range,

the effectiveness decreased. This observation is in excellent agree

ment with our previous finding (42) from acid degration studies of

X-carrageenan from 6^. p istilla ta . I t should be noticed that ic-carra-

geenan from G. pistillata fell outside this plot but that hydrolysis

was able to improve the performance.

Overall, these results indicate that the apparent relationship

between the viscosity and the stabilizing ability of carrageenan is a secondary effect reflecting the molecular size differences. This

is important because such a relationship emphasizes the polymer size

and conformation as controlling factors in stabilization of calcium

sensitive casein and may well be an useful guide for the selection of appropriate stabilizers. To the best of our knowledge this is

the firs t time that.this relationship has been proved.

4. Concentration effects.

The stability of the interaction products between calcium

sensitive proteins and hydrocolloids may be expected to vary, not

only with the relative hydrocolloid concentration, but also with the

absolute concentration of proteins. 6 7

FIGURE 13. RELATIONSHIP BETWEEN MOLECULAR WEIGHT OF CARRAGEENANS AND ITS STABILIZING ABILITY

(0.15% as-casein, 0.0375% carrageenan, pH 7.2 and 0.01 M CaCla) 1. K-Carrageenan from Chondrus crispus (Batch A)

2. K-Carrageenen from Chondrus crispus (Batch B)

3. K-Carrageenan from Chondrus crispus (Batch C)

4. Carrageenan extract from Eucheuma spinosum

5. K-Carrageenan from Chondrus crispus (Batch D)

6. Carrageenan extract from Gigartina stellata

7. K-Carrageenan from Gigartina pistillata 6 8

100

MOLECULAR WEIGHT (xl0“4) 69

(a). Hydrocolloid concentration.

Figure 14 shows the stabilization of different levels of as- casein by increasing amounts of K-carrageenan. It was evident that maximum stability occurred uniformly at the ratio of carrageenan/ protein of 0.2. Therefore, the stabilization depends not on the absolute concentration of hydrocolloid, but on the amount supplied relative to the protein. This information further supports the con cept that the mechanism of stabilization involves a binding of hydro colloids to proteins in specific amounts and definitely not viscosity e ffe c ts . However, i t was apparent th a t maximum s t a b ilit y was dependent upon the absolute amount of protein, therefore, this effect was examined in more d e ta il.

(b). Protein concentration.

Figure 15 shows the plot of ag-casein stability against protein concentration at the constant ratio of carrageenan/protein of 0 . 2 . A rapid decrease o f the s t a b ilit y o f the complex was observed when the protein concentration was increased. At the highest concentration,

only 20% of the protein remained soluble. In the case of the native

protective colloid, K-casein, an in itia l decrease of 2 0 % sta b ility was

observed a t 0 . 6 % protein concentration followed by increasing stabi

lity when the total protein concentration was further increased. The

observed difference o f K-carrageenan and K-casein in response to

protein concentration changes firs t of all indicates a difference in

their mode of action but also suggests that the concentration effects

are operative on the hydrocolloid rather than on the as-casein. FIGURE 14. STABILIZATION OF ag-CASEIN BY K-CARRAGEENAN AT THREE DIFFERENT PROTEIN CONCENTRATIONS.

(pH 7.2 and 0.01 M CaCl 2 .) 100

0.3% as-Casein

S 3

0.6% as-Casein I

0.1 0.2 0.3 0.4 RATIO OF K-CARRAGEENAN/as-CASEIN (w/w) FIGURE 15. EFFECT OF ag-CASEIN CONCENTRATION ON THE STABILIZING ABILITY OF K-CARRAGEENAN AND k- casein.

(Hydrocol1oid/as“ Casein = 0.2, pH 7.2 and 0.01 M CaCl2.) 100

5 S

G 0.3 0.6 0.9 1.2

CONCENTRATION OF as-CASEIN (%) 74

5. Sedimentation velocity study.

In order to study i f the complex formation between K-carrageenan and ag-casein occurs in the absence of calcium ions, sedimentation velocity experiments were performed on ag-casein, K-carrageenan as well as mixture of two. The corrected S2o,w otg-casein (0.94%) was 7.34 S and for K-carrageenan (0.15%) 4.40 S. For the mixture of as-casein (0.94%) and K-carrageenan (0.15%), two peaks were clearly in evidence as shown in Figure 16. The broad fast moving peak had a S20,w of 7.96 S corresponding to as-casein, whereas the sharp, slow moving peak had a S2 q,w of 4.39 S equivalent to that of K-carrageenan.

I t is evident that complex formation does not occur in the absence of

calcium ions. These results are in agreement with our previously

reported free boundary electrophoresis experiments on sim ilar system

(30); however, in lig h t of newer data, we may now reject the possibi

l i t y that a minor component participates in this interaction.

C. Interaction between synthetically sulfated polysaccharides and as-casein. The stabilizatio n of calcium sensitive casein by protective

colloids other than native K-casein was f ir s t reported (30) to be

lim ited to the carrageenan type polysaccharides. However, further

studies revealed that some synthetically sulfated polysaccharides also

possessed this a b ility .

1. Comparison of chemically derived polysaccharide sulfates.

Figure 17 shows the stabilization of as-casein by five polysaccha

rides sulfated by chemical means. I t was evident that both sulfated FIGURE 16. SEDIMENTATION VELOCITY PATTERNS OF K-CARRAGEENAN, Og- CASEIN AND MIXTURE OF TWO IN THE ABSENCE OF CALCIUM IONS.

A. K-Carrageenan after 72 minutes at 44,000 RPM.

B. ag-Casein a fte r 80 minutes at 44,000 RPM.

C. Mixture of two a fter 64 minutes at 44,000 RPM. 76

B FIGURE 1 7 . STABILIZATION OF as-CASEIN BY SELECTED CHEMICALLY SULFATED POLYSACCHARIDES.

(Protein concentration 0.15%, pH 7.2 and 0.01 M CaCl 2 ) SLBG 100

Alkali Modified SLBG 0 --

SG6

Sul fated cellulose .Q ______o------O

Sul fated starch

0.1 0.2 0.3 0.4

RATIO OF HYDROCOLLOID/as-CASEIN (w/w) 79 cellulose and sulfated starch were totally ineffective. Sulfate guar gum gave a 85% stabilizing ability at the highest ratio (0.4) of hydrocolloid/protein tested, however, its effectiveness was poor at the low concentration of hydrocolloid. In contrast, sulfated locust bean gum (SLBG) showed a remarkably high performance (85%) even at the ratio of hydrocolloid/protein of 0.067. This performance at low concentrations was even better than that of K-carrageenan from Chondrus crispus. Complete stabilization was achieved at the ra tio of hydrocolloid/protein of 0.2. The turbidity of the resulting solution was more transparent than that of the K-carrageenan/protein systems indicating a smaller particle size of the complex.

Alkali modification of SLBG impaired its stabilizing ability from 10 - 60% with the damage more prominent at the low concentration of the hydrocolloid used. This response was drastically d iffe re n t from that observed fo r X-carrageenan from Chondrus crispus which we reported showing improvement in the stabilizing ability after alkali modification (42),

These results demonstrate that the stabilizatio n of calcium sensitive casein is not lim ited to the carrageenans, but is a property which may be shared with polysaccharide sulfates from other sources.

However, the reaction is nevertheless s t ill specific since not all polysaccharide sulfates promote sta b iliza tio n . I t is not lik e ly that the mode of action o f these other polysaccharides parallel the action of carrageenans in every d e ta il. However, i f the hypothesized mechanism fo r K-carrageenan (41) holds true, the prediction may be made that other effective suifated polysaccharides would also exhibit unique conformational characteristics where the sulfate groups occur in specified positions. A comparison of the chemical structure of th e ir parent materials is given in the following table:

POLYSACCHARIDES GLYCOSIDIC LINKAGES MOLECULAR WEIGHT (Mw)

K-carrageenan a-1,3, 3-1j4 107,000

Locust bean gum 3-1,4, branch a-1 ,6 310,000 ( 2 )

Guar gum 3-1,4, branch a-1 ,6 2 2 0 ,0 0 0 ( 2 )

Cellulose 3-1,4 1 ,0 0 0 ,0 0 0 (73)

Amylose a-1,4 2,400,000 (74)

Amylopectin a-1,4, branch a-1 ,6 1 0 ,0 0 0 ,0 0 0 (74)

2 . £H effect.

The effect of pH on the stabilizing a bility of SLBG is shown in

Figure 18. I t was evident that complete stabilizatio n occurred over the entire pH range with only a s lig h t reduction at very low pH values. This remarkable acid tolerance was a property to ta lly d iffe r ent from that o f the carrageenans and of K-casein. These results are an indication that the mode of action of SLBG is different from carra geenans and the conformation of the polysaccharides may be a co n tro ll ing factor in the complex formation. I t does not seem possible that the sulfate groups are d ire ctly involved in this reaction because at the pH corresponding to the pKa of ester sulfate o f -^2 .0 , the loss of stability was only slight. FIGURE 18. EFFECT OF pH ON THE STABILIZING ABILITY OF SLBG.

(Curves of K-casein and K-carrageenan are includ ed fo r comparison. Protein concentration 0.15%, SLBG concentration 0.0375% and 0.01 M CaClg.) 100

■Carrageenan

0 2 4 6 8 10 3. Heat e ffe c t.

The effect of heating time at 8 5 is given in Figure 19. For

SLBG at the higher ratio of hydrocolloid/protein tested, heating up to one hour did not show any change in the s ta b ilit y o f the complex.

In the case of the lower ratio (0.1) of SLBG/protein tested, the firs t

30 minutes heating did not affect the effectiveness of the protective colloid, and subsequent heating up to one hour resulted in only 1 0 % loss in stability. Therefore, the heat tolerance of the SLBG/protein was also greater than that for carrageenans.

Figure 20 shows the effect of heating temperature at constant heating time on the stabilizing ability of SLBG. It is apparent th a t maximum effectiveness o f SLBG was w ith in the range o f 60 - 80*0 and, outside th is range the s t a b ilit y decreased. These results are in marked contrast to the findings fo r K-carrageenan in which case the effectiveness decreased linearly with temperature. Therefore, there is a possibility that hydrophobic bonding may be involved in the SLBG s ta b iliz a tio n . For carrageenans, however, th is s itu a tio n is different.

4. Concentration effect.

Figure 21 shows the effect of hydrocolloid concentration on the s t a b ilit y o f the complex at three levels o f protein concentrations.

It was evident that sta b ility depended not only on SLBG concentration but also on protein concentration. When protein concentration was

increased to 0.6%, maximum s t a b ilit y obtained was only 40%. This

concentration dependency was fu rth e r demonstrated by the p lo t o f the FIGURE 19. EFFECT OF HEATING TIME AT 85 C ON THE STABILIZING ABILITY OF SLBG AND SGG.

(Protein concentration 0.15%, pH 7.2 and 0.01 M CaCl 2 . Note the values in parentheses denote ra tio o f hydrocolloid/protein.) SLBG ( 0 .2 5 ) 100 f

SLBG (0.1)

SGG (0.25)

g i SGG (0.1)

0 10 20 30 40 50 60

HEATING TIME (MIN. AT 85”C) FIGURE 20. EFFECT OF TEMPERATURE ON THE STABILIZING ABILITY OF SLBG.

(Experimental conditions were same as given in Figure 19 except heating time was fixed for 15 minutes.) 100 Ratio of SLBG/protein =0.1

Ratio of SLBG/protein = 0.05

100

TEMPERATURE (°C) STABILIZATION OF as-CASEIN BY SLBG AT THREE DIFFERENT PROTEIN CONCENTRATIONS.

(pH 7.2 and 0.01 M CaCl 2 -) 100

0.6% as-Casein I

0 0.1 0.2 0.3 0.4

RATIO OF SLBG/as-CASEIN (w/w) 90 s ta b ility o f the complex vs protein concentration as shown in Figure

22. A near lin e a r decrease in s t a b ilit y was observed when protein con centration was increased. At 1.2% protein concentration, only 20% was stable in the presence o f calcium. These re su lts were id e n tica l to the response o f K-carrageenan demonstrated previously (Figure 15),

and, suggested that although the type of bonding involved in the

complex formation may be d iffe re n t as revealed by the pH and heat

effects, there are other factors involved which leads to identical

response to the concentration e ffe c t in both systems.

Another approach to study the concentration e ffects was freeze-

drying the stable supernatant from SLBG/protein (0.15%) complex

followed by redissolving the dry complex in water a t several concen

tra tio n s . The re su lts as summarized in Table 3 indicated a very poor

re s o lu b ility o f the complex a t a ll concentrations tested and demon

strated th a t the loss o f s ta b ilit y due to high complex concentration

is an irreversible process. The irreversible loss of stability was

further confirmed by an experiment which did not involve freeze-drying

but simply d ilu tio n o f a concentrated complex to a lower concentration.

The results presented in Figure 23 indicated that not only was no

improvement in s t a b ilit y observed, but a s lig h t decrease appeared as

the result of dilution. FIGURE 22; EFFECT OF as-CASEIN CONCENTRATION ON THE STABILIZING ABILITY OF SLBG.

(SLBG/as-casein = 0.2, pH 7.2 and 0.01 M CaClg.) STABILITY OF as-CASEIN (%) TABLE 3. SOLUBILITY OF FREEZE-DRIED STABILIZED COMPLEX FROM SLBG/as-CASEIN SYSTEM.

Sample description Ca++ Total os-casein Soluble concentration as-casein

M (%) (%)

Before freeze drying 0 .0 1 0.15 98.6

0 .0 1 0.30 83.8

0 .0 1 0.60 43.1

0 .0 1 1 .2 0 18.7

After freeze drying 0 .0 1 0.15 6 . 2

0 .0 1 0.30 3.6

0 .0 2 0.30 3.4

0 .0 1 0.60 1.4

0.04 0.60 2.3

0 .01 1 .2 0 1.5

0.08 1 .2 0 1 . 8

** Freeze dried samples prepared from stabilized complexes of 0.15% protein. FIGURE 23. EFFECT OF DILUTION ON THE STABILITY OF THE SLBG/ as-CASEIN COMPLEX.

(Curves of 0.15% and 0.6% proteins are included fo r comparison. pH 7.2 and 0.01 M CaCl 2 .) 100 r -

0.6% then diluted to 0.155

.1 0.2 0. 0.4

RATIO OF SLBG/as-CASEIN (w/w) DISCUSSION

The concept that food stabilizers interact in some unique manner with other constituents in food was given substantial support in previous studies (30, 41) when i t was found that carrageenan could promote micelle formation in casein. This discovery was a ll the more sta rtlin g because fo r the f ir s t time i t became possible to lin k the gum with a definite function other than those related to viscosity.

I t was found previously (42) that only sulfated polysaccharides with alternating a-1,3 and g-1,4 glycosidic linkages possessed this proper ty. A requirement with respect to the optimum position of sulfate in the polysaccharide were derived by considering the findings of a number of polysaccharides fo r which details of structure were already known. The effectiveness of C-4 or C-2 sulfates in contrast to the antagonistic effect of C -6 sulfate suggested steric effects. Evidently, the ester sulfate groups served as binding sites and to some extent the binding was mediated through divalent ions, since a complex fo r mation was not detected in the absence of calcium ions.

Although the requirements with respect to the structure fo r the carrageenan have been determined (42), details of the interaction mechanism have remained largely unknown. Some studies (12) have

pointed to the importance of the formation of a three-dimensional network of the polysaccharide fo r interaction through entrapment of

96 97 the protein, but s till nothing has been known about the binding sites which would be involved. Another study (58) has implicated hyaluronic acid as a possible component in the casein which could s p e c ific a lly interact with carrageenan. It is of interest to notice that hyaluronic acid possesses points o f s im ila r ity w ith carrageenan in it s primary structure since it is composed of alternating 3-1,3 and 3-1,4 glyco sidic linkages. The possibility that the specific binding could be one o f a hybrid double h e lix between hyaluronic acid and carrageenan must, the re fo re , not be overlooked. However, since ag-casein has been

reported (55) to contain no carbohydrate at a ll, this theory can not

be developed further until the presence of carbohydrate in ag-casein

has been firm ly established.

The matter of the stabilization mechanism of carrageenan was

considerably complicated in the present study firs t by our results

which revealed a gross heterogeneity of K-carrageenan and next by our

findings that sulfated locust bean gum also could stabilize ag-casein.

Although it was pointed out that some degree of stabilization may be

achieved through the restriction of flow due to viscosity, our results

from the polymer size studies have discounted the possible involvement

of viscosity as the controlling factor in the stabilization mechanism

because there was no re la tio n s h ip between v is c o s ity and the e ffe c tiv e

ness o f the hydrocolloid.

The observation that one of the most effective carrageenans, the

K-fraction, was heterogeneous suggested two possible mechanisms for

its mode of action in stabilizing calcium sensitive caseins: (a) the 9 8 independent action of one of the constituents or (b) the combined action of the constituents through association leading to quarternary structures. The results of studies from isolation and properties of carrageenan fractions revealed that none of the sub-fractions was as effective as the original K-carrageenan. Therefore, the possibility that the stabilizing ability resides in a single component of the

K-carrageenan must be rejected. However, the concept that its stabilizing function may relate to a quarternary structure of the polysaccharide is s t ill questionable because K-carrageenan was better than the combination of the subfractions. Nevertheless, the results from zone electrophoresis showed evidence of concentration dependent polymerization which gave indirect support for the concept of in te r action between subfractions. Further evidence for possible interac

tions were noted in the IR spectra for mixtures of carrageenan frac

tions in which K-carrageenan apparently changed the IR characteristics

of the X-fraction.

Polysaccharide-polysaccharide interaction for sulfated galactans

have been firmly established by published work (53) and may relate directly to the observation made in this study. According to X-ray

diffraction studies and conformation analysis of carrageenan, Rees (53)

concluded that double helix formation was possible for carrageenan

systems. He related the mechanism of gelation of these polysaccharides

to this conformation, particularly to the creation of junction zones

involving tertiary and quarternary structures. The double helix for

mation according to Rees depends primarily on the chemical structure 99 of the sulfated polysaccharides and i t is of interest to notice that • the requirements are identical to those we have found necessary for stabilization of calcium sensitive caseins (42). Furthermore, Chakra- borty and Hansen (12) observed in the electron microscope the ultra structure of carrageenan/as-casein systems and found evidence of a structural network. They observed that ag-casein existed as electron dense aggregates (1,000-1,500 A) entrapped by interconnecting strands of relatively electron transparent K-carrageenan. The ag-casein aggregates were distributed as discrete particles in this three dimen sional network with none of very lit t le protein in evidence in the polysaccharide strands between the particles. A shematic diagram is given in the following to illustrate the postulated involvement of double helix in the stabilization mechanism. The fundamental concept

Random coil of K-carrageenan Gel I Gel II

Og-casei n aggregate.

carrageenan double helix bundles.

ULTRASTRUCTURE OF K-CARRAGEENAN/gg-CASEIN SYSTEM (12). TOO of this model is that the loose ends of the double helices serve as junction zones to connect other strands of polysaccharides to form the three dimensional network. The interconnecting bridges are bundles of K-carrageenan double helices which possess l i t t l e or no protein reactivity as the result of self-aggregation. Unattached loose ends serve to "trap" as-casein micelles and keep them apart, so that further aggregation of protein under the influence of calcium w ill be prevented.

The present investigation has produced further support fo r the model proposed by Chakraborty and Hansen (12). Our findings that optimum performance in stabilizatio n was associated with a definite range of the molecular weight of the carrageenans from 1 0 0 ,0 0 0 -

300,000 can be best explained by the proposed model. On the assump tion that physical separation of ag-casein by K-carrageenan is the major criterion fo r a stable complex, low molecular weight fractions w ill have "short arms" (double helix bundles) which w ill not be able

to keep the ag-casein particles s u ffic ie n tly separated to prevent

aggregation; thus, the resulting complex w ill have poor s ta b ility .

In the case of high molecular weight fractions, the in e rt regions

of the double helix bundles become too long, and as a consequence,

the available reactive zones become proportionally fewer; therefore,

the stabilization becomes inefficient. On the basis of this model, one would expect that increasing

concentrations o f protective colloids would confer increased s ta b ility

to the casein by providing more reactive zones. This, however, was 101 not the case in our studies because concentration-dependent polymeri zation of the polysaccharides became a lim iting factor. The results from polymer/monomer tra n s itio n studies by electrophoresis have revealed that at high concentrations, the polysaccharide polymerizes in such a way as to preclude electrophoretic migration, either due to a loss of net electrical charge or due to mass gelation. For neutralized or gelled protective colloids, stabilizing ability would c e rta in ly be poor because they would lack su ita b le binding s ite s .

The results from the studies involving concentration effects on the stabilizing ability or the ag-casein were consistent with this view.

The observed difference between K-carrageenan and K-casein in response to concentration changes suggested that these effects are operative on the polysaccharide rather than on the ag-casein, giving additional support to the concept that carrageenan conformation is the controlling fa c to r.

A drastic decrease in stabilizing a b ility of K-carrageenan was revealed when the pH was lowered and below pH 3.0 the carrageenans were completely in e ffe c tiv e . Since double h e lix formation is highly dependent on pH, it is within expectation that a shift in pH value would affect the stabilizing ability of carrageenans. Lehninger (39) has reported that poly-L-glutamic acid exists as a random coil at neutral pH because it s carboxyl groups are charged and tend to repel each other; however, below pH 4.0 the d issociatio n o f the carboxyl

groups is suppressed and poly-L-glutam ic acid a tta in s e xclusively the a -h e lix form. I t is tempting to draw an analogy from th is example to 102 explain the pH effect on the performance of K-carrageenan in our study.

Below pH 3.0, the ester s u lfa te groups o f carrageenan are s ig n ific a n tly less charged; thus, double helix formation may be so favored that the inert double helix regions become too long leading to an inefficient stabilization as described earlier.

Another important factor affecting the double helix formation is temperature. In general, heat does not favor hydrogen bonding (39), the re fo re , the double helices s ta rt to unwind during increasing temperature and eventually become random coils. The fact that the protective a b ility of K-carrageenan was impaired by increasing temper ature would therefore also be consistent with the possible involvement of a double helix conformation in the stabilization mechanism.

The result from free boundary electrophoresis (30) and analytic u ltra c e n trifu g a tio n showed no complex form ation in the absence o f

calcium ions suggesting that the major role of calcium ions is to

neu tralize charge groups o f ester s u lfa te so th a t the charge repulsion

forces are eliminated permitting double helix formation. This effect

may be f a ir ly s p e c ific , because based on the X-ray d iffra c tio n

studies, Rees (53) has observed that double helix formation for k -

and i-carrageenan was favored in the presence o f cations which cause

gelation o f solution such as K"^, NH^"*", Cs"*" and Rb"*" but not Na"^ and Li'*’ .

Although Rees did not include Ca'*”*’ in his study, this divalent ion

has a potent gelling effect on both K-carrageenan and on our isolated

Kg-carrageenan. Therefore, it is very likely that the presence of

Ca'*"*' w ill promote double h e lix form ation. 103

Thus, the findings from the studies involving factors affecting the s ta b iliz a tio n o f ag-casein by carrageenan point to a mechanism in which the most im portant fa c to r is the physical structure o f the carrageenan. This mechanism, we believe, is in sharp contrast to the action o f the native system o f K-casein in which hydrophobic forces between d e fin ite groups may be involved(23, 24).

It is important to realize that the stabilization of calcium sensitive caseins by carrageenan is f a ir ly sp e c ific and th a t most native as well as chemically sulfated polysaccharides are totally without stabilizing effect. The group of ineffective sulfated poly saccharides includes fucoidan, chondroitin sulfates and heparin as

demonstrated previously (42) and also sulfated cellulose and sul fated

starch as found in the present study. The only sulfated polysacchar

ides besides the carrageenans which were found to be e ffe c tiv e in

stabilizing calcium sensitive caseins were the synthetically sulfated

lo cust bean gum (SLBG) and sulfated guar gum (S 6 G). The s ta b iliz a tio n

or protection of ag-casein against the precipitating influence of

calcium ions remains, therefore, a unique reaction and it is relevant

to search for points of sim ilarity as well as dissim ilarity among the

effective protective colloids. Table 4 contains a concentrate of the

stabilizing properties of known stabilizers of ag-casein.

The stru ctu ra l ch a ra cte ristics o f the e ffe c tiv e galactomannans

are quite different from those of the carrageenans. They consist

mainly of mannose joined by B-1,4 glycosidic linkages with galactose

units branching at specific positions along the mannose chain. These TABLE 4. COMPARISON OF THE STABILIZING PROPERTIES OF KNOWN STABILIZERS OF ois-CASEIN.

STABILIZER/ SOLUBLE STABILIZERS as-CASEIN as-CASEIN TYPE OF INTERACTION (wt/wt)

Detergents: (24)

Cety U r i methyl- Dissociation o f caseins followed by ionic ammonium bromide 1.5 97 interaction. No turbidity or micelle formation.

Tween 20 23.0 53 Dissociation of caseins followed by hydrophobic interaction. No turbidity or micelle formation.

B rij 35 Same as Tween 20.

Proteins:

K-casein 0.25 100 Prim arily hydrophobic interactions. (23)

Para-K-casein/BSA Probably equal to K-casein. Not reported. (81)

Gelatin (23) 5.5 86 Ionic interaction, possible occlusion in gel network.

Polysaccharides:

K-carrageenan Binding o f caseins to the three dimensional network formed by junctions of double helices o f carrageenan. Specific binding not known.

Sul fated Entrappment o f caseins in the three dimensional galactomanna network formed by the branched galactose sub (SLBG & S6 G) units. Specific binding possibly hydrophobic. 105 units of galactose are a- 1 ,6 linked and appear fo r every four mannose units in SLBG. In S 6 G, a sim ilar unit branch appears at every other mannose subunit. Based on the chemical structure reported in the lite ra tu re (2), electron microscopic observation ( 1 2 ) and the results from the present

study, a model is proposed in the following diagram to explain the

interaction mechanism between SLBG and calcium sensitive caseins.

SLBG

Branch subunit (Galactose 6 -sulfate)

as-casein aggregate

PROPOSED SEGMENT OF THE SLBG/ag-CASEIN COMPLEX.

The basic concept of this model is that the branched galactose

units of SLBG serve as junction zones to lin k other strands of SLBG

to form a three dimensional network. Small particles of as-casein

aggregates (100 A) participate in forming these junction zones, pos

sibly through hydrophobic forces. The ester sulfate groups in the

main chain of SLBG would possibly form intra-molecular linkages with 106 adjacent subunits through the mediation of calcium ions; this would assist in reducing electrostatic tensions. The arms of the resulting network would be somewhat rig id because of the numerous junctions.

I t is also possible that the strands are aggregated to some extent and th is would give added rig id ity to the segments. Apparently, such calcium interaction may be hindered to some extent in SGG because o f closer branching and may explain the low protective action o f this hydrocolloid. This model share the same principle as we have postu lated fo r the carrageenan stabilized systems, namely that the confor mational arrangement of the polysaccharides is the controlling factor for creating segments with a sufficiently rigid structure to resist collapse. However, they d iffe r in the mechanism o f formation.

The assumption that physical separation o f ag-casein aggregates is the major requirement fo r a stable protein system is same in both the K-carrageenan and the SLBG models. The fact that both types of complexes responded in an identical manner to change in the protein

concentration suggests the v a lid ity of the assumption. When the

protein concentration increases, the chance fo r a collapse of the

entrapped protein particles becomes greater, and as a result, the

s ta b ility of the complex decreases.

The mode o f action for the SLBG system explains why the tu rb i

d ity of the stabilized protein system is less than fo r K-casein or

K-carrageenan: there are many more protein reactive centers in this

complex in which the aggregates of a^-casein are trapped, therefore,

the individual protein particles are smaller. The proposed model 107 would also explain the observed improved sta b ilizin g a b ility with increasing temperature fo r SLBG. I t is lik e ly the segments of the polysaccharide strands may be hydrogen-bonded to form a closely packed aggregate with l i t t l e opportunity for forming an extended three dimensional network. One might picture this model as a wet chicken feather in which the details of a network are not prominent.

On drying, the feather opens up and exposes the individual branches.

The closed hydrogen-bonded structure apparently is less conducive fo r the formation of the required three dimensional network.

The results from s ta b ility tests revealed that removal of 6 - sulfate by a lka li modification greatly impaired the protective action of SLBG. This observation is consistent with the proposed model.

Since the branch galactose 6 -sulfate is involved as the junction zones to form the three dimensional network, a removal o f sulfate and re placement with 3 ,6 -anhydrogalactose would certainly hinder its a b ility to attain this stabilized conformation.

I t is more d iffic u lt to reconcile the model with the observation that pH had no appreciable e ffe ct on the s ta b ility o f the complex.

The only reasonable explanation seems to be that the sulfate groups

do not participate directly in the interaction with protein and that

the binding may occur through hydrophobic forces in a sim ilar way to

K-casein. This is supported by the improvements due to heat tre a t

ments. One might then speculate on the possible role of sulfate in

SLBG since sulfation is d e fin ite ly required. In the absence of

knowledge to the distribu tion or to the location of sulfates, i t would 108 seem that the function relates firs t of all to a change in the solu b ilit y properties of this polysaccharide, and secondly to th e ir involvement as the in it ia l sites for cross linkin g. SUMMARY AND CONCLUSION

This study was undertaken to improve the basic knowledge con cerning the interactions between sulfated polysaccharides, particu la rly carrageenan and sulfated locust bean gum, and calcium sensitive caseins. The results support the concept that not only is the chem ical structure of the stabilizers important but th e ir conformational characteristics exert a profound influence on the physical function.

The importance of polymer size was well documented by the re sults of the sedimention equilibrium study which revealed that maxi mum stabilizatio n occurred in the molecular weight range of 1 0 0 ,0 0 0 -

300,000 fo r carrageenans.

Heat, pH and concentration studies a ll pointed to the involvement of setting up a three dimensional network as the major requirement for a stable complex between proteins and protective colloids. How ever, while the carrageenans form gel network through the preformed double helices, SLBG and SGG form gel networks probably through the branching subunits. I t is believed that this difference in gel net work formation mechanism leads to the specific responses to pH and

heat effects o f carrageenan and SLBG systems.

The results of this study suggest that the stabilization of

calcium sensitive proteins may serve as one of the c rite ria in

evaluation o f the performance o f sulfated stabilizers. In this

respect, the results have confirmed previous findings that polymer

109 no size, conformation as well as chemical characteristics of the protective colloid are important fo r the stabilizatio n of these calcium sensitive proteins. The finding was made in this study that chemically sulfated locust bean gum reacted with calcium sensitive proteins in much the same way as the natural carrageenans but with greatly improved effectiveness. Thus, the search fo r the highly effective stabilizers may well continue along the lines of chemical modification. 1. Al ai s, C., Kiger, N., and Jolies, P. 1967 Action o f Heat on Cow K-Casein. Heat Caseinoglycopeptide. J. Dairy Soi., 50: 1738.

2. American Chemical society. 1;954 Natural Plant Hydrocolloids. Adv. in Chemistry Series, No. 11, Published by ACS.

3. Anderson, B., Hoffman, P., and Meyer, K. 1953 A serine-linked Peptide of Chondroitin Sulfate. Biochim. Biophys. Acta 74:309.

4. Andrew, T. R. 1971 Private Communication.

5. B e ll, G. H., Davidson, J. N., and Scarborough, H. 1968 Textbook of Physiology and Biochemistry. 7th Edition. The Williams and Wilkins Co., Baltimore, p. 448.

6 . Bettelheim-Jevons, F. R. 1958 Protein-Carbohydrate Complexes. Adv. in Protein Chemistry, 13:35.

7. Biggs, R., Douglas, A. S., and Macfarlane, R. G. 1953 The Action of Thromboplastic Substances. J. Physiol., 122:554.

8 . Black, W. A. P., Blakemore, W. R., Colquhoun, J. A., and Dewar, E. T. 1965 The Evaluation of Some Red Marine Algae as a Source of Carrageenan and of Its k- and X-Components. J. Sci. Fd. A gric., 16:573.

9. Blihovde, N. 1952 Process for Stabilizing Foodstuff and Stabi liz in g Composition. U.S. Patent 2,604,406.

10. Bourgade, G. 1871 U.S. Patent 112,535. 11. Brimacombe, J. S., and Webber, 0. M. 1964 Mucopolysaccharides: Chemical Structure, Distribution and Isolation. BBA Library Vol. 6 .

12. Chakraborty, B. K., and Hansen, P. M. T. 1971 Electron Micro scopy of Protein/Hydrocolloid Interacting System. M27. J. Dairy S ci., 54:754. 13. Chang, J. C. 1968 Analysis of Food stabilizers in Milk Products. M.S. Thesis. The Ohio State University. 112

14. Cheeseman, G. C., and Knight, D. J. 1970 The Inte ra ctio n o f Bovine M ilk Caseins w ith the Detergent Sodium Dodecyl Sulphate. I . The Relationship between the Composition and the Size o f the Protein-Detergent Aggregate. J. Dairy Res., 37:245.

15. Cheeseman, G. C., and Knight, D, J. 1970 The In te ra ctio n o f Bovine M ilk Caseins w ith the Detergent Sodium Dodecyl Sulphate. II. The Effect of Detergent Binding on Spectra Properties of Caseins. J. Dairy Res., 37:259.

16. Christensen, 0. 1964 Carrageenan, a Useful Food A d ditive. Food Manuf., 39:(3):49.

17. Cook, W. H ., Rose, R. C ., and C olvin, J. R. 1952 E lectrop horetic, Sedimentation and Diffusion Properties of Carrageenin. Biochim. Biophys. Acta. 8:595.

18. Cornwell, D. G., and Kruger, F. A. 1961 Molecular Complexes in the Isolation and Characterization of Plasma Lipoproteins. J. Lipid Res., 2:(2):110.

19. Doane, W. M., and W h istle r, R. Y. (1963 Comparison o f the A n ti coagulant Activity of Three Polysaccharides Sulfates. Arch. Biochim. Biophys., 101:436.

20. Douglas, A. S. 1956 The Action of Heparin in the Prevention of Prothrombin Conversion. J. Clin. Invest., 35:533.

21. Edelstein, S. J ., and Schachman, H. K. 1967 The Simultaneous Determination of Partial Specific Volumes and Molecular Weights with Microgram Q uantities. J. B io l. Chem., 242:306.

22. Glicksman, M. 1969 Gum Technology in the Food Industry, p. 223 Academic Press, N.Y.

23. Green, M. L. 1971 The S p e c ific ity fo r K-Casein as the S ta b iliz e r o f as-Casein and B-Casein. I. Replacement o f K-Casein by Other Proteins. J. Dairy Res., 38:9.

24. Green, M. L. 1971 The S p e c ific ity fo r K-Casein as the S ta b iliz e r o f ag-Casein and 3-Casein. I I . Replacement o f K-Casein by Deter gents and Water Soluble Polymers. J. Dairy Res., 38:25.

25. Greenberg, D. M. 1944 The In te ra ctio n between the A lk a li Earth Cations, P a rtic u la rly Calcium, w ith Proteins. Adv. in Protein Chemistry. 1:121.

26. Grindrod, J., and Nickerson, T. A. 1968 E ffe ct o f Various Gums on Skimmilk and Purified Milk Proteins. J. Dairy Sci., 51:834. 113

27. Gui s e ley, K. B. 1971 Private Communication.

28. Gurd, F. R. N ., and Wilcox, P. E. 1956 Complex Formation be tween M etallic Cations and Proteins, Peptides and Amino Acids. Adv. in Protein Chemistry 11:311.

29. Hansen, P. M. T. 1966 D is trib u tio n o f Carrageenin S ta b iliz e rs in Milk. J. Dairy Sci., 49:698.

30. Hansen, P. M. T. 1968 S ta b iliz a tio n o f ag-Casein by Carragee- nan. J. Dairy S c i., 51:192.

31. Hidalgo, J ., and Hansen, P.M. T. 1969 Inte ra ctio n between Food Stabilizers and g-Lactoblobulin. J. of Agr. & Food Chem., 17:1089. *

32. H ill, R. D. 1970 The Effect of the Modification of Arginine Side Chains in Casein on the Coagulation o f Rennin-altered Casein. J. Dairy Res., 37:187.

33. H i ll , R. D., and Craker, B. A. 1968 The Role o f Lysine in the Coagulation of Casein. J. Dairy Res., 35:13.

34. Howell, W. H. 1924-25 The P u rific a tio n o f Heparin and Its Presence in Blood, Am. J. P hysiol. , 71:553.

35. Jensen, R., Snell man, 0., and Sylven, B. 1948 On the Inhomo geneity o f Commercial Heparin Preparations from Physicochemical Point o f View. J. B io l. Chem., 174:265.

36. Kenkare, D. B. 1966 Changes in the a-Casein Complex a t Elevat ed Temperatures. Ph.D. D isse rta tio n , The Ohio State U n iversity.

37. Lasker, S. E ., and S tiv a la , S. S. 1966 Physicochemical Studies of Fractionated Bovine Heparin. I. Some Dilute Solution Pro perties. Arch. Biochim. Biophys., 115:360.

38. Laurent, T. C. 1961 Studies on Fractionated Heparin. Arch. Biochem. Biophys., 92:224.

39. Lehninger, A. L. 1970. Biochemistry: The Molecular Basis o f Cell Structure and Function. Worth Publishers, Inc.

40. Liberti, P. A. and Stivala, S. S. 1967 Physicochemical Studies of Fractionated Bovine Heparin. II. Viscosity as a Function of Ionic Strength. Arch. Biochem. Biophys., 119:510.

41. L in , C. F. 1968 S ta b iliz a tio n o f Casein M icelles by Carragee- nans. M.S. Thesis. The Ohio State U niversity. 114

42. L in , C. F ., and Hansen, P. M. T. 1970 S ta b iliz a tio n o f Casein M icelles by Carrageenan. Macromolecules 3:269.

43. Mackinlay, A. G., and Wake, R. G. 1964 The Heterogeneity of K-Casein. Biochim. Biophys. Acta. 93:378.

44. McKenzie, H. A. 1967 M ilk Proteins. Adv. in Protein Chem., 22:55.

45. McMeekin, I . L ., and P o lis, B. D. 1949. M ilk Proteins. Adv. in Protein Chemistry. 5:202.

46. Meyer, K. H ., Pi roue, R. P., and Odier, M. E. 1952 Les Derives de Lacide Chondroitine-sulfurique et leur Action Anticoagulante sur les Polysaccharides Amines IV. Helv. Chim. Acta. 35:574.

47. Moirano, A. L. 1969 Dessert Gel and Composition thereof. U.S. Patent 3,445,243.

48. Parry, R. M., Ford, L. W., and Carroll, R. J. 1969 Interaction o f a g i-casein and ic-casein in the Absence o f Calcium ions. Abstract paper presented before the 64th Annual ADSA Meeting.

49. Pepper, L., and Thompson, M. P. 1963 Dephosphorylation o f as- Casein and K-Casein and Its E ffe ct on M icelle S ta b ility in the K-ctg-Casein System. J. Dairy S c i., 46:764.

50. P orter, P ., P orter, M. C ., and Shanberge, J. N. 1967. Heparin Cofactor and Plasma Anti thrombin in Relation to the Mechanism of In a c tiv a tio n o f Thrombin by Heparin. C lin. Chim. Acta. 17 :(2 ): 189.

51. P o rter, P ., Porter, M. C., and Shanberge, J. N. 1967 Interaction of Heparin with the Plasma Proteins in Relation to Its Anti thrombin Activity. Biochem., 6:1854.

52. Putnam, F. W. 1948 The Interaction s o f Proteins and Synthetic Detergents. Adv. in Protein Chemistry. 4:79.

53. Rees, D. A. 1969 Structure, Conformation, and Mechanism in the Formation o f Polysaccharide Gels and Networks. Adv. in Carbohy drate Chemistry and Biochemistry. 24:267.

54. Rigney, J. A. 1970 Chemical In vestigation o f Chondrus crispus. Progress Report, 1969, fo r In d u s tria l Development Branch, Fisheries Services, Dept, of Fisheries and Forestry, Ottawa, Canada.

55. Roden, L. 1964 S tructural o f Chondroitin 4-Sulfate Glycopeptides. Isolation of Glucuronosyl Galactose. Federation Proc., 23:484. 56. Rose, D. et. a l. 1970 Nomenclature of the Proteins of Cow's Milk: Third Revision. J. Dairy S ci., 53:1.

57. Shanberge, J. N., Sarelis, A., and Regan, E. E. 1959 The Effect of Heparin on Plasma Thromboplastin Formation. J. Lab. Clin. Med., 54:501.

58. Sharma, K. K., and Hansen, P. M. T, 1968 Action of Hyaluroni- dase on Casein. Abstract Paper Presented before 63rd Annual ADSA Meeting,

59. Shaw, 0. J. 1966 Introduction to Colloid and Surface Chemis try. Butterworths, London.

60. Smith, D. B., and Cook, W. H. 1953 Fractionation of Carragee nin. Arch. Biochem. Biophys. 45:232.

61. Steinhardt, J., and Beychok, S. 1964 Interaction of Proteins with Hydrogen Ions and Other Small Ions and Molecules, in the Proteins Edited by Neurath. 2:139. Academic Press, N.Y.

62. Stivala, S. S., and L ib e rti, P. A. 1967 Physicochemical Studies o f Factionated Bovine Heparin IV. Cu ( II ) Binding in Relation to pH, Molecular Weight, and Biological A ctivity. Arch. Bio chim. Biophys. 122:40.

63. Stivala, S. S., Yuan, L., Ehrlich, J., and Liberti, P. A. 1967 Physicochemical Studies of Fractionated Bovine Heparin. I I I . Some Physical Parameters in Relation to Biological A ctivity. Arch. Biochem. Biophys. 122:32. 64. Stoi o ff, L. 1958 Preparation of Flavored Milk Drinks, U.S. Patent 2,834,679.

65. Thompson, M. P., and Pepper, L. 1962. Effect of Neuraminidase on K-Casein. J. Dairy S ci., 45:794.

6 6 . Van Hoi de, K. E. 1967. Sedimentation Equilibrium. Fraction 1967, No. 1, Beckman Instruments, Inc.

67. Veil us, L., Nomine, G., and Mathieu, J. 1959 Investigation on Heparin. The Antilipemic Heparides. Bull. Soc. Chim. Biol. 41:415.

6 8 . Walton, P. L ., Ricketts, C. R., and Bangham, D. R. 1966. Heterogeneity of Heparin. B ritish J. Haematol. 12:310.

69. Warner, D. T., and Coleman, L. L. 1958 Selective Sulfonation of Amino Groups in Amino Alcohols. J. Org. Chem. 23:1133. 116

70. Warner, R. C., and P o lis, E. 1945. On the Presence o f Proteo ly tic Enzyme in Casein. J. Am. Chem. Soc., 67:529.

71. Waugh, D. A. 1954 P rotein -protein In te ra c tio n . Adv. in Pro tein Chemistry. 9:325.

72. Waugh, D. F ., and Von Hippel, P. H. 1956. K-Casein and the S ta b iliz a tio n o f Casein M icelles. J. Am. Chem. Soc., 78:4576.

73. W h istle r, R. L . , and Smart, C. L. 1953. Polysaccharide Chemistry. Academic Press In c ., New York.

74. W histle r, R. L . , and Paschal1, E. F. 1965 Starch: Chemistry and Technology. Academic Press In c ., New York.

75. W ilcox, D. F. 1958. Process fo r Preparing S tabilized Concen tra te d M ilk and Product Produced Thereby. U.S. Patent 2,845,350.

76. Wolfrom, M. L . , Shen, T. M., and Summers, C. 6 . 1953 Sul fated Nitrogeneous Polysaccharides and Their Anticoagulant A ctivity J. Am. Chem. Soc., 75:1519.

77. Wolfrom, M. L., Vercellotti, J. R., and Horton, 0. 1963 A Second Dissaccharide from Carboxyl-reduced Heparin. J. Org. Chem. 28:279.

78. Wolfrom, M. L . , Wang, P. Y ., and Honda, S. 1969 D is trib u tio n o f Sulfate in Heparin. Carbohydrate Res., 11 :(2 ):179.

79. Woychik, J. H. 1965. Preparation and Properties o f Reduced k- Casein. Arch. Biochem. Biophys., 109:542.

80. Woychik, J. H. 1969 Preparation and Properties on Trifluoro- acetylated K-Casein. J. Dairy Sci., 52:17.

81. Woychik, J. H ., and Wondolowski, M. V. 1967 Formation o f Soluble Para-K-casein by Disulfide Coupling with Bovine Serine Albumin. J. Dairy Sci., 50:949.

82. Woychik, 0. H ., and Wondolowski, M. V. 1969 N itra tio n o f Tyrosyl Residues in k - and asl-Caseins. J. Dairy Sci., 52:1669.

83. Z it t le , C. A. 1961 S ta b iliz a tio n o f Calcium Sensitive (as) Casein by K-Casein: E ffe ct o f Chymotrypsin and Heat on k - Casein. J. Dairy Sci., 44:2101.

84. Z ittle , C. A. 1965 Some Properties of Photooxidized K-Casein. 0. Dairy Sci., 48:1149. 117

85. Z ittle , C. A. 1969 Influence of Heat on K-Casein. J Dai ry S ci., 52:12.

8 6 . Z ittle , C. A. 1969 Influence of Heat on K-Casein: Effect of as-Casein and Concentration of Calcium Chloride and Sodium Chloride. J. Dairy S ci., 52:1356.

87. Zittle, C. A., and Custer, J. H. 1963 Purification and Some of the Properties of as-Casein andK-Casein. J. Dairy S c i., 46: 1183.

8 8 . Zittle, C. A., and Walter, M. 1963 Stabilization of g-Casein by K-Casein against Precipitation by Calcium Chloride. J. Dairy S ci., 45:1189. A FORTRAN IV PROGRAM

FOR THE ANALYSIS OF EQUILIBRIUM ULTRACENTRIFUGATION DATA. 119

This program was written for the analysis of equilibrium ultra centrifugal data from the high speed method (meniscus depletion method). The program will provide a calculation of molecular weight or molecular weight and partial specific volume simultaneously from records of the interferograms produced by the Raleigh Optical system.

The basic equation for this program was adapted from Edelstein and

Schachman (21):

2RT d In c M(l-vp) = . üj2 d

V 2RT d In c KM(1 - _ p 0 2 0 ) = _ _ ( ------5-) K 0)2 d y2 OgO

where R is the gas constant, T is the absolute temperature,

0) is the angular velocity of the rotor in radians per sec, c is the concentration of the redistributed solute, y is the distance from the axis of rotation, v is the partial specific volume, p is the

density of the buffer, K is the deuterium exchange constant, and M

is the molecular weight. The term d(ln c)/d y2 is obtained from

the slope of the plot In (fringe number) vs y2.

A. Out-put of ^ program. The out-put contains the following information:

1. Tabulated information of the experimental conditions used in the

equilibrium ultracentrifugation. 2. A table showing y» y^. In (fringe number) and molecular weight o f each corresponding fringe and read-out from the microcomparator.

3. Polynominal equations from zero to second order given for the plot of In (fringe number vs) y2.

4. A plot of In (fringe number) vs y^. The symbol H is used for the H 2O system. The symbol D is used for the DgO system.

An example o f the out-put is given in Table 5.

B. Preparation of th£ control cards.

The following control must be prepared in the order liste d :

1. / / (5000,500),CLASS=C 2. //STEPl EXEC PROC= FORTRANG,PRAM.CMP='MAP,10 ',TIME.CMP=(,15),

3. / / REGION.CMP=240K

4. //CMP.SYSPUMCH DO SYS0UT=B

5. //CMP.SYSIN DO *

6 . //STEP2 EXEC PROC=RUNFORT,PARM.LKED='XREF',TIME.LKED=(,15),

7. / / TIME.G0=(1,30)

8 . //LKED.SYSLIB DD DSN=SYS1.FORTLIB,DISP=SHR OSU SUBROUTINES

9. //LKED.SYSLIN DD DSN=*.STEPl.CMP.SYSLIN,DISP=SHR

10. //GO.SYSIN DD *

These control cards are designed to u tiliz e the computer

lib ra ry provided by the OSU computer center and are subject to change

at any time, therefore, i t is necessary to check with the consultant

about the control language before using the program. TABLE 5. AN EXAMPLE OF THE COMPUTER OUT-PUT. i’ n i

iNli* : ! :

I 2 2 ' • ^ , ! jiip ly iH jiij I

I ill:- !!!!!!!!!! | Hi > PLOT OP R-SQUARE VS LOG.E. PRINCE NUMBER « 1.600E 00 ♦

R 6.800E-01 ♦ ■

E 2.200E-01

-2.A00E-O1

^.OOOÉ-OÏ"* ■ H ♦ ♦ ♦ ♦ ♦ ». 0 3.540E 01 3.574E 01 3.608E 01 3.642E 01 3.676E 01 3.710E 01 SQUARE OP DISTANCE FROM ROTATION CENTER TO A GIVEN POINT R IN SAMPLE COLUMN. CH**2 124

C. Preparation of the data decks.

The fo llo w in g cards must be prepared and assembled in the prescribed manner. (See Table 6 for examples.)

1. Sample card; Format (5A4, 15A4).

The inform ation punched in th is card w ill appear as printed and may be used for identification. The firs t 20 columns are reserved for the name of the operator and the next 60 columns are reserved fo r the description o f the sample tested.

2. Count card; Format (I10,2F10.4).

This card supplies information on the number of experimental sets of data in the data deck and also instructions to solve for either molecular weight alone or solve for both molecular weight and p a rtia l s p e c ific volume. The number o f experimental data sets should be punched in columns 9-10, rig h t hand adjusted. Maximum number allowed is 30. Columns 11-20 are reserved fo r p a rtia l s p e c ific volume. Supply realistic value to the program, if only molecular weight calculatio n is wanted. Supply 0.0 to the program, i f both molecular weight and p a rtia l s p e c ific volume calculatio n are needed.

Columns 21-30 are reserved fo r deuterium exchange constant. Supply

0 . 0 for solving only molecular weight, otherwise supply a realistic

value. (For protein 1.0155, for polysaccharides this value have not yet been established and may vary for different species.)

3. Date card; Format (8A4,2A4,I10).

First 32 columns are reserved for the date of the experiment.

Columns 33-40 are reserved fo r the run number. Column 50 is TABLE 6 . AN EXAMPLE OF THE IN-PUT DATA CARDS. CAKRAGEENAN EXTRACT FROM PULŸIOES ROTUNÜUS tSCOTLANU SAMPLE CHII-FA LIN 12 O.SOOO

1 H SODIUM CHLORIDE IN WATER

IN PUT °*2 *(j.o c 2C.C00 1.C402 0.2000 13 0.0 7.3800 C.5000 8.69 70 1.COOO 9.1920 1.5000 9.4510 2.0000 9.6130 2.5000 9.7550 3.0000 9.8500 3.5C00 9.9410 4.0000 10.0090 4.5000 10.0780 5.0000 10.1400 1 1 1 . m o 10.2140

IN PUT 20.0C0 1.0402 0.1000 H 0.0 17.2460 . . 0.5000 20.7370 1.COOO 21.3280 1.5000 21.5770 2.00C0 21.6910 2.5000 21.7880 3.0000 21.8550 3.5000 21.9370 4.0000 21.9860 111.1110 22.0460

IN PUT 20.000 1.0402 0.0500 6 0.0 29.C690 0.5000 33.3480 I . 0000 33.4920 ...... 1.5000 33.52 80 111.1110 33.8630

. i 4 ^ r ; . N

IN PUT Qg 20.000 1.0402 0.2000 l< 0.0 5.9000 C.5000 7.4310 1.0000 8.7550 1.50C0 9.2040 ...... ------2.0000 9.4890 2.5000 9.6240 3.0000 9.7710 3.5000 9.8650 4.0000 9.9680 4.5000 10.0410 O.P 36.27)0 4 («CN.I 6 (OAYI 1970 (YEAR! L-84 — KPM — MIN. 8.240 RPM 1485 MIN IN-PUT OATA 8240.00 20.000 1.0402 0.1000 0.0 17.2620 C.5000 20.8220 1.0000 21.3620 1.5000 21.5680 2.0000 21.6830 2.5000 21.7980 3.0000 21.8610 111.1110 22.1460 0 .0 36.2750 4 (MON.I 6 (DAY) 1970 (YEAR) L-84 — RPM — MIN. 8,240 RPM 1485 MIN IN-PUT DATA 8240.00 20.000 1.0402 0.0500 0 .0 29.0810 0.5000 32.2290 1.0000 33.3820 1.5000 33.5200 ' 2.0000 33.6270 111.1110 33.8320 0.0 36.2750 4 (HON.) 6 (DAY) 1970 (YEAR) L-84 — RPM — MIN. 8,240 RPM 2835 MIN ...... IN-PUT OATA 6240.00 20.000 1.0402 0.2000 0.0 5.9050 0.5000 7.6310 1.0000 8.7860 ... . 1.5000 9.2180 2.0000 9.4790 2.5000 9.6410 3.0000 9.7670 3.5000 9.8630 4.0000 9.9680 4.5000 10.0480 111.1110 10.9750 0 .0 36.2240 4 (MON.) 6 (DAY) 1970 (YEAR) L-84 — RPM — MIN. 8,240 RPM 2835 MIN IN-PUT DATA 8240.00 20.000 1.0402 0.1000 0.0 17.2510 0.5000 2C.9C00 1.COOO 21.3690 1.5000 21.5830 2.0000 21.7080 2.5000 21.8070 3.0000 21.8940 111.1110 22.8810 0 .0 36.2240 4 (MON.) 6 (DAY) 1970 (YEAR) L-84 — RPM — MIN. 8,240 RPM 2835 MIN IN-PUT DATA 8240.00 20.000 1.0402 0.0500 0 .0 29.0340 0.5000 32.6190 1 2 8 m .u io 0.0 3 (,.2240 4 (MON.) 6 (DAY) 1970 (YFAR) 1-H4 I — KI'M — MIN. H./40 KMH 2HJ!> MIN

IN-PUl OATA 8240.00 20.000 1.0402 0.2000 12 0.0 5.8890 0.5000 7.5450 J.COCO 8.7500 1.5000 9.1990 2.0000 9.4650 2.5000 9.6240 3.0000 9.7580 3.5000 9.0550 4.0000 9.9460 4.5000 10.0250 111.1110 11.0420 0.0 36.2270 4 (MON.) 6 (ÜAY) 1970 (YEAR) L-84 2 — RPM — MIN. 8,240 RPM 2835 MIN

IN-PUT DATA 8240.00 20.000 1.0402 0.1000 9 0.0 17.2330 .0.5000 20.8480 l.OOCO 21.3600 1.5000 21.5790 2.0000 21.7050 2.5000 21.8030 3.0000 21.8800 111.1110 22.5530 0 .0 36.2270 ...... 4 (MON.) 6 (DAY) 1970 (YEAR) L-84 3 — RPM — MIN. 8,240 RPM 2335 MIN

0.0 29.0230 0.5000 32.3510 1.0000 33.3650 1.5000 33.5400 2.0000 33.6340 111.1110 33.8860 0.0 36.2270 129 reserved for channel number of the cell when the Yphantis cell is used.

4. Speed card; Format (10A4).

The f ir s t 20 columns are reserved for the sweep speed and time.

I f no sweep speed is used, leave i t blank. The next 20 columns are reserved fo r the equilibrium speed and time. Information punched in this card is not used in the calculation and w ill appear as printed.

5. Buffer card; Format (8A4).

The type o f buffer may be punched in columns 1-32 of this card. The information is not used for the calculation.

6 . Experimental conditions card; Format (F15.2,F10.3,2F10.4,I10).

The f ir s t 15 columns are reserved fo r the equilibrium speed in

RPM (only numerical value). Columns 16-25 are reserved fo r tempera

ture o f the experiment. Columns 26-35 are reserved fo r solution

density. Columns 36-45 are reserved fo r sample concentration in per

centage. Columns 46-55 are reserved fo r the number of fringes or

h a lf fringes plus three (meniscus, cell bottom, reference edge.).

The interger number should be punched in columns 54-55 rig h t hand

adjusted. Maximum number allowed is 40.

7. Fringe, read-out cards; Format (2F10.4).

The fringe reading obtained from the microcomparator should be

punched in here. Each card contains a fringe number (columns 1-10)

are the corresponding read-out (columns 11-20). The fringe number at

meniscus is 0 .0 , whereas the fringe number at cell bottom and refer

ence edge are not used in the calculation and can be any value. For

example; 111.111 and 222.222 may be chosen. The total number of cards 130 should equal to the interger appearing in columns 54-55 of data card

6 . Conversion of fringe reading is done by the program from the reference edge value. Center to reference wire is 5.62 cm and is supplied in the program.

8 . Data cards fo r the second set and so on;

Only cards 3,4, and 6 are needed to proceed fo r a set of data describing a single experiment. The total number o f data sets in the deck should equal to the interger appearing in columns 9-10 of card 2. For solving molecular weight and partial specific volume simultaneously, besides observing the requirements given in card 2 i t is necessary that the sets are supplied as pairs of H 2O and DgO analyses with the H 2O -set supplied f ir s t. Note that i t is necessary to supply the appropriate data cards 3,4,5 and 6 prior to the^ second set of data (the f ir s t set of D 2 O). From the th ird set on, card 5 must be omitted. The interger appearing in columns 9-10 of card 2 is the total number of data set (including H 2O and D2O) divided by two. PROGRAM SOURCE LANGUAGE

FORTRAN IV G DATE • 71167

DEUTERIUM EXCHANGE CONSTANT (DXCHAGI IS USED TO OETERMINE WHETHER THE PROGRAM WILL SOLVE ONLY MOLECULAR WEIGHT OH MOLECULAR HEIGHT AND V-BAK VALUE SIMULTANEOUSLY WHEN (DXCHAGI IS SUPPLIED AS ZERO, THE PROGRAM WILL SOLVE MOLECULAR WEIGHT (V-BAR VALUE SHOULD BE PROVIDED TO THE PROGRAM I WHEN (DXCHAGI IS LARGER THAN ONE,TEE PROGRAM WILL SOLVE MOLECULAR height and V-OAR VALUE SIMULTANEOUSLY (ZLRO IS PRUVIOED TO V-BARI DOUBLE PRECISION SUMX2, SUMY2, SUHXV,SUMX.SUMY, SLOPEtCOEFP DIMENSION RPM(33,2l,TbHP(30,2l,DENSTYU0,2l,CONC(30.2lfKOUNTll3O,2 ll,iLOGCR(A0,3C,2lf(lPERAT(5l,SAMPLE(15I.CHANEL(30,2I.BUFFER(B,2l DIMENSION FRINGE(4Ü ,3 0 ,2 1,REDOUT(4C,30 ,21,R(AO,3 0,21,R 2(4 0,3 0,2i DIMENSION SUMX(3 0 ,2 1 ,SUMY(iO ,2 », SUMX2I30,2 I , SUMY2(30 ,2 1, SUMXYI30,2 1I,CONSTA(3C,2I,COUNT(30,2I,XH(40I,YH(40I,X0(401,YDI40),X(4CI,V(40I DIMENSION SLOPE(30 .21,XUAR(3 0 ,2 1 ,YHAR(30 ,2 1, CEPT(30,2 I,COEfF(3 0 ,2 1 DIMENSION K*MWT(40,30,2l,4SLUPE(40,30,2),VUAR(3r>l,mWT(JO),iMZ(30, 121,IARRAY(1428I,H(14I,A(22I,0(BI DIMENSION DATL(8,3C,2I,RUNNO(2,30,2),SWEEP(S,3 0 ,2 1,EOUILI( S ,30,2) DIMENSION 8L0CK3(4CI,BLOCK4(40),YCAR(40I,RMT(3I,VAT(3I,CXY(3,3) CATA ChARH/' H '/,CHAR0/' D '/ DATA H /' • , • « , • * * * ' , 'P LO T', ' OF • , 'R-SO*, 'UARE■,• VS ','L O IG .'.'E . F','RING','E NU'.'MBEH',* *•••/ DATA 0/' ','LOO.','E. F','RING',*E NU'.'MBER'/ OATA A/' '.'SQUA'.'RE 0','F Dl','STAN','CE F ','ROM '.'ROTA'.'TI ION',' CEN'.'TER ','TO A ',' GIV'.'EN P'.'O INT',' R l','N SA'.'MPLE* 2,' COL'.'UMN.',' C','M»*2'/ , DATA SMHT/0.0/,C0NSTB/5.62/ READ (5,101 (0PERAT(I0I,I0-1,5I,(SAMPLE(IS),IS«1,15I 10 FORMAT I5A4,I5A4I WRITE (6,101 (OPERAT(101,IO "l,S I,( SAMPLEIISI.IS-I.IS) IN-PUT MO. OF SET OF EXPERIMENTAL DATA,V-BAR VALUE AND DEUTERIUM EXCHANGE CONSTANT. THESE VALUES ARE USED AS TEST VALUE LATER .READ (5,1 1) NOFSET.SVHAR.OXCHAG 11 FORMAT ( llO ,2 F n .4 ) WRITE (6,1 1) NOFSET.SVBAR.OXCHAG DO 3000 J«1,NCFSET Dl) 2000 K-1,2 IN-PUT THE EXPERIMENTAL CONDITIONS READ (5,20) |DATE(ID,J,K),I0«1,8),(RUNN0(IR,J,KI,1R-1,2I,CHANELIJ,

20 FORMAT (8A4,2A4,110) WRITE (6,20) (OATE(IO,J,KI,tO>l,B),IRUNNOIIR,J,K),lR-1,21,CHANEL!J *READ (5,211 (SWEEP(IW,J,K),IW-1,5) ,IEQUIL1IIE,J,K),IE"1,5) DATE • 71167

WRITÊ^(6’ 2 Îi’ îsHEEP,IB«lt8> 22 FORMAT I8A4] 900 REÀo^lstÉn'RPK*! J^R>!-TEMPu ! k | Î dI nSTYIJ,KJ tCONCIJ.K) .KOUNTK J,KI 23 FORMAT (F 15.2,F10.3,F10.4,F 10 .4 ,1 ICI WRITE (6,241 RPM(J,K l,TEMP!J,K l,DENSTY(J,K),CONC(a,K l,KOUNTKJ,K) 24 FORMAT ( /• ',T2«'IN-PUT DATA'/* • , H 5.2,F10.3,2F10.4,I101 K4«KQUNT1(J,KI KB»KUUNT1(J,KI-l KC'ROUNTK J,K I-2 C IN-PUT FRINGE NUMBER AND CORRESPONDING READING FROM COMPARATOR DO ICOO I'l.K A REAU (5,251 FRINGE(l,J,KI,RFOOUT(I,J,KI 25 FORMAT (2F10.4I WRITE (6,251 FKINGE(I,J,KI,REOOUT(I,J,KI 1000 CONTINUE ^ CONSTA(J,K1.(7.30-CONST8I/REUOUTIKA,J,K1 C THE FOLLOWING SECTION CALCULATE THE LESSSUUARE SLOPE OF LINE LOG. C E.(FRINGE NUMBER) VS R-SOARE COUNT!J,K)=O.C SUMX!J,KI>C.O SUMY(J,KI«0.0 SUMX2(J,K)=0.C SUHY2(J,KI»0.0 SUHXY!J,K)=0.0

1200 l»H-l R(I,J,KI«REDOUTII,J,KI*CONSTA(J,KI-fCONSTB R2(I,J,K1*RII,J,KI**2 IF (FRINGE!I,J,K).EO.O) GO TO 1200 . »L0CCR(I,J,K1»AL0G(FRINGEII,J.KI I IF (FRINGE!I,J,KI.LT.II GO TO 1200 SUMX! J,K)-SUMX(J,KKR2(I,J,KI SUHX2( J,KUSUMX2(J,KKR2(I,J,KI*»2 SUMY!J,K)»SUMY(J,KI*tLOGCR(l,J,KI SUMY2! J,K1=>SUMY2(J,K)*»L0GCR(1,J,KI**2 SUMXY!J,K1-SUMXY(J,K)»R2(I,J,K)**LCCCRCI,J,KI COUNT!J,KI«COUNT(J,Klf1.0 IF II.LT.KC) GO TO 1200 ^ slope(J,K|.(COUNT!J,K|.SUHXY(J,KI-SUHX(J,K)*SUHYIJ,KI|yiCOUNT(J,K) 1*SUHX2(J,KI-SUMX(J,KI**2I XBARIJ,K)«SUMX(J,K)/COUNT(J,K) FORTRAN IV G LEVEL 20 MAIN DATE ■ 71167 16/23/38 0067 VrtARtJ.KI-SUHYlJtKI/COUNTU.K» 0068 CEPT(J,KI»VBARIJ,K)-SLO?E(J,Kj*XaAR(J,K» 0069 CiJLFMj .KI'ICOUNTI J,K)*SUHXYIJtKi>SUHX(JfKI«SUNV(J,KII/OSQRTl tCOUM 1T(J,K»«SUMX2(JtKI-SUMX(J,K>«*2)*(CCUNT{J,K)*SUMY2(J,KI-SUHY(J>K)** 0070 IF (ÜXCHAG.EQ.O) GO TO 2100 0071 2000 CONTINUE

0072 VHARUI»(DXCHAG -SLOPE U ,2 1/SLOPE! J , 1 ) )/IüENSTV(J,21-DENSTVIJ, 1 » 1*SLÜPE(J,2)/SLÜPEIJ,1I) 0073 GO TO 2200 0074 2100 V8AR!JJ-SVOAR 00 75 2200 tMUT(JI-!2.0*83.l3*(273.16+TEHP(J,Ill*SLOPE(Ji1)l/I!1.0-VBARIJI*DE INSTYIJ,Il 1*10.104724KPMIJ,11/1000.01**2) 0076 IF (UXCMAG.EO.OI GO Tü 3000 0077 SVHAR»SVDAR*VOAR(JI 0078 SMhT'SMWTfSMWTIJI 0079 3000 CONTINUE

0080 IF (OXCHAG.EQ.Oi GO TO 3020 0081 AVUAR>SVBAK/NOFSËT C082 AMWT-SMWT/NOFSET 0083 00 TO 3030 0084 3020 AVDAR=SVHAR 0085 3030 00 60C0 J«1,NGFSET 0086 01) 5000 K=I,2 0087 KC*K0UNTl(J,K|-2 0088 KC2-KC-2 0089 00 3100 I2-1.KC2 0C90 1=12*2 0091 X !I2 l-R 2 ( IfJ ,K ) 0092 Y!IZ)-$LOGCR(I,J,K) 0093 3100 CONTINUE C .THE FOLLOWING SECTION HANDLES THE POLYNOMINAL CURVE FITTING FOR PLOT C OF THE LOG.E.(FRINGE NUMBER) VS R-SOUAKE 0094 H-1.0 0095 LA»0 0096 LEOH-1 0097 LAMAX*2 0098 NK=KC2 0099 3200 CALL PCFIIX,Y,H,LE0H,NK,LA,LAHAK,BLGCK3,BL0CK4,CXY,YCAR,RMT,VATI OICO LA«LA*1 0101 IF ILA.LE.LAHAX) GO TO 3200 FORTRAN IV G LEVEL 20 MAIN DATE ■ 71167 16/23/38

0106 $SLDPE?l!3?KjScxVI3,2)+2.0*CXV(3f3l*R2(IfJiKI 0105 W$HWTJI,J,K)=FACTOR*»SLOPE(ItJfKJ 0106 3300 CONTINUE C PRINT THE TABLE HEADING AND EXPERIMENTAL CONDITIONS 0107 WRITE 16,301 0108 30 FORMAT l ' l ' , T 3 0 , 'ANALYSIS OF SEDIMENTATION EQUILIBRIUM D A T A '///' '

0109 WRITE (6,311 J,K 0110 31 FORMAT (/• ',T2,'J ••,I2,T10,*K «',12,/' •) OUI WRITE (6,321 (DATE(ID,J,K),IO«l,el,(OPERAT(IO),IO«l,St,(RUNNO(IR,ô l,K ),IR «1,2 ),(SAMPLE(ISI,1S«1.151 0112 32 FORMAT ( / / ' ',T2,'l)ATfc :',B A 4 ,T 4 2 ,'OPERATOR J ' ,5A6,T82, ' RUN NUMBER 1 : ' ,2A4,//' *,T2,'SAMPLE :',15A4) 0113 WRITE (6,3 3) CÜNCIJ,K|, ( BUFFER( IB ,K ),IB « 1 ,8 1 ,UENSTV(J,K),VBAR(JI 0114 33 FORMA) ( / ' ' , T2,'CONCENTRAT(ON : ' , E8. 3 , T24, ' X ( W/V)' ,T 4 2 ,'BUFFER 1 : ' ,8A4,T82,'DENSITY : ' , F 6 . 3 , / / ' ', 1 2 , 'V-BAR VALUE :',F B .3 ) 0115 WRITE (6 ,3 4 ) TEMP(J,K) , ( SWEfcP( IW, J ,K) , IW»1, 5 ) , IE UUILI( lE , J ,K I, IE «l 1 ,5 ) ,CHANEL)J,K),CONSTAIJ,K),CüNSTB 0116 34 FORMAT ( / ' ' , 1 2 , ' TEMPERATURE : ' ,F6.1,2X,T23,*C * ,T 4 2 ,'SWEEP SPEED l:',5 A 4 ,T 8 2 ,'EQUILIBRIUM SPEED I ', 5 A 4 , / / ' ' ,T 2 ,'CHANNEL NO. (IF ANY 2) : ', 1 2 , T42,'DISTANCE CONVERSION FACTOR : ' , F 8.5, T82,'ROTOR CENTER 3 TU REFERENCE WIRE : ' ,F 5.2 ,1 X, T120, ' CM') 0117 WRITE 16,35) SLOPE( J ,K ) ,COEFF(J,KI ,SMWT(JI 0118 35 FORMAT ( / ' ' ,T2,'SLOPE : ' ,F 8 .4 ,T42, 'LINEAR CORRELATION COEFF. J ',F 1 9 .5 ,1 8 2 ,'MOLECULAR WEIGHT : ' , F l l . l ) 0119 WRITE (6,3 6) 0120 36 FORMAT ( / / / ' ' ,T 1 1 ,'FRINGE', T25,' REAO-OUT',T43,' R *,T57,*R 2*,7 7 0 ,*L lOG.E(F)',T86,'V-BAR',T100,'W-AVE M.W.') 0121 WRITE (6,3 7) 0122 37 format (/' ',T12,'WIRE',T26,'0.000') C PRINT THE CALCULATED RESULTS 0123 WRITE (6,3 8) FRINGE(1 ,J,K).REDOUT( I , J ,« ) ,R (1, J ,K I,R 2 (I,J ,K ) 0124 38 FORMAT ( / ' • ,F 1 4 .2 ,3F15.3) 0125 00 4000 (»2,KC 0126 WRITE (6,3 9) FRINGE(I, J,K).REDOUT( I,J , K ) ,R ( I, J ,K »,R2CI, J ,K ), SLOGCR l(l,J,K),AVQAR,H*MWriI,4,KI 0127 39 FORMAT ( / ' • , F14.2 ,5F1 5 .3 ,F17.1) 0128 IJNE«I-1 0129 IF (K.EQ.2) GO TO 3800 0130 XH(I0NE)=R2(I,J,K) 0131 YH(inNE)=$LOGCRII,J,K) 0132 GO TO 4000 0133 3800 XD(IDNE)»R2(I,J,K) 0134 V0(IONE)=*LOCCR(I,J,K) 0135 4000 CONTINUE 135

HIKIHAN IV U LkVLL 20 MAIN UATfc • lllb T

0136 ^ KA = KOUNTU J,K) 0137 KU»KlluNTl(JfKl-l 0138 WHITE (6,401 RfcUOUTIKB,J,KI 0139 40 FORMAT ( / • ' .F29.3* 0140 WRITE (6,401 REDOUT(KA,J,KI C PRINT THE POLYNOMINAL EQUATION OF THE LINE LOG.E.(FRINGE NUMBER 1 C vS R-SQUARE 0141 WRITE (6,41) 0142 41 FORMAT ( / / • ' ,T6,"POLYNOMINAL EQUATION: Y - A ♦ 1 H*X ♦ C*X**2*,//* STIS.'ORDER*/* M 0143 DO 4100 LC-l.LA 0144 LO'LC-1 0145 WRITE 16,42) LD,(CXY(LC,LR),LR«l,LA) 0146 42 FORMAT ( / • • , 116,15X,5F15.3) 0147 4100 CONTINUE 0148 IF (OXCHAG.EQ.OI GO TO 5710 0149 5000 CONTINUE c THl FOLLOWING SECTION HANDLES THE PLOT OF LOG.E.(FRINGE NUMBER) C VS R-SQUARE 0150 KBh=KCUNTllJ,l)-3 0151 KBO=KOUNTl(J,2)-3 0152 IF (X H Il).C E .X D (l)) GO TO 5100 0153 IXH-XH(l) 0154 XL«IXH 0155 CO TO 5200 0156 5100 IXC=XD(1I 0157 XL=IXO 0158 5200 IF(XH(KHHI.CE.X0(KB0)) GO TO 5300 0159 JXU=XC(KRD)+1 0160 XR=JXO 0161 GO TO 5400 0162 5300 JXh=XKKDH) + l 0163 XR.JXH 0164 5400 YTESr=YH(l)-YO(l)+lO.O 0165 IF ( YTEST.GE.10.0) GO TO 5500 0166 KYH=VH(1 1*10.0-1.0 0167 YL=KYH/10.0 0168 GO TO 56C0 0169 5500 XYC=YD(1)*10.0-1 .0 0170 YL»KYD/10.0 0171 5600 YTEST=YH(KBH)-Y0(K80)+10.0 ...... 0172 IF (YTtST.GE.lO.O) GO TO 5700 0173 LVD»VO(KOO)*10.0 0174 YU«LYD/10.0 0175 GO TO 5800 136

FORTRAN IV C LEVEL 20 MAIN DATE • 71167 0176 5700 LVH-YH(KBHI*lO.O*l.O 0177 VU»LYh/10.0 0178 GO TO 5800 0179 5710 K8H-KQUNTl(Jfl»-3 0180 IXH-XHC1)*10.0 -1 .0 0181 XL-IXH/IO.P 0182 JXh>XH(KRH)*10.0«1.0 0183 XR»JXH/10.0 0184 KYH«YM<1)»10.0-1 .0 0185 YL*KYH/10.0 0186 LYHnYh(KUH»*10.0+1.0 0187 YU»LYH/10.0 0188 5800 0=0 0189 CALL PLOTAIIARRAY,XL,XR,YL,YU,DI 0190 CALL PLOTfllXHtYH.CHARH.KBH) 0191 IF IOXCHAG.EQ.OI GO TO 5810 0192 CALL PLOTB(XO,VO,CHARO,KBO) 0193 5810 NWh=14 0194 NW0»8

C198 ^ IF (OXCHAG.EQ.OI GO TO 8000 C THF FOLLOWING SECTION SUMMARIZES THE RESULT OF PARTIAL SPECIFIC C VOLUME ANU MOLECULAR WEIGHT 0199 WRITE (6,601 ( SAMPLE( I S I,1 S -1 ,151 0200 60 FORMAT C l ' , / / / / ' ' , T27, ' SUMMARY OF PARTIAL SPECIFIC VOLUME AND MO ILfcCULAR WEIGHT O A TA ',/' • ,T3C,'O F',15A4I 0201 WRITE (6,611 0202 61 FORMAT (T28,'V-»AR VALUE', T60, ' W-AV6RA0E M .W .'I 0203 DO 70C0 J=1,NCFSET 0204 .WRITE (6,621 VBAR( J I , $MWT(JI 02C5 62 FORMAT ( / ' ',F 3 3 .4 ,F 3 5 .il 0206 7000 CONTINUE 0207 WRITE (6,631 AVBAR,AMWT 0208 63 FORMAT ( / / ' ' ,T 1 0 ,' AVERAGE - ' ,F 1 6 .4 ,F 3 5 .II C2C9 GO TO 9000 0210 8C00 WRITE (6,711 (SAMPLE(ISI,IS=1,15I 0211 71 FORMAT C l ' , / / / / - ' ' , T27, ' SUMMARY OF MOLECULAR HEIGHT D A T A ',/' ',7 2 17,'OF',15A4I FORTRAN IV G LEVEL 20 MAIN DATE • 71167 16/23/38 0215 72 FORMAT (/• T20,5A4,T43,'CHANEL',12, 5%,FIO.3,T66,'««,FIS.Il 0216 8100 CONTINUE 0217 9000 STCP 138

THIS PKClGKArt IS WRITTEN FOR THE ANALYSIS OP ULTRACLNTRIFUGAL OAT* FROM HIGH SPEED METHOD (OR MENISCUS DEPLETION METHOD» DEUTERIUM F.XCHANÙE CONSTANT (DXCHAG) IS USED TO OLTERMINF WHETHER THE PP.CGRAM WILL SOLVF ONLY MOlFCULAN WEIGHT OK HfUrCULAR WEIGH ANÜ V-BAR VALUE SIMULTANEOUSLY WHEN IDXCHAGI IS SUPPLIED AS EERO, THE PROGRAM WILL SOLVE HULECULA HEIGHT (V-BAR VALUE SHOULD BE PROVIOEO TO THF. PROGRAM» WHEN (OXCHAG» IS LARGER THAN ONE,THE PHUGRAM WILL SOLVE MOLECULAR WEIGHT AND V-HAR VALUE SIMULTANEOUSLY (ZERO IS PROVIOEO TO V-BA

DOUBLE PRECISION SUMX2, SUHY2,SUMXY, SUMX,SUMY.SLOPE.COEFF DI.MENSU'W KPMI TC,2»,TLMP(iC,2I.DLNSTY( 30.2) .CONC( 30 .2» .KO UNT 1 ( 30 .2 I . SLOGf.R ( AC .30 .2 I . OPER AT ( 5 », SAMP LE ( 15 » .CHANEL ( 30 .2l.tlUFFE RI8.2l

DIMENSION SUHXnO.2» ,SUMYt33.2», SUMX2130. 2»’.SUMY2(30.21 .S UMXY(30,21 .CONSTATjC ,21,CUUNT(30.2I.XHIAOI.YHIAOl.XOI40». YU|4C).X(40»,Y(40I DIMENSION SLOPE(30.21,

,'R-SO','UARE',

p'R IN G '.'E NU*.

r'SWUA'.'RE 0 STAN' ,'CE F'.'ROM ' CEN'.'TER • GIV». *£N P '.'O 'N T 'i IA'.'HPLE*.' ( C ','M » « 2 '/ DATA SMWT/0.O/.C0NSTB/5.62/ 139

RtAO (5,10» <0PERAT(I01,!0«1,5I,ISAHPLE(IS»,IS-1,15» FORMAT (5A4.15A4I

WRITk (6,101 (UPCRAKIÜI,(0-l,5»,(SAHPLfc(ISI.IS-1.151 FORMAT (5A4,15A4)

READ (5,111 N0FSET,SVBAK,I)XCFAG FORMAT ((1C ,ZF10.4)

~WRfTE ( 6 , ' il l N0FSfT,SVHAR,DXCHAG FORMAT (I1 0 ,2 F 1 0 .4 I

Oil UNTIL 2000

-PUT THE FXPER(MENTAL CUNDITIUNS

FORMAT (8A4,2A4,I10) FORMAT iaA4,?A4,H0»__

READ (5,211 (SMttPdW, JtK lt IH=1,5 ),(EOUILKie.J.K), IE-1.5

FORMAT (5A4.5A4I_____

WRITE (6,21» (SWtEPdW, J,K),IW -l,5»,(E0UILI(IË,JfK».IE-l,

...... format (5A4.5A4I ......

I GO TO 900 I

RCAO (5,221 (BUFFERdfltKI.IS-lff FORMAT (8A4)

WRITE (6,221 (»UFFER( I( t,K ) ,IR -l,a i FORMAT (8A4I

FORMAT (F15.2,F10.3,F10.4,F10.4,I10»

FORMAT ( /• ',T 2 ,'IN -P U T DATA'/' • ,F 15.2,F 10,3,2F10 . 4 ,110» 141

» KUUNTUJ.K» KB • KOUNTlCJ.KI-l KC = KOUMTHJ.KI-2 PUT FRINGK NUiBtR « T CURRESPONUING READING FKCN COMPARATOR

READ (5,2 5) rRINGE( I , J,K).REDOUT( I

FORMAT (2FI0.A)

WRITE (6,2 5) FRINGE(I,J,K),REDOUT( I, J , ) FORMAT (2F10.4)

• { CONTINUE )

CaïSTÀ('j ,K ) V I r . 30-CÜNSTB)/RE()OUTfKA, J ,k ) OWING SECTION CALCULATE THE LESS SQUARE SLOPE OF LINE LOG, INGE NUMBER) VS R-SQARE CUUNT(J,K) • 0.0 SUMX(J,K) ."O'.O'' SUMV(J,K) - 6 . 0 0.0 142

SUHYZtJfK) ■ 0.0 SUMXYUtKI - 6 .0

m .J .K ) » REDOUT«l,J,KI*CONSTA(J,K»«.CONST3 R2tI,J,K) « I

(FRINGE» I,J.K I.E O .O )

tLOGCRdf J.K I > ALOGiFRtNGEdi J.KM

I GO TO 1200 I 143

SUMX( JtK » SUMX(J,KI*R2IIfJiKI SUMX2(J,K) • SUMX2«J,KI*R2(IfJtKI**2 iUMYtJ.KI : SUMYIJ,XI+$LOGCRIItJtKI SUMY2( 'SUHY2(j;V» + iLÜCCRl t .J # k ï**2 SUKXYI J.K I • SUMXY(J,K)*R2n,JtKI*»LrCCRI ItJ»K I COUNT(J,R) = C nu N TIJtKl^l.C I

I GO TO 1200 I

XBARtJ.K» SUMX(J,K»/COUNT(JiKI. .SUMY( J , KI/COUNTt J ,M _ rjJ .K ) » YOARIJ, -SLOPfcT J,K»*XOArt( J,K»' CCEFFCJ.KI = (fOUNT< J,X)*SUMXY( J,M-SUMX( J,K I*S U M Y IJfK n/ DSORK (COUNTI J,KI*SUMX2I J,K)-SUMX(J,K)**2I**SU MY2(J,K»-SUMY(J,KI*.2») ; ......

I GO TO 2100 I > (DXChAG-SLÜPE(J,2l/SL0PE( J.in/lDe;4STY(J«2l-O EN I STY* J, l)*SLOPt:( JtZi/SLO P EIJin I |

_L. GÜ.TO 2200.1

VBAR(J) » SVOAR

_ GO TO 3000 I

SV'SAR » SVttAR+VBARCjl

SMMT » SMWT*»MWT(J) 145

^ ( OXCHÀG«ËQ*0) ------.- false . . ..

i " ” i

1 GO ro 3020 1 i -- ..... — • ------

_SyflAR/Np.FSET._ SMWT/NÜFSET

1 GC TO 30iO I

A.yBAR.-.SyBAR no ONT I!. 50C0 K,LA,LAMAX,BLOCKS,BL0CK4,C%Y,VCAR,R I

I GO TU 3200 I

iS DP E ll.J.K I = CXY«3.2I*2,C«CXY(J,31*R2(I,. M*MWT(I,JiKI » FACroH 148

NT THE TABLE HEADING AND EXPERIMENTAL CONDITIONS

WRITE (6,311 J.K "FORMAT 17'" ',T2",'J'=',I2,T10,'K ,12,/'

FORMAT ( /• *,T2,'CONCENTRATION : • ,F8. 3 , T2A,' X IW/VI',T42 , 'BUFFER BAA,Tfl2,'DENSITY S ',F o .3 ,// ' ',T2,'V -0A R VALU

FORMAT I / ' ' ,T2,'TEMPERATURE : ' ,F 6 .I, 2X,T?3, ' C ',TA2,'SWE EP SPELO ,5AA,T32,'EQUILIBRIUM SPEED :',5A A ,//' ',T2,'C

T120,'CM ') . .

WRITE (6,351 SLQPEIJ,KI,COEFF(J,K),tHHT(J) FORMAfl/^ ......

WRITE (6,36) 149

WRITE 16,371 — POMMAT'(/» ' S 'Ili!,’W lBE'ïl26; ' 0';C00''T" Hf f.ALCULATEl) RESULTS WRITE 16,33» FRINCtIl,J,KI,RE00UT(l,J,K),RIl,J,KI,R2ll,J,

FURMAT (/' ' ,F14.2,3F1S,3» .

• 1 WRITE (6,391 FR !I.GE( I, J,KI,REOOüT(l ,J,K» ,R< I,J,K» ,R 2(I, J, I R»,tLUGCR(I.J.KI,AVOAR,WtMHT(I ,J,K ) I ■ FORMAT (/'"' rF l4.2 ,5FÏ5. j ,F17TÎT j "

I GO TO 3800 I IllONEI « VlUCCKIIiJ.K

I GO TO 4000 I

xonoNE»RZIItJ.j' IIIOIMEI • tLOGCRd,.

ITE_(6.401 RE00UT(KB,J,K> FORMAT ( /• '.F29.3)

(TE (6,401 RËOOUT(KA,J, ' T0RMÂfl7*~'TF 29'.7r~ WRITE «6,421 I ICXVILC,tRI,LR*l,LA) FORMAT «/ ,I15,15X,5F15.3»

I GO TO 5710 I VNOLES THE PLOT OF LOG.E RINOE NUMBER»

KHH » R0UNTHJ,l)-3 KBO • ROUNTl(J,2»-3

I GO TO SlOO I

IXH > XH(1>

I GO TÙ S20Ô I

1X0 > X0(1) _J GO TO 5300 I

JXO • XO(KBO>*i XR = JXO

1 GO TO 5 ^ 0 1

~VT¥5T— VHlïl-YOUÏtiÔTO KYH - YH(l)»lO.O-l.C YL ■ KYH/10.0

I GO TU 5600 I

KYD » YUI1I*10.0-1.C YL Ï KYD/10.o' YTEST a YH(KQH)-Y0(KB0I+10.0

- L .

(YTEST.GE.1 0 .0

LYO • YD(kaD>*10.0 YU » LYU/10.0 155

I CO TU 9800 I

T yh- - " yh'( kqh » ♦ÏÔVCVÜO

YU ■ LYH/10.0

I GO TO 5800 I

■ KOH - KUUNrUJ,ll-i IXH « XH(1)*10.0-1.0 XL • IXH/IO.C " jXH~ï XIH KHH I *10 . 0*1.0 XR = JXH/IO.O KYH « YH(11*10.0-1.0 KYH/iOlO LYH » YH(K8HI*10.0*1.0 YU • LYH/10.0

1 CALL PLOTAIIARRAY,XL,XR,YL,YU,0) I

. PLOTU(XH,YH,CHARH,KBHI I I CO lU 5810 I 157

I «SAHPLt(tS»,lS-l

WRITE (6,611 FORMAT IT28,' IK VALUE'fT60t'M-AVERAGE M.l

WRITE <6,621 V8AR(JI,SHWT(Jl FORMAT I/' ',F33.4,FT5.n

-=^rCQNTlNÜE"T~

______WRITE .16,631 AVBAR,A«WT. (SAHPt.Ë(lS>.IS«l .1») ,////' ',T27,

' ,T20,5AA,TA3,'CHANELSI2t5X.Fl0.3,T66,**',Fl5

■ < CONTINUe") 159

FUitMAT STATEMENTS ......

ALL FORMATS HAVE ALREADY BEEN DISPLAYED