THE CHEMISTRY OF HÆOGLOBIN AND

MYOGLOBIN IN RELATION TO THE COLOR

OF MEAT

DISSERTATION

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

By

HOWARD NED DRAUDT, B.Sc., M.Sc.

The Ohio State University

1955

Approved by:

Adviser The Department of Agricultural Biochemistry TABLE OF CONTENTS

Page

INTRODUCTION ...... 1

LITERATURE SURVEY ...... 3

Myoglobin and Hemoglobin...... 3

Methemoglobin or Metmyoglobin Formation ...... 7

The Action of Nitrites on Hemoglobin and Myoglobin ... 11

The Effect of pH on Curing ..... 18

The Effect of Reducing Agents in Meat Curing ...... 19

Heating and Hemochrome Formation...... 21

Color Loss in Cured Products ..... 2k

EXPERIMENTAL PROCEDURE ...... 27

Obj ect of the Investigation ...... 27

The Effect of Heating on Color Fixation and Color Stability...... 29

Isolation of Metmj'-oglobin ...... 37

Qualitative Experiments on the Effect of Possible Meat Components on Discoloration ...... 39

Spectroscopy of the Pigment ...... 54

Manometric Experiments ...... 6l

Preparation of Samples ...... 62

Warburg Experiments with Heat Fractionated Pigment ... 65

Gas Uptake in the Presence of Sodium Pyruvate ...... 70

Results of Warburg Experiments with Purified Pigment in which Nitrate and Nitrite were not Determined ..... 72

ii Page

The Effect of Oleic Acid on Oxygen Uptake ...... 74

Nitrate and Nitrite Production . «...... 80

Results of Warburg Experiments in which Nitrate and Nitrite were Determined ...... 83

DISCUSSION...... 91

SUMMARY AMD CONCLUSIONS ...... 95

BIBLIOGRAPHY ...... 97

AUTOBIOGRAPHY...... 10$

1 1 1 List of Tables

Table Title Page

1 Visual Color Scores for Cured Meat Irradiated in the Presence and Absence of Potassium Ferricyanide . 34

2 Visual Color Scores for Cured Meat Irradiated in the Presence and Absence of Potassium Ferricyanide Experiment 2 ...... 35

3 Color Score After Shaking Pigment with Fat Derivatives ...... « ... 45

4 The Effect of the Acid Group on Visually Scored Color Loss for Cured Meat Pigment ...... 47

5 Color Scores for Heat Denatured Globin Myohemichrome Irradiated in the Presence of Surface Active Agents and Acetone ...... 51

6 The Effect of Fatty Materials on Discoloration of Denatured Globin Myohemichrome in the Light and in the Dark ...... 53

7 The Effect of Fatty Materials on Denatured Globin Myohemichrcane Discoloration ...... 54

8 The Effect of Oxygen Pressure on Rate of Oxygen Uptake ...... 67

9 Oxidation of Denatured Globin Nitric Oxide Myo- honochrcme in the Presence of Sodium Pyruvate... 71

10 The Effect of Oleic Acid on the Oxygen Uptake of the Heat Denatured Pigment ...... 76

11 The Effect of Oleic Acid on Oxygen Uptake..... 78

12 Nitrate and Nitrite Produced and Oxygen Taken Up J.n the Presence of Light with KOH in the Wells ...... 84

13 Nitrate and Nitrite Produced and Oxygen Taken Up in the Dark with KOH in the Wells ...... 85

14 Nitrate and Nitrite Produced and Oxygen Taken Up in the Presence of Light without KOH in the Wells ..... 89

IV List of Illustrations

Figure Title Page

1 Absorbance Versus Wavelength for Denatured Globin I'lyohemichrome and for Denatured Globin Nitric Oxide %rohemochrome After Irradiation ...... 57

2 Absorbance Versus Wavelength for Pigment Samples Prepared as for the Manometric Experiments ...... 58

3 Absorbance Versus Wavelength for Pigment Samples Prepared as for the Manometric Experiments (Curve 2) ...... 59

4 Rate of Net Gas Uptake for Partially Purified Denatured Globin Nitric Oxide I^oheraochrome ...... 68

5 Rate of Oxygen Uptake for Partially Purified Denatured Globin Nitric Oxide Myohemochrome in Pure Oxygen and in Air ...... 69

6 Rate of Net Gas Uptake for Denatured Globin Nitric Oxide Myohemochrome ...... 73

7 Rate of Oxygen Uptake of Denatured Globin Nitric Oxide Myohenochrome in the Presence of Methyl Oleate in Pure Oxygen ...... 77

8 Rate of Oxygen Uptake of Denatured Globin Nitric Oxide Myohemochrome and Denatured Globin Myo­ hemichrome in the Light and in the D a r k ...... 79

9 Visually Observed Rate of Color Loss in the Light (a ) and in the Dark (B) ...... 86

10 Rate of Color Loss and Rate of Oxygen Uptake of Denatured Globin Nitric Oxide Hemochrome in the Light in Air ...... 87

11 Rate of Nitrate and Nitrite Production from Denatured Globin Nitric Oxide >îyohemochrome ..... 88 ACKKOV.JLEDGEMSNTS

The author wishes to express his appreciation to Professor

F. E. Deatherage, under whose direction this work was carried out, for his helpful suggestions. The author is indebted to The Ohio

State University for the facilities provided.

The financial help provided the author by Kingan Incorporated made it possible for the author to carry out this work and is gratefully acknowledged.

Helpful suggestions were provided by Dr. R. E, Morse and Dr.

M. C. Brockmann of Kingan Incorporated as well as by Professor

J. E. Varner of the Department of Agricultui'al Biochemistry of The

Ohio State University.

VI INTRODUCTION

In the presence of oxygen and light, the nitric oxide deriva­

tives of cured meat, either nitric oxide myoglobin or denatured

globin nitric oxide myohemochrome, are oxidized to the ferric form

with the production of undesirable color changes from red to brown.

This oxidative process has received remarkably little study

considering the economic loss to the meat packing industry and

eventually to the consumer. This process has become especially

important since the advent of self service merchandising and pre­

packaging of cured meat products. Examples of this oxidation may be

observed at almost any meat counter and may include dark brown

weiners, bologna that has lost its fresh appearance and similar

products that have lost their appeal to the potential consumer due to

discoloration. This undesirable color change involves the nitric

oxide derivative of either myoglobin or denatured myoglobin. These

derivatives are formed by curing, a process involving nitrite salts

either added as such or formed by bacterial reduction of nitrate

salts and reducing activity either associated with the enzyme systems

of the tissue or with reducing groups developed during heating.

The art of curing is an ancient one but has still not yielded

ccanpletely to scientific study. An understanding of curing necessarily had to come after some knowledge of hemoglobin was gained.

The period from about 186$ to 19OO marked an active period in the study of hemoglobin (1). The first comprehensive work on the nature of the cured meat pigment and a model, nitric oxide hemoglobin came in 1901. At that time, Haldane (2) observed and explained many of

1 2 the facts now known about meat curing.

While curing is concerned with formation of the nitric oxide derivative of the heme pipients, the work to be discussed here is largely concerned with the destruction of these derivatives. The chemistry of curing is thus of interest, inasmuch as it gives an insight into the possible chemistry of cured meat discoloration and to the related question of preservation of color.

Lack of published work on the chemical reactions involved in fading has been a serious handicap to technologists who wish to inçrove the color stability of cured meat products.

Lack of information in this field is perhaps due more to a lack of adequate experimental methods suitable for attacking this problem than to any other cause.

Meat is a complex biochemical system and the molar quantity of myoglobin is very low. Also, in practice many meat products are heated during their manufacture in which case the pigment becomes water insoluble. It was thus desirable to study the chemical nature of purified heat denatured pigment with the objective of gaining some insight into the reactions that may be involved in discoloration. LITERATÜHE SURVEY

Myoglobin and Hemoglobin

Although the hulk of the scientific work done with pm-ified

pigments has been carried out with hemoglobin, it would appear to be

at best a good model for the pigment, myoglobin or muscle hemoglobin.

Most of the blood is removed from the carcass at the time of slaughter

and thus most of the pigment of meat is myoglobin.

Hemoglobin is perhaps one of the most thoroughly studied of all

organic biological substances, yet myoglobin had received very little

attention until Theorell (3) prepared it in crystalline form in 1932.

Since the literature on hemoglobin is so voluminous and since it is

covered comprehensively in two recent books {k) (5), it will be

covered only briefly here.

Myoglobin has a m.olecul.ar weight of approximately 17,000 and one

hane group whereas hanoglobin has a molecular weight of 68,000 with

four heme groups. Although they are similar in many of their chemical

properties, such as the ability to undergo reversible oxygenation,

the myoglobin molecule cannot be considered to be simply one quarter

of a hemoglobin molecule.

In the live animal much more hemoglobin than myoglobin is present. Drabkin reported that an entire cow contained 1786 gm. hemoglobin and 274 gm. nyoglobin (6). Shenk, Hall and King (7) found that in beef only about 10^ of the pigment remaining is hemoglobin.

As early as 1865, Kuhne (8) showed that the red coloring matter of muscle was due to the same coloring material found in blood. In

1897, Morgan (9) recognized that the solubility of myoglobin is much

3 4 greater than that of hemoglobin. This property has proven useful in techniques for separating the two pigments. Kennedy and Wipple (lO) in 1926, found little evidence that the twp pigments were different.

Gunter (ll) pointed out on the basis of a slight difference in the absorption bands, that muscle hemoglobin was not identical with myoglobin.

With Theorell's (3) crystallization of pure myoglobin from horse heart in 1932, a marked increase in activity in the study of myoglobin began. Theorell*s (12) early value of 34,000 for its molecular weight was based on ultracentrifuge studies. Later this figure was revised fran 16,900 to 17,600 by Poison (13) and this is approximately the value (17,000) generally accepted at the present time.

Early indications pointed to the fact that the differences between hemoglobin and myoglobin were probably due to differences in the globin of the molecule rather than to the hone group (I4 ). The prosthetic group of both hemoglobin and myoglobin is iron protoporphyrin or heme, a structure which wfas elucidated by H. Fischer

(4 ) through synthesis of the molecule. The protoporphyrin molecule is flat, has a high degree of stability and absorbs light in the visible range.

Lonberg and Legge (4 ) have reviewed the evidence for a hemoglobin structure in which the globin, or protein of hemoglobin and myoglobin, is attached to the iron of the heme group through the imidazole nitrogen of a histidine group of the globin. A number of earlier workers, Kuster (15), Conant (I6 ) and others, had considered the 5

linkage was probably through the histidine imidazole.

The free titratable groups in protein, such as the histidine

imidazole group, theg -amino group in lysine, and others, show

characteristic acidity constant (pK) values as well as different heats

of ionization (17). Ionizing groups thus may be identified from the

shape of the acid base titration curve and from studying such curves

made at several different temperatures. From the acid base titration

curve of carboxyhemoglobin, Cohn, Green, and Blanchard (18) postulated

the existence of thirty-three histidine imidazole groups in hemo­

globin. This agreed exactly with that found by determination of the

amino acid histidine (19). % m a n (20) found that the heat of

ionization of these groups agreed with the known values for the

histidine imidazole group. Viyman (20) determined the heat of oxygena­

tion of hemoglobin and found that the heats of dissociation of

hydrogen ions involved vdien hemoglobin is oxygenated is 6$00 cal. and

reasoned that the histidine imidazole group was involved. Haurowitz

(21) attempted to study the hemeprotein linkage by enzymatic hydrolysis

of part of the globin but it was found that after such hydrolysis the

original linkage was destroyed.

Perhaps the most characteristic property of hanoglobin and myoglobin is their unique ability to undergo reversible oxygenation without undergoing oxidation of the hane iron. Pauling and Coryell

(22) noted that hemoglobin undergoes a remarkable change in magnetic properties on oxygenation. This change is frcan the paramagnetic form

characteristic of ionic bonds, to the diamagnetic form characteristic of non-ionic bonds (22). 6

There eure some differences in behavior on reversible oxygena­ tion, which in general have been attributed to heme-heme interaction in hemoglobin and lack of such interaction in myoglobin (17). The plot of log % oxygen saturation versus oxygen pressure yields a sigmoid curve with hemoglobin, whereas such a plot for myoglobin yields a straight line. The affinity of hemoglobin for oxygen is minimum at pH 6.1 and increases on either side whereas pH has little effect on myoglobin oxygenaticai (23). Myoglobin has a much greater affinity for oxygen than has hemoglobin.

Porter and Sanger (24), using the 1, 2, 4 - fluro-dinitrobenzaie end group method, found that horse hemoglobin has 6 terminal valine groups, while horse myoglobin has 1 terminal glycine group. However, for the cow they found two valine and two methionine end groups in hemoglobin.

Rossi-Fanelli and Travia ($) observed differences in the amino acid composition of myoglobin and hemoglobin, particularly in the arginine, tyrosine and lysine content. Tristam (25) compiled the amino acids composition data available up to 1943. His table shows a wide difference for some amino acids. Large differences are noted for arginine, lysine, methionine, glutamic acid, and serine.

Kiese emd Kaeske (26) noted that myoglobin foms hemochrcmogens easier than hemoglobin, and interpreted this as indicating a weaker hemeprotein linkage in the case of myoglobin.

Unless direct study of tissues was used, in a large part of the reported work directly concerned with meat color investigation, hemo­ globin rather than myoglobin has been used. Thus, in the inter­ 7 pretation of work done on hemoglobin compounds, consideration must be given to the above differences. Methods now available for preparing pure myoglobin, although tedious, decrease the justification for using a model even though the model may be a good one (3) (2?). In the case of heat denatured compounds, there is a possibility that these differences become less important. There is, however, little evidence on this point.

Methemoglobin or Metmyoglobin Formation

The widely studied question of metmyoglobin or methonoglobin formation through autoxidaticm or through the action of nitrites is of particular interest to this work since at least in the case of the unheated cured meat meünyoglobin the undesirable brown product is formed.

Hoppe-Seyler, as early as 1864, recognized the existence of methemoglobin (28). Neill and Hastings (29) observed that the rate of methemoglobin formation increased with decreasing oxygen pressures.

Brooks (30) found that the maximum rate of methemoglobin fonoation occurred at 20 to 25 millimeters oxygen pressure at 30° and this was in line with earlier work by Conant and Fieser (31) Wio found much greater quantities of ferri-cyanide was required to completely oxidize oxyhemoglobin than was required for hemoglobin. They suggested the following possible pathways for formation of methemoglobin: 1.

Spontaneous decomposition of oxyhemoglobin into methemoglobin water and oxygen, or hydrogen peroxide. 2. The oxidation of reduced hemoglobin by oxyhemoglobin. 3. Oxidation of reduced hemoglobin by oxygen.

Brooks (30) considered the reaction consisted of the oxidation of 8 reduced hemoglobin by oxygen. Qualitatively a high rate of methe­ moglobin formation at low oxygen pressure might be explained on the basis that as more oxyhemoglobin is formed with increased oxygen pressures less hemoglobin is free to react. Brooks (32) studied the kinetics of the hanoglobin autoxidation and to explain his data he postulated that in addition to the effect on the concentration of oxyhemoglobin and hemoglobin an inhibitory effect of oxygen was present. Brooks (32) considered the reaction first order with respect to hemoglobin concentration. Legge (33), in the case of hemoglobin, preferred to interpret the apparent inhibition effect Brooks found as due to breakdown of a postulated one half oxygenated oxyhemoglobin intermediate of the type Hb/^(Û2)2 with the formation of methemoglobin.

Brooks found that myoglobin exhibits a maximum rate of autoxidation at approximately 1+ millimeters of oxygen pressure. Recent work by George and Stratmann (34) confirmed Brooks’ observation. They found that the maximum rate of metmyoglobin formation is at about 3 millimeters oxygen pressure.

To explain the stoichiometry of his theory Legge (33) postulated that a hydrogen donor must be present in the protein part of the mole­ cule. Brooks (35) suggested that instead of inhibition of methemo­ globin formation by oxygen, this result might be explained on the basis of an F8202 conpound acting as a catalyst for the formation of hemoglobin. George (36), in a reinterpretation of Brooks’ work suggested that hemoglobin but not methemoglobin has a group able to readily undergo oxidation or reduction. He postulated that in its reduced form it can reduce methemoglobin back to hemoglobin, George 9

and Stratmann (37) found that for myoglobin the formation of metmyo­

globin by autoxidation is first order with respect to unoxidized

myoglobin. At 30° in 0.6 M. phosphate buffer at pH 3.69, they obtained a rate constant of 0.2?. They also found that 1.5 moles of

oxygen in addition to the erne mole present in oxymyoglobin, was used

up for each mole of metmyoglobin formed^ or one mole of myoglobin plus

2.5 moles of oxygen gave one mole of metmyoglobin. As these authors

point out, in the autoxidation of a ferrous compound one mole of

oxygen would be esqpected to oxidize 4 moles of iron. The authors

suggested that hydrogen donor groups form the protein molecule itself

must be involved to account for all the oxygen. It had already been

shown by Mirsky and Anson (38) that tryptophan and tyrosine in

proteins are easily oxidized. According to Tristam (25), myoglobin

contains two molecules each of tyrosine and tryptophan. As has

previously been pointed out, Lemberg and Legge (4) also postulated

oxidation of groups in the protein molecule. George and Stratmann (34)

maintain that the reaction with the hydrogen donor groups of protein

alone do not explain the two and a half moles of oxygen required for

the formation of each mole of metmyoglobin. They suggest that a

coupled oxidation is involved.

In a later paper George and Stratmann (37) found a transition in

kinetics in the reaction between high and low oxygen pressures. They calculated the activation energy at 760 millimeters to be 25 kilo-

calories per mole, whereas at four millimeters pressure the activation

energy was 19 kilocalories per mole. They explain this difference in

activation energy by assuming that additional energy is required to 10

break the iron-oxygen bond in oxymyoglobin. George and Stratmann (37)

were able to account for the kinetics of the reaction in terms of a

complicated free radical mechanism which thar describe in detail but

t h ^ were still unable to account for the high oxygen requirement in

the reaction.

Brooks (39) recognized that the addition of sodium chloride to

meat increased the rate of metmyoglobin formation. He noted that the

rate of methemoglobin formation could be increased four to five times

by adding sufficiently large amounts of sodium chloride. Although he

was at a loss to explain part of this effect, he suggested that the

increase in ionic strength of the solution in the tissues, lowers the pH of the buffering materials present. Coleman (40) found that at

atmospheric oxygen pressures sodium chloride solutions do not effect

the oxygen hemoglobin equilibrium. Coleman (40) concluded that a

separate mechanism must be involved in the acceleration effect cm

methemoglobin formation of sodium chloride and for the accelerating

effect of low oxygen pressures. According to Brooks (41) decreasing

pH below the normal range of tissue increases the rate of methemoglobin

formation by oxyg&i. Brooks (39) found that the rate of oxidation in

meat was decreased at low temperatures. Freezing frequently retards

formation of metmyoglobin but after thawing the rate of metmyoglobin

formation is more rapid than in the case of fresh unfrozen meat (39).

Meat retains indefinitely a certain amount of respiratory activity

(41), and at the interior of meat the oxygen pressure is zero. Even in

cured meat such as bacon some residual oxygen uptake is retained; how-

evw, in this case the rate of oxygen uptake of tissue is very small 11 compared with that of fresh meat (41). This fact in combination with the fact that the maximum rate of metmyoglobin formation takes place at low oxygen pressures, gives rise to the often observed phenomenon in fresh meat of a brown area situated 3 to 5 millimeters under the surface. Generally the outer surface is a bright red while under the brown zone of metmyoglobin the purplish color of myoglobin is observed. Brooks (41) observed that the oxygen uptake of tissues slowly decreases with time and therefore the brown zone will move towards the center of the piece of meat during storage.

Due to this oxygen uptake of the tissues and the fact that the interior of meat is devoid of oxygen, the interior portions of both fresh and cured meat are protected frcxn autoxidation. As the aut­ oxidation has a marked temperature dependence the rate of oxidation of meat is minimum at low ten^eratures. Brooks (41) noted that at

-4° centigrade beef shows little discoloration due to metmyoglobin formation up to 45 days after slaughter. He also noted that partial drying of the surface of meat tends to slow the rate of metmyoglobin formation. Even though the maximum rate of metmyoglobin formation takes place beneath the surface of the tissue it nevertheless con­ tinues slowly at the surface. In time, this will be evidenced by a brown surface characterized in the industry by the term ’♦loss of bloom” .

The Action of Nitrites on Hemoglobin and Mvoglobin

Numerous papers have appeared concerning the reaction of nitrites and blood. There has been wide disagreement on whether or not a compound is formed between methanoglobin and nitrite. The stoi- 12 chicmetry of the reaction between nitrite and oxyhemoglobin has been in question for a good many years, although some agreement has now been attained. To quote Darling and Houghton (42), "The action of nitrite on hemoglobin is extremely complicated. It varies with the molecular ratio of nitrite to hanoglobin, pH, presence or absence of oxygen, and reducing agents, and possibly other factors. Among the products of reactions found under varying conditions in vitro are methemoglobin, nitric oxide hemoglobin, and nitric oxide methemoglobin."

Gamgee (43)» in 1868, observed the action of nitrites on blood and the formation of methemoglobin. Gamgee held that the product in the reaction between nitrite aind blood hemoglobin was a combination of nitrite and methemoglobin rather than the mixture. Other workers in the 1800*3 believed that the reaction product was simply methemo­ globin. Gamgee (l) in his extensive review on hemoglobin in 1898, indicated his acceptance that methemoglobin was the product formed.

Haldane (44) pointed out that the spectrum indicated that a mixture of methemoglobin and nitric oxide hemoglobin was obtadned by the action of nitrites on hemoglobin. Haldane (44) held that if the oxygen in the blood is removed by evacuation, almost pure nitric oxide hemoglobin and cxily a little methemoglobin is formed. Haldane also found that methemoglobin gave nitric oxide hemoglobin and methemo­ globin when treated with nitrite. Hartridge (45) tested the idea that the products were a mixture of nitric oxide hemoglobin and methemo­ globin by superimposing two solutions, one of methemoglobin and one of nitric oxide hemoglobin, and observing the absorption spectra. He found that this way he could not reproduce the spectrum of a mixture 13 of the two and concluded that a ccsnpound must be formed between nitrite and methemoglobin. Haurowitz (46) held the view that the products formed by the action of nitrite on reduced hemoglobin were nitric oxide hemoglobin and methemoglobin.

Meier (47) indicated that in the absence of oxygen nitrites react with reduced hemoglobin giving nitric oxide hanoglobin and methemoglobin. Brooks found that in the absence of oxygen but with a reducing agent present, only nitric oxide hanoglobin is formed by the action of sodium nitrite on hemoglobin. He, however, found that in the absence of both oxygen and a reducing agent, for each mole of NaN02 one mole each of NOHb and metHb is formed.

HNO2 •+* 2Hb MetHb -f" NOHb

In strong acid solutions when oxyhemoglobin was treated with nitrite,

Meier (4?) found the product was methemoglobin, whereas in weak acid solutions (pH 5), or in neutral or alkaline solutions, NO-hemoglobin and methemoglobin were fonned.

Marshal and Marshal (48) found that in the reaction of oxyhemo­ globin with nitrite to form methemoglobin the reaction proceeds in essentially three stages involving an induction period, a reactionary period, and a stationary period. They worked with an extremely low nitrite concentration, for example, at pH 6.2 and .00033 molar in nitrite, they obtained a lag period of one minute. They also found that pH had a very striking effect on the reaction. They found a very marked increase in reaction rate when pH decreased below 8.2. Marshal and Marshal suggest that the lag phase indicates that perhaps nitrite as such, is not the active agent transforming the oxyhemoglobin to 14 methemoglobin.

Marshal and Marshal also found that at low concentration of nitrite largely methemoglobin is produced while at relatively high concentrations of sodium nitrite demonstrable quantities of nitric oxide hemoglobin were produced from oxyhemoglobin. They studied the effect of nitrite on methemoglobin and found that this reaction was highly dependent on pH. The lowest concentration of sodium nitrite at which the formation of nitric oxide hemoglobin was observed at pH 6.2 was at 0.001 molar. At pH 7.2 sodium nitrite concentrations below

0.01 molar did not produce nitric oxide hemoglobin from methemoglobin in the absence of added reducing agents. Marshal and Marshal (48) considered that the production of nitric oxide hemoglobin involves reduction to hemoglobin before the nitric oxide hemoglobin is formed.

These authors demonstrated that considerable methemoglobin formation took place when nitric oxide hemoglobin was treated with nitrite.

They believe the action of nitrite on blood is that of the undissociated nitrous acid rather than simply that of the NO2 ion.

Haurowitz (45) considered the reaction with oxyhemoglobin to be as follows.

2 Hb02 'h HNO2 = HbOH + HbNO + O2

Meier (47) however, considered the overall reaction to be

4 HbOg •+• 4HNO2 + 2H2O = 4HbOH + 4HN0g 4* 0

Jung and Remmer (49) reported in 1949, that by the use of a very large excess of sodium nitrite a compound of nitrite and methemoglobin was formed.

Considerable disagreement has been noted in the literature for the 15

stoichicmetry of the formation of methemoglobin by the action of nit­

rite on Qjgiiemoglobin. Earlier work indicated a ratio of methemo­

globin formed to nitrite utilized from 0.5 to 2. Greenberg, Lester

and Haggard (50) found that in vitro two moles of methemoglobin are

formed frcm two moles of oxyhemoglobin by the action of one mole of

nitrite. They found that the ratio was independent of temperature,

concentration, and pH; however the rate of the reaction was dependent

on these factors. Watts (5I) found some indication that the

oxidation by nitrite does not stop with oxidation of hemoglobin to

methemoglobin. Jung and Remmer (49) confirmed this work both with

respect to the ratios and effects of pH, temperature and concentration.

Jung and Remmer (49) noted that the concentration of hemoglobin has

very little effect on this reaction rate.

Barnard (52) revived the idea that nitrite forms a campound with

methanoglobin. On the basis of oxidation-reduction experiments he

believed a combination with the iron of methemoglobin was involved.

The compound he postulated was of the type methemoglobin-NOg, an un­

dissociated nitrite conpound. Barnard considered that reaction of

nitrite and globin might take place with dénaturation of the globin.

He also suggested that the nitrite ion might react with seme site of the heme radical other than that of the iron. Marshal and Marshal (48), in their investigation of the action of nitrite on hemoglobin, did not find a nitric oxide methemoglobin or a methemoglobin nitrite complex.

Keilin and Hartree (53) discovered that an unstable NO-methemo-

^obin compound is formed by the action of nitric oxide on methemo­

globin. This compound was however, obtained with a large excess of 16 the oxidizing agent, potassium ferricyanide.

It is not known whether any of these nitric oxide or nitrite methemoglobin compounds are intermediates in the formation of nitric

oxide hemoglobin in curing. In view of the fact that the mechanisms of the reactions in the autoxidation of hemoglobin and myoglobin are

still incompletely known, it is understandable that the complex reaction between nitrite and hemoglobin derivatives are still not

satisfactorily explained.

Haldane (2) in 1901, identified the red coloring matter of cured meat as nitric oxide hemoglobin from the absorption spectra of a water extract of cured meat. He extracted cooked cured meat with acid and ether and was able to obtain a weak solution of what he thought was nitric oxide hemochrcmogen. He found that in contrast to carbonmonoxide hemoglobin, which is quickly converted to methemoglobin by sodium ferricyanide, nitric oxide hemoglobin is much more difficult to oxidize. He observed the changes on boiling and found the nitric oxide hemochrcmogen formed was stable, whereas other hemoglobin com­ pounds that might be present gave a brown color in the presence of air. He found that this compound corresponded to that which was obtained frcm hematin on treatment with nitric oxide. Haldane (2) recognized that nitrite was probably the responsible agent in curing rather than nitrate as such, and also that bacteria were probably responsible for reduction of nitrate salts to nitrite salts. In earlier work, he had noted that in the tissues of animals poisoned with nitrite salts, methemoglobin is first formed and that after a period of time reducing substances present in the tissue or in bacteria 17 bring about conversion to nitric oxide hemoglobin. He recognized that this case was similar to that found in meat curing. Hogland (54) in

1914, extended the work of Haldane and confirmed that nitrite was generally the responsible agent in curing and that sodium nitrate as such produced no cured color.

Hoagland found that nitric oxide henoglobin can be extracted from many uncooked cured products with water but in some cases he found that the pigment could not be extracted with water but only with ether in the presence of acid or alcohol. He considered this non-water extractable coloring matter to be either a breakdown product of nitric oxide hemoglobin or a derivative of nitric oxide hemoglobin. Hoagland believed that in this case the coloring matter was probably the same as that found in the cooked cured meat, Poliak (55) in a practical study of meat curing, found that nitrite salts could be used advantageously in the place of nitrate salts in curing. Lewis (56) conducted an extensive practical study on the curing of meat products with the cooperation of the meat packing industry. He confirmed that nitrite when used carefully could replace nitrate in meat curing.

Brooks, Haines, and Moran (57) reported that nitrite salts alone could not be used in curing bacon and suggested that the presence of bacteria is not essential for development of a good bacon flavor. Brooks» observation is confirmed in commercial practice by the highly successful use of 24 to 48 hour curing by means of injection of pickle in bacon with a multiple needle system, and the fact that flavor-wise this bacon is indistinguishable fron bacon cured by the older long time methods (57). In 1925, the U. S. Bureau of Animal Industry finally 18 recognized the value of sodium nitrite and since that time it has been a permitted curing ingredient in this country. Sodium nitrate is, however, often used along with sodium nitrite as a reserve source of nitrous acid, produced through the mechanism of bacterial action to aid in preventing "undercuring" of local areas of the meat.

The Effect of pH on Curing

Brooks (41) found that in the presence of reducing agents and sodium nitrite the rate of formation of nitric oxide hemoglobin from reduced hemoglobin depends on pH. Velocity varies inversely with pH, being highest at low pH. Duisberg and Miller (59) however, found no relationship between pH and color of cured meat above pH 5»0. They found that at low pH values (4.4 to 4.9) poor color fixation was obtained and recommended a pH of 5.2 as a practical minimum. It appears that this may be due to loss of nitrous acid at low pH values.

Such losses may possibly occur by the Van Slyke reaction of nitric oxide with protein amino acid nitrogen. Nitric oxide may also possibly be lost as a gas to react with oxygen at the surface of the meat or in the pickle solution, with formation of nitrogen dioxide and consequently nitric acid. This may be a possible explanation for

Gibbens* and Dyson*s (50) observation, that immediately after curing, bacon with a low pH has a relatively small amount of sodium nitrite left. The above work suggests that for rapid curing techniques the use of a lower pH would depend on retaining a favorable ratio of rate of nitric oxide hemoglobin formation to the rate of reactions contributing to nitric oxide loss. Recently, Johnson and Bull (6I) studying an accelerated curing system based on the use of higher than normal 19 temperatures reported good color fixation, although in all of their work the curing solutions were above pH 7»1« Watts (62) found that decreasing pH increased the rate of formation and the amount of HbNO formed when ascorbic acid was used in curing.

Effect of Reducing Agents in Meat Curing

Considering the fact that conminuted products during the course of their manufacture are usually devoid of red color, it appears likely that the reported reaction between nitrite and methemoglobin is not a factor in color development (53). Thus, at least a major portion of the methemoglobin present probably requires reduction before nitric oxide hemoglobin is formed. In solid products this reduction is probably accomplished by the reducing action of the tissues but in conminuted products such as weiners and bologna, the reducing agents apparently are developed during the cooking and smoking process. In a very recent paper. Watts (63) has presented evidence that sulfhydryl groups liberated on heating are responsible for reduction during heating.

A pai't of the respiratory activity normally associated with muscle can be noted in the ability of tissue to take up oxygen long after death of the animal (41). The interior portions of meat are thus under reducing conditions. Brooks (41) found that in meat an oxidation-reduction potential of approximately -0.2 volts is main­ tained. At pH 7 the oxidation-reduction potential of the myoglobin- metmyoglobin system has been reported at O.O46 volts, pH 7.0 and

30° (64). Eggleton (65) found that glutathione added to destranatized heraolysates decreases methemoglobin formation. A mixture of glutathione 20

and ascorbic acid is reported to reduce methemoglobin in vitro (66).

Lemberg and Legge (6?) noted however, that ascorbic acid under some

conditions could, in the presence of glutathione, cause destruction of

hemoglobin. It has been reported that reduction of methemoglobin by

glutathione is less active in the presence of nitrite (68). Cysteine is also able to reduce methemoglobin (69).

Lemberg and Legge (4) have revieived the literature on numerous

methaaoglobin reduction pathways in the erythrocyte. The enzyme

system of the erythrocyte is appau*aitly much simpler than that of the

red muscle cell. Thus it seems doubtful that work on this

extensively studied reduction system can be used for explaining the

meianyoglobin reducing action of the muscle cell.

Ascorbic acid has received considerable attention as a possible

reducing agent for use in meat curing (51), (62). Ascorbic acid has

the ability to reduce methemoglobin but under some conditions causes a

series of reactions leading to green choleglobin and to methemoglobin

formation (4), (51). High temperatures, high ascorbic acid content,

and freezing cause the undesirable reaction. Lemberg emd Legge have

reviewed the mechanism of this reaction which has received extensive

study (4).

Watts (51) found that nitric oxide hemoglobin is easily formed by nitrite when ascorbic acid is used as the reducing agent. She further found that although the green pigments are formed in the absence of nitrite, when nitrite is present nitric oxide hemoglobin rather than choleglobin is formed (51). Increasing salt content increased the rates of nitric oxide hemoglobin formation. 21

Gibson (69) noted the catalytic effect of metals (copper and iron)

on the reduction of methemoglobin by ascorbic acid. Weiss (?0)

recently reported that copper (2-5 p.p.m.), iron (5-10 p.p.m.), and

zinc (10-20 p.p.m.) increased the rate of nitric oxide hemoglobin

formation in hemoglobin solutions treated with nitrite. In his work

Weiss used a metal complexing agent, ethylenediaminetetraacetic acid,

to remove trace metals present in the hemoglobin solutions.

Although under certain conditions the use of ascorbic acid appears

to be effective, a reducing agent causing fewer undesirable side

reactions is to be desired.

Heating and Hanochrome Formation

In practice most cured meat products are heated during their

manufacture. In some cases (comminuted products) most of the color is

developed during this heating process, while in solid products such as

bacon a marked brightening of the color is observed and the color

becomes "fixed" and more stable. Experience teaches this is a function

of time and temperature. In normal practice desired color may be

developed in bacon in six to eight hours at approximately 130°F. house

.temperature.

On heating the protein probably undergoes dénaturation. Sulf-

hydryl groups are known to be present in globin and bec

dénaturation (63). Watts has recently shown that formation of the

nitric oxide derivative can take place in the presence of nitrite due

to sulfhydryl groups liberated on heating the protein of meat (63).

Hartridge (71) reported that nitric oxide hanoglobin undergoes heat

dénaturation at the relatively low temperature of 50°C. (122°F.). 22

After heat denatiaration the coloring pigment of cured meat is probably

no longer nitric oxide myoglobin but probably is denatured globin

nitric oxide hemochrcsne. The true nature of the water insoluble pigment

in products heated to low temperatures only, as for example in bacon,

has not been elucidated.

From cooked cured meat with acid and ether both Haldane and

Hoagland extracted a material which had a spectrum similar to nitric

oxide hemoglobin (4), (55). On the basis of solubility it is

improbable that this was the denatured globin NO-hemochrome which is

believed to be the coloring material of cured cooked meat.

Anson and Mirsky (72) first arrived at the correct nature of the

compounds termed hemochromogens in 1925. They found these conpounds

were composed of heme combined with nitrogenous organic substances.

In the hemochromes the iron, in addition to being bound to the

four porphyrin nitrogens as it is in heme, is also attached to two

additional nitrogenous substances by two covalent bonds. The

structure is characterized by a strong spectral band at 550 milli-

microms (?2). A considerable number of nitrogen-containing com­

pounds form hemochromes and these include pyridine, amines, the

imidazole nitrogen of histidine and nitdc oxide.

Hemochromes are also formed with certain accessible nitrogen

groups in denatured protein. The affinity of these groups is reported to be higher than that of most simple nitrogenous hemochrome forming groups (73).

Anson and Mirsky (72) recognized the close similarity between hemochromes containing denatured globin and those containing simpler 23

groups as well as the fact mixed hanochrcmes can be formed having two

different nitrogenous groups. Myoglobin is reported to form mixed

hemochi'omes more easily than hemoglobin (74)• It appears that the

coloring matter of cooked cured meat is probably of the mixed

hemochrcme class. Carbon monoxide forms a mixed hemochrome type com­

pound but one of the hemochrcme forming nitrogens is replaced by CO

(74). This conpound is of interest because it has a structure similar

to the NO derivative and has an absorption spectrum similar to carbon

monoxide hemoglobin.

Lysine and arginine reportedly do not form hemochromes (76).

Keilin (75), however, found that many amino acids under some conditions

form hemochromogens. Keilin suggested that groups other than the

histidine imidazole in denatured proteins, such as free amino groups,

might also form hemochromes. In an earlier paper Keilin found serum

albumen and GO formed a compound of the hemochrome type. Additional

evidence that more than the imidazole nitrogen is involved was given

by Holden and Freeman (77) who treated denatured globin with nitrous

acid and found a decreased ability to form hemochromes. Since the

Imidazole nitrogen of histidine does not undergo the Van Slyke reaction,

their work indicates other groups may be involved. A recent paper on the acid dénaturation of CO-hemoglobin (78) indicated that thirty-six basic groups are liberated, and that all groups become available at

one time,-that is, the reaction is an all or nothing phenomenon. In

this case it was noted that methemoglobin was denatured more rapidly by acid than is CO-hemoglobin. It is therefore not known whether or not the heme linkage rmnains with the same group or even whether or not 24

the heme is linked to some other protein.

No information on the light absorption characteristics of the

pigment in heated cured meat as such was found in the literature.

Further investigation of this heat-denatured pigment was much to

be desired, for this and not nitric oxide hemoglobin was thought to be

the coloring material of cooked cured meat.

Color Loss in Cured Products

Dehydration of the surface, bacterial discoloration, autoxidation

and light-induced oxidation are the principal known causes of dis­

coloration in cured meat products. Prevention of bacterial discolora­ tion will not be considered here. Surface dehydration is subject to control by the selection of proper packaging materials. Autoxidation in the absence of light is not a serious problan if products are heated sufficiently during the course of their manufacture and if low temperatures are maintained. Due to modern merchandising methods in which the product is displayed under lights, the light-induced reaction leading to discoloration has become a serious problem.

Covering the product to exclude light could be a solution but hardly one in keeping with merchandising methods in use. Decreasing the amount of light reaching the product yields some benefit since the reaction is said to be a function of foot candle hours (79)* Kraft and

Ayres (80) found less fading took place with intensities of 30 to 35 foot candles than at 50 to 60 foot candles. These authors also found less fading with ultraviolet light than with white light but they note that the observed difference could have been due to absorption of ultraviolet light by the packaging material. Urbain and Ramsbottcm 25 found that with boiled ham color retention was best obtained by excluding light or oxygen (81).

Even the small amount of available published material that does exist on this problem provides few clues as to the mechanism of the fading reaction.

Watts very recently shov/ed that after exposure to light in the presence of nitrite, the color of cured meat is partly regenerated in samples giving a positive nitroprusside test for sulphydryl groups.

Weiss, Green and Watts (70) noted that metal ions, particularly copper and iron, accelerated the formation of nitric oxide hemoglobin.

These authors also noted that copper, iron and zinc in the presence of ascorbic acid retarded fading.

With a molecule as large as that of denatured globin nitric oxide myohemochrome, it appears that the heme group need not undergo reaction due to the primary absorption of a quantum of energy. It appears possible that energy might be transferred to the protein part of the ft molecule. Bucher and Kaspers (82) have shown that energy absorbed by the protein part of the molecule at 280 m was as effective in splitting carbon monoxide from carbon monoxide myoglobin as was light absorbed by the heme group at 546 . The quantum efficiency of this reaction was one. It has already been noted that the work of George and Stratmann

(34) indicated that myoglobin upon autoxidation to metmyoglobin prob­ ably undergoes oxidation of some groups in the globin.

Weil, Gordon, and Buchert (83) found that tyrosine, tryptophan, histidine, methionine, and cysteine readily undergo photochemical oxidation in the presence of methylene blue as a sensitizer and in 26 visible light. They further found that proteins ^-lactoglobulin and others) undergo a similar reaction (.83), In both cases (with amino acids or with proteins) hydrogen peroxide or organic peroxides are products. It is well known that H2O2 can oxidize hemoglobin compounds to the met or ferric form. It seems unlikely that catalase is present, due to the effect of nitrite and of heating. It was thought possible that the above could be a possible mechanism in which oxidation of the denatured globin NO-hemochrome with loss of NO, or perhaps oxidation of NO by hydrogen peroxide might be involved. The fate of the nitric oxide in the fading reaction was unknown although it was thought that it might react with oxygen to form NO2 and finally give the nitrate and nitrite ions. EXPERIMENTAL PROCEDURE

Ob.iect of the Investigation

The purpose of this work was to attempt to find out what chenical changes take place during discoloration of the heated purified cured meat pignent and especially what changes taking place in the presence of light and oxygen are inherent in the nature of the pigment itself. It was also desired to explore the related question of what components of meat other than the pigment itself can be involved in meat discoloration.

Nitric oxide myohanochrcme prepared from metngroglobin isolated from beef was used in most cases for two reasons - first, to eliminate possible interference by other substances in observations of reactions ccaicerning the pignent itself and, second, because of the extremely low concentration of this pigment in meat.

Since most meat products are heated during the course of their manufacture the heat denatured pigment was used in almost all of the work. This precluded the use of spectrophotometry, the classical tool in this field, for quantitative work. For this reason and because of the extremely low molar quantities of material involved a mancmetric technique was selected as the major tool.

The fact that the pigment was a solid greatly complicated the possibility of obtaining rate information in the Warburg work due to the heterogeneous nature of the system, but the fact that the pigment was solid had one important advantage. It could be washed free of nitrite salts and reducing agents used in its prepsuration. A relatively simple systan could thus be studied.

27 28

It was desired to relate oxygen uptake and any other factors that could be studied since oxygen is known to be in^ortant in meat discoloration. It was especially desirable to find out if the oxidation of this pigment could be related to published work on the oxidation of myoglobin. Oxygen uptake was studied with a Warburg apparatus especially constructed for this photochemical work. George and Stratmann (34) had proposed that peroxide formation takes place in the oxidation of metmyoglobin. Sodium pyruvate was added to Warburg samples in an attempt to detect peroxide formation by CO2 liberation.

It was especially desirable to find out what happened to the nitric oxide lost from the pigment since it was possible that preservation of color may involve regeneration of color. Several experiments were carried out in a nitrogen atmosphere and oxygen uptake was observed in the presence of extraneous protein in order to find out if nitric oxide may be lost by a reaction involving free amino groups.

Nitrite and nitrate were determined in the supematent liquid in which pignent was suspended during the Warburg runs to find out if these ions are produced. In order to find out whether or not nitric oxide can be lost as a gas the KOH in some of the wells was analyzed for nitrite or nitrate.

It was desired to find out if substances in meat other than the pigment itself can cause discoloration of the pigment in light and oxygen. Pbr this purpose the pignent was vigorously shaken with many possible ccmiponents of meat and loss of color was observed compared to controls in water.

Fatty acids were found to cause darkening when shaken with the 29 pigment in the light and in air and thus an attempt was made to study oxygen uptake of the pigment in the presence of fatty acids.

In bacon "color fixation" or production of a water insoluble o pigment takes place in about 45 minutes at 48.6 C. A much higher temperature is required to render the purified pigment insoluble.

An attonpt was made to find out if free fatty acids or surface active agents affect temperature at which the pigment becomes water insoluble.

The heated pigment has been assumed to be a hemochrome.

Spectrophotometry was used to obtain some evidence on this point.

Since it was necessary to work with the solid pigment the method employ­ ed, at best, left much to be desired.

The affect of Heating on Color Fixation and Color Stability

It was desired to confirm the practical observation that the pigment of heated cured meat is water insoluble. Two samples of bologna (different brands) and two samples of bacon (different brands) were blended with 5 parts water to 1 part meat. On addition of

K3Fe(CN)6 and KCN to the water extract no red cyanometmyoglobin was observed. In later experiments at the laboratory of Kingan Incorp­ orated, it was noted that bacon that had a cured color which was stable in the dark for at least two days generally showed little or no soluble myoglobin by the above test with potassium cyanide. In tests carried out at Kingan, Inc., single layers of bacon were placed in aluminum foil polyethylene lined packages. The packages were vacuum sealed. These were placed in a water bath for different lengths of time. Approximately 45 minutes at 120°F. (48.6°G.) was required to

"fix" color to render it insoluble. This is far below the temperature 30

at which the purified pigment is denatured.

It was desired to find out something about the relative ease of

precipitation of myoglobin and hemoglobin by heat and to find out if

free fatty acids or surface active agents could possibly modify the

temperature required for precipitation in an arbitraurily chosen period

of time. Fatty acids are known to increase the stability of serum

albumin to heat precipitation (84). Anionic detergents are said to be

effective in denaturing proteins (85). It was thought possible that

free fatty acids might occur in meat and that they might affect the

precipitation. A secondary objective was to find a way to denature

the protein without precipitation in order to prepare a sample for

absorption spectroscopy. A solution of a water extract of beef and a

hemoglobin solution were used. liJhere the nitric oxide derivative was prepared 4.0 ml. of 0.5^ NaN02 solution and 4 ml. of a 1% Na2S20j|^

solution were added per 100 ml. of beef extract or hemoglobin. Where

fatty acid is indicated 1 gram of lauric acid and 10 gm. methyl

laurate were added. In each case the mixture was shaken before

sampling. Samples were used at the pH of the stock solution and were not buffered since it was desired to observe pH changes on heating.

Ten ml. samples were heated 30 minutes in each case at different tsaperatures in a water bath.

At the end of the heating period, the samples were cooled in the cold roOTi and were centrifuged and filtered. Two ml. aliquots were taken for determination of cyancmetymyoglobin. In this case 0.1 ml.

0.5 M pH 7.0 phosphate buffer, 0.2 ml. 0.06 M K^Pe(GN)^, and 0.2 ml. KCN was added. Since a slight turbidity developed in some cases before 31

determining the absorbance, readings were taken at the absorbance

minimum of 500 millimicrons as well as at 540 millimicrons, and the

difference between these readings was taken as the index of the

solubility.

Treatment

A - Mb*

B - NCMb

C - Mb Dreft

D - NOMb Dreft

E - Mb Lauric Acid

F - NOMb Lauric Acid

*Either hemoglobin or a heat fractionated water extract contain­

ing myoglobin.

There was not a clear cut difference in the behavior between

samples with fatty acid or Dreft and between paired samples. There was

not a clear difference between the met form and the nitric oxide

derivatives, but in all cases heat dénaturation was largely complete in

30 minutes at 65°C. This was somewhat below the tanperature at which

the purified pigment was precipitated and yet much higher than the

temperature at which the pigment is "fixed" or made water insoluble in

bacon.

It is still unexplained how color is "fixed" and nitric oxide myoglobin is made insoluble at the low temperatures encountered in

practice. This question is of the greatest importance since a know­

ledge of the nature of color fixation in such cases could lead to a knowledge of the true nature of pigment in heated cured meat. Since 32 color "fixation” takes place rather sharply as temperature is raised it appears possible that color fixation may involve proteins other than the myoglobin itself. It would seen possible that this might involve an interaction between acid grovçjs liberated on heat dénatura­ tion of protein and the basic groups known to be present in the myo­ globin protein. Further work in this area is desired. According to

ELotz (86), "The precipitation of anionic proteins by cationic proteins or by protamines or histones has been recognized for over 50 years as being salt like in nature."

Early in this work it was desired to determine the quantum yield of the fading reaction since this could give some clue as to whether this reaction was likely to involve a chain catalysis, whether the rate of the reaction was directly dependent on the primary photo­ chemical process or whether secondary reactions limited the rate.

Bucher and Kaspers (82) found that for CO-myoglobin and CO-myoherao- chrerne the quantum efficiency was one.

A Marburg experiment was carried out using the specially con­ structed Warburg flasks with side windows. The actincmeter of Warburg and, Schocken with protoporphyrin as the sensitizer was used and is described in another section of this paper. The number of light quanta striking each flask was 1.0 micromoles quanta/minute in which case for a 4.6 micromole pigment sample complete fading would be expected in 4.6 minutes if the quantum yield were one. Based on the average O2 uptake in 254 minutes approximately I64 light quanta were required per mole O2 or 41 quanta per electron transfer. The observed rate of loss of red color could not be put on a quantitative 33 basis but certainly corresponded to a low quantum efficiency.

According to Keilin and Hartree the dissociation of nitric oxide hemoglobin is slow even in the presence of ferricyanide. The ferricyanide however reacts rapidly with hemoglobin. It was there­ fore thought that it would be possible to find out if the low observed quantum yield was due to a recanbining of 1K> split off of the molecule by light or whether seme other explanation must be needed.

In this experiment, it would be expected that ferricyanide would trap free myoglobin produced.

The visual color scores given in tables 1 and 2 are based on an arbitrary value of five given to the untreated control sample of each individual meat product and zero for samples faded to such an extent that all redness was visually judged to be gpne. 34

Table 1

Visual Color Scores for Cured Meat Irradiated in the Presence and Absence of Potassium Ferricyanide

Dark Light Light

K3Fe(CN)^ K^Fe(GN)^ Water

Time in Time in Time in minutes minutes minutes

Sample 15, 45, 90 15, 45, 90 15, 45, 90

Ham loaf 4, 4, 3 3, 1, 1 4, 2, 1 duplicates unheated

Ham loaf 3, 3, 3 4, 3, 1 3, 3, 1 heated

Boiled ham 5, 5, 5 2, 2, 0 5, 3, 2 heated

Boiled ham 5, 5, 5 2, 1, 0 4, 2, 0 unheated

Bologna 1, 1, 2 2, 1, 0 4, 2, 1 unheated

Bologna 5, 5, 4 3, 2, 0 4, 2, 1 heated

It was also desired to find out if heating increased stability

toward oxidation by weak ferricyanide solutions. In this case a

0.0075 M solution of K^FeCCN)^ was used in all cases. 35

Table 2

Visual Color Scores for Cured Meat Irradiated in the Presence and Absence of Potassium Ferricyanide Experiment 2.

Dark Light Light

K3Fe(CN)^ K^Fe(CN)^ Water

Time in Time in Time in minutes minutes minutes

Sample 0, 20, 60 0, 20, 60 0, 20, 60

Ham loaf 3, 3, 3 3, 0, 0 5, 3, 1 unheated

Ham loaf k, 4, 3 4, 1, 0 5, 4, 2 heated

Bologna 2, 2, 2 2, 1, 1 5, 4, 3 unheated

Bologna 3, 2, 2 3, 1, 1 5, 3, 3 heated

Bologna 1, 2, 2 1, 1, 0 3, 3, 3 unheated Brand B

Bologna 2, 2, 2 2, 1, 0 5, 3, 2 heated Brand B

Bacon 5, 4, 3 5, 3, 1 5, 3, 2 unheated

Bacon 5, 4, 3 5, 2, 0 5, 3, 2 heated

Slices of different cured meat products were either used as they

came from the package or slices were placed in test tubes under Ng and heated in a water bath at 85° for 20 minutes. One fourth slices of product were dipped in water or in .0075 M potassium ferricyanide solution. All samples were placed in paper plates and were covered 36 with saran to prevent dehydration. Samples were irradiated with a

General Electric 150 watt tungsten spotlight at 4 ft. Controls dipped in water were held in the dark at 4°C. Each sample was compared to the control rated 5, thus these color scores are relative to the individual controls only. Color was scored at 15, 45 and 90 minutes.

With most products, heating to 85° for 20 minutes increased the stability toward oxidation by the ferricyanide to sane extent. It is not apparent from the above tables but heating increased the redness of the samples in most cases. Heating did not, however, appear to have much effect on samples treated with water alone.

In a few cases there appeared to be an increased rate of color loss in the light in the presence of ferricyanide but in general this experiment indicated that the rate of loss after an initial rapid color loss in some cases was the same in the presence and absence of ferricyanide. This experiment suggests the possibility that the low queuitum yield cannot be explained on the basis of a splitting off and recombining of NO. The experiment is conplicated by the fact that sulfhydryl groups were probably reacting with the ferricyanide.

Essentially all excess ferricyanide may have been reduced in most cases.

In many cases, this experiment suggested that additional sulfhydryl groups are produced on additional heating of ccmmercial products.

Cured ground beef was heated to 60°, 75°, and 82°C. respectively for 5 minutes. In the presence of .0075 M K^Fe(GN)^ the samples heated to 60° lost its red color in 1 minute in room light. Without added ferricyanide in room light, it lost nearly all of its red color in 1 37

hour.

Beef heated to 75° for 5 minutes lost about 1/2 of its color in

1 minute when covered with 0.0075 M K^Pe(CN)^. No color was lost at

roan temperature in one hour. The color was lost after 2 days at 4°G.

in the dark. The sample heated at 82°C. lost only a barely

perceptible amount of color in 1 minute in 0.0075 M K^Fe(CN)^ and

lost its red color only eufter 3 days in the dark at 4°C. This

indicated that heating in some manner stabilized color. In view of

recent work by Watts (63) the above earlier results can be interpreted

to be due possibly to production of sulfhydryl groups on dénaturation

of the protein.

Isolation of Metmyoglobin

In most of the work carried out purified metmyoglobin was used

for preparing samples of denatured globin nitric oxide myohemochrome,

the pigment studied. In some of the early Warburg work, however,

partially purified samples were prepared by heat fractionation alone.

Also several myoglobin preparations were made in which NaN02 was

added. It was thought possible that preparation of the pigment as the

nitric oxide derivative might help prevent undesirable oxidation during preparation of the sample. This seemed to offer no advantage and the method was abandoned. In some of the early work, myoglobin was

isolated by a method essentially that of Theorell and hemoglobin was not removed. In later experiments including all experiments involving nitrite and nitrate determinations metmyoglobin was isolated by a combination of the methods of Theorell (3) and of Morgan (87) and the method used was similar to that described by Ginger and Schweigert (88). 38

A number of preparations were made at different times, there was also some difference of details.

Twenty lbs. of beef round was ground 6 times in a meat grinder using a fine plate. This was mixed with one part water and was held overnight at 4^C. The mixture was filtered. Basic lead acetate was added to the mixture until no more protein precipitated as determined by removing small portions of the mixture and testing by addition of lead acetate. The mixture was filtered. The lead was removed by adding a small amount of solid KH2P02^ and K2HP0^ alternately, with a constant check on pH. The point at which all of the lead was precip­ itated was checked by centrifugation of small amounts after each addition of potassium salts. The mixture was filtered. After all of the lead was removed, the solution was made 3 molar in phosphate.

According to Morgan (87) this precipitates the hemoglobin. The pH was held in the range of 6 to 7 throughout this work. The addition of salts was controlled to reach a final pH of 6.6. The filtrate was dialyzed against distilled water at 4°C. for one day to remove most of the phosphate. This material was then dialyzed against saturated ammonium sulphate which had been adjusted to pH 6.6 with a small amount of KOH solution. A few drops of 2% KOH solution was added to a thick slurry of solid ammonium sulphate and water to give a pH of

7 .0 . The dialysis tubing containing the filtrate was placed in this slurry for several days. The pH was never allowed to go below 6.0.

This treatment decreased the volume of the filtrate and concentrated the pigment. Dialysis was carried out for several days against saturated ammonium sulphate. The precipitate was centrifuged off at 39

approximately 8,000 g. The pigment was extracted with successive small portions of 82% saturated ammonium sulphate until the color of the remaining material began to become a light grey. This solid was

discarded. The myoglobin solution was dialyzed against saturated ammonium, sulphate adjusted to pH 6.6 for about 3 days. The precip­

itate was centrifuged off and was mixed with saturated ammonium

sulphate, about 10 parts to one part pigment. The mixture was again

centrifuged. The solid vjas extracted with very small portions of 82%

saturated ammonium sulphate. The solution was dialyzed 3 days

against distilled wiater vjith agitation at 4°C. Addition of BaCl2

solution shov/ed it to be free of sulphate. In each case the amount

of metmyoglobin present was determined as cyanometmyoglobin by the method of Drabkin and Austin (89). Potassium ferricyanide and potassium cyanide were added to give .0006 molar and .0008 molar

concentrations respectively. Phosphate buffer of pH 6.0 was added to give concentrations between 0.01 and 0.0025 molar. The reagent absorb­ ance is negli g ible at 540 m and in each case the absorbance was measured v/ith a water blanlt at 540 m . Protein was determined for some preparations and comparison of the amount of metmyoglobin with the protein found gave apparent molecular weights from 17,000 to

17,900.

Qualitative Experiments on the Effect of Possible Meat Components on Discoloration

In i/arburg experiments it appeared that the red color was more stable than would be expected considering the rate of color loss knov.n to take place in cured meat products. It was thus thought possible 40 that seme canponents of meat might be involved in producing fading which were absent in the purified pigment suspensions.

In view of the fact that a peroxide mechanism involving oxida­ tion of groups in the myoglobin has been proposed (34), it was thought possible that oxidation of protein other than that of the myoglobin or the hemochrcme might be involved in color loss. The pigment was prepared as described later for mancmetric experiments except that in general washing and centrifugation was carried out at room temperature. In view of later work, the material present in these samples was probably largely the myohemichrcme. The heat denatured pigment (0.79 micromoles) prepared from purified myoglobin, was vigorously shaken with 1 ml. of 1% solutions of tyrosine, tryptophan, methionine, glycine, and cysteine in 0.05 M. phosphate buffer in approximately 5 ml. of solution. In two hours all samples and the control had lost seme color in the light. In 48 hours with an intensity corresponding to 400 Weston, all samples in the light were nearly colorless but the samples in the dark had lost very little color. All of the samples in the light lost color at the same rate. In view of later work there is little doubt that the pig­ ment was largely in the form of the myohemichrcme at the start of this experiment. It can be concluded that the discoloration of the myohemichrcme is not effected by added amino acids, but no conclusions can be made regarding the oxidation of the nitric oxide myohemochrcme.

It was thought possible that metals such as iron might catalyze the destruction of the pigment in view of the findings by Weiss (?0) since metals increased the rate of formation of the nitric oxide 41 hemochrome.

In a qualitative experiment using 20 x 150 m.m. test tubes con­

taining approximately 0.5 micromoles of purified pigment and 0,2,5 and

100 parts per million of iron, samples were shaken eleven hours in a

cold room at l°C. under tungsten illumination. There was little

color loss in the dark and all samples in the light lost color at

about the same rate.

A Warburg run was carried out with no added iron and with 50 parts per million of iron (as Fe) added in the form of ferrous sulphate. In

624 minutes in pure oxygen in the presence of iron 52 microliters of

oxygen was taken up in the dark and 93 microliters of oxygen was

taken up in the light. Without iron, the uptake was 45 microliters in the dark and 82 microliters in the light.

An early Warburg experiment was carried out to find out if soluble materials in meat would effect the oxidation of the pigment. Pigment partially purified by heating a water extract of beef to 63°G. for 30 minutes was used. The extract of heated meat was prepared by heating the 63°C. filtrate to 80°C. for 20 minutes and filtering. This filtrate was added back after preparation of samples by addition of sodium nitrate, heating and washing by centrifugation. The samples were irradiated 56O minutes. The rate of oxygen uptake was higher than normal for all samples, but little difference was noted between samples and the control. No difference in color loss was observed between the sample and the control in the light.

Soluble materials and amino acids did not give an increased rate of color loss with the pigment which was at that time considered to be 42

the pigment of cured meat. On reviewing the technology of the manufac­

ture of cured meat products it appeared that the long heating operation

in the presence of water might lead to an initially increased lipase

activity with formation of free fatty acids. It was also thought

possible that chemical hydrolysis of fat might take place during cook­

ing operations. It thus seaned possible that a small amount of free

fatty acid might be present due to chemical hydrolysis.

Purified pigment, at that time believed to be nitric oxide

myohemochrome, but now known to be partly the denatured globin

myohemichrcmie, was shaken vigorously at about 200 oscillations per

minute, under tungsten illumination using an Ansco heat filter.

Color was scored against a similarly prepared control held in the dark

and given a score of 10. In the first exploratory experiments, the

fatty materials were added dropwise. This was justified since the

system was a heterogeneous one and differences in dispersion and

adhesion to the side walls of the vessel would far outweigh small

variations in amount of material added.

In the first experiment, 1.3 micrcmoles of purified pigment in

5 ml. of solution containing in most cases 2% of sodium chloride was

used. After irradiation for 60 minutes, the control in the light

scored 9» Oleic acid alone scored 3. Oleic acid in a 2^ solution of

NaCl scored 2. In the dark, oleic acid scored 10. A sample contain­

ing 5 drops of commercial corn oil scored 8 after irradiation in the

light.

In the second experiment to observe the effect of oleic acid, the color score at 90 minutes was observed using 1.3 micromoles of 43

pignient and one drop oleic acid in 5 nil. of solution. In the light

alone, the pigment scored 9. Samples containing oleic acid alone or

oleic acid in combination with 10 parts per million iron, with 0.5 M.

pH 6.4 buffer, with 1 ml. k% sodium pyruvate and with 1 ml. of \%

cysteine had a color score of 1. Four drops of oleic acid alone gave

a color score of 0. Oleic acid shaken with the sample in the dark

with 10 parts per million of iron had a color score of 10.

Since color was rapidly destroyed when oleic acid was shaken

with the pigment (probably largely the hemichrome) it was desired to

find out if the effect was due to the acid grouping, to the double

bond, to peroxides that might be present in the oleic acid used, to

linoleic acid that might be present as an impurity in the oleic acid,

to surface activity alone or to a pH effect. About thirteen

additional qualitative tests were conducted in a similar manner to

attempt to establish how the fatty acid is involved in discoloration.

In the next experiment, the effect of oleic acid and methyl

oleate was compared. All samples were shaken in the light. Sodium

chloride had little effect on samples containing oleic acid or methyl oleate. Three samples containing respectively one drop methyl

oleate, one drop methyl oleate plus NaCl and one containing 10 drops

of methyl oleate all scored 9 after 60 minutes irradiation. Oleic acid plus methyl oleate score 4. Two samples with oleic acid scored 3»

It thus appeared that the acid group must be involved in some manner although involvanent of the double bond was not eliminated.

In order to find out if this was due to pH effect alone, three pigment samples in pH 5*70, 6.04 and 7.20 0.05 M. phosphate buffer hh were shaken in the light for 40 minutes. After 40 minutes, these had scores of 8, 9 and 5 respectively. The sample with oleic acid in pH 6.40 buffer had a score of 3* This experiment suggested a pH effect, but it did not seem to be the major factor.

It was desired to find out if surface activity might be a factor and to find out if oxidized fat could cause discoloration of the pig­ ment. In this case, the sample used had been held three days in the cold room after preparing and washing out reagents. In the light of later information, this was undoubtedly almost entirely the myo- honichrome. The oxidized fat had been prepared by blowing air through corn oil for 3 days with the sample held approximately 10 hours under ultraviolet light. The starting score for all samples was 10. 45

Table 3

Color Score After Shaking Pigment with Fat Derivatives

Time Shaken

Treatment 10 minutes 70 minutes 180 minutes

Pigment only light 10 8 7 dark 10 10 10

Oleic acid light 9 6 3 dark 10 10 10

Tween-80 in Methyloleate light 9 7 5 dark 10 10 10

Oxidized Fat light 7 3 2 dark 7 6 5

Oxidized fat Tween-80 light 1 0 0 dark 1 0 0

Tween-80 light 10 7 6 dark 10 10 10

Oleic acid Tween-80 light 6 3 1 dark 8 6 6

This shows some effect on color loss due probably to the double

bond in Tween-80, but Tween-80 enhanced the rate of color loss in the

presence of oleic acid. Tween-80 greatly enhanced the color loss in

the presence of oxidized fat.

Lecithin was known to be likely to cause difficulty due to

peroxides, so several exploratory experiments were carried out to test

this possibility. After shaking 90 minutes in the light in the

presence of one drop lecithin alone the color was 7 compared to a con­ trol scoring 9. The pigment plus oleic acid alone scored 4 and in the hk presence of lecithin scored 8 at pH 6.4*

In another experiment after irradiation for 60 minutes, lecithin and methyl oleate together produced a score of 1, while the sample with methyl oleate alone scored 1 and the control scored 9*

In one experiment, approximately 0.5 micromole samples of the denatured pigment alone were shaken 60 nanutes with 4 drops of fresh melted lard. After 60 minutes shaking in the light, the'pigment alone still had a score of 10. Two pigment samples with lard and phosphate buffer only and one with lard and pigment only had a score of 4 after shaking for 60 minutes. Samples ifith lard and lauric acid and with lard and oleic acid had scores of 0. In the dark, lard and lauric acid had a score of 8 while lard in the presence of oleic acid scored

9. Lard and methyl oleate scored 9*

The results with lard could not be reproduced with another lard sample. In another experiment the addition of lard did not effect the amount of color loss after 30 minutes when it was added to samples of pigment containing lauric acid, oleic acid or methyl oleate. It is likely that in the first case peroxides were present.

In order to check on whether the acid group alone of the free fatty acid is involved, lauric acid dissolved in methyl laurate was used. The lauric acid had a zero iodine number and the methyl laurate used was prepared by refluxing this acid with methanol. Pigment sam­ ples containing 0.36 micromoles of heated pigment were shaken in the light under the usual conditions for 75 minutes. In this case oleic acid destroyed color more rapidly than did lauric acid, indicating kl the possibility that both the acid grouping and the double bond or peroxides may be involved. There was sane suggestion in this experiment that Tween-80 was increasing the color loss. Peroxides were probably not associated with the Tween-80 since there was no color loss in the dark with lauric acid and methyl laurate.

Table 4

The Effect of the Acid Group on Visually Scored Color Loss for Cured Meat Pigment

Treatment Score (75 minutes)

Pigment only 9

Pigment only 10

Oleic Acid $0 mg., - Methyl laurate 150 mg., Tween-80 1

Lauric acid 50 mg.. Methyl laurate 150 mg., Tween-80 5

Lauric acid 52 mg.. Methyl oleate 140 mg., Tween-80 3

Methyl laurate I50 mg., Tween-80 7

Methyl oleate 142 mg., Tween-80 7

Lauric acid 50 mg. Methyl oleate I6O mg., Tween-80, Dark 5

Lauric acid 50 mg. Methyl laurate I50 mg., Tween-80 10

Lauric acid 5O mg.. Methyl oleate I50 mg. 3

Lauric acid 50 mg.. Methyl laurate I48 mg. 7

In order to eliminate possible ambiguity due to possible peroxides associated with the oleic acid and especially the possibility that linoleic acid could be present in the oleic acid, pure oleic acid was 48 prepared by low temperature crystallization (90) stai'ting with 225 grains of Malinkrodt U.S.P. oleic acid. This was crystallized 4 times at -60°C. in 3500 ml. of acetone. Saturated acids were removed by holding over night at -20° in 2000 ml. of acetone. The oleic acid was distilled under high vacuum and had a sharp melting point of

12.0°C. Methyl oleate was prepared by estérification using C. P. methanol.

In another eatperiment carried out under the usual shaking and lighting conditions, 48 mg. of lauric acid in 48 mg. methyl laurate produced a color score of 3 in 60 minutes. A score of 3 was also obtained with purified oleic acid (46 mg.) and methyl laurate (45 mg.).

Lauric acid (45 mg.) in methyl oleate (42 mg.) also gave a color score of 3* In the latter sample the score in the dark was 8.

It was also desired to explore further the effect of an anionic surface-active agent. Dreft was one possibility. One ml. of 2%

Dreft gave a color score of 9 compared to the control with 9 in 60 minutes. A score of 9 was also obtained in the dark. Dreft added along with methyl oleate gave a color score of 3 after 60 minutes shaking in the light. Dreft (1 ml. 2%) plus oleic acid gave a score of 2 after shaking in the light. From this experiment, it appeared that Dreft was effective in causing rapid color loss in the presence of methyl oleate or oleic acid.

In another experiment, oleic acid (22 mg.) in 45 minutes gave a color score of 6 in the dark and 3 in the light. The control scored

9 in the dark, but dropped to 6 in the light. In the light of later work, this indicated that in this case a considerable portion of thé' 49 pigment was in the form of the nitric oxide derivative at the start, since fading to the hemichrome stage is relatively rapid and further fading of the hemichrcme is slower.

In order to observe the effects of a nonionic surface active agent and especially one that would probably not be readily oxidizable

Brij-30 (20 mg.) was used in sane of the samples and was compared with

Tween-80. Color was lost in the presence of 40 mg. of oleic acid at the same rate with 20 mg. Brij-30 and 20 mg. of Tween-80. In the presence of 60 mg. of methyl laurate however the color score in the light was 8 with 20 mg. of Brij-30 and 4 with Tween-80. Color loss was slightly more rapid in the presence of Brij-30 alone than for the control.

It was desirable to compare the effect of an ionic detergent,

(Dreft) and a nonionic detergent (Brij-30). After shaking samples for

30 minutes in the light, the control sangle had a color score of 9«

The sample with 1 ml. 2% Dreft and with 0.1 ml. methyl oleate scored

8. Dreft along with 0.1 gm. lauric acid 0.2 gm. methyl laurate as well as a sample with methyl oleate scored 8. Dreft along with

0.1 gm. lauric acid 0.2 gm. methyl laurate had a score of 1. In this case the fat layer had a brown color. Na2S20^ was added and it was found that the red color was regenerated. A sample with 0.10 gn. lauric acid and 0.2 gm. methyl laurate had a score of 6 after shaking

30 minutes. Again a small amount of Na2S20^ regenerated the color.

It was, however, noted that much of the red color was in the fat layer and the protein had lost much of its color. In the same experiment,

Brij-30 plus methyl oleate had a score of 7 after 30 minutes. Brij-30 50 plus methyl oleate had a score of 7 after 30 minutes. Brij-30 and methyl laurate scored 6 while Brij-30 plus lauric acid and methyl laurate also scored 6. Lauric acid plus methyl laurate plus 0.1 ml. methyl oleate gave a score of 6 in 30 minutes.

At about the time the next experiment was carried out, the mixed nature of the pigment being used in the experimental work was recognized. In view of the fact that most of the earlier samples of pigment were mixtures of denatured globin myohemichrome and denatured globin nitric oxide myohemochrome, it was desirable to check seme of the results with the purified hemichrome prepared by heating 0.54 micranoles of metmyoglobin at 85° for 15 minutes. In 60 minutes in the light, both oleic acid (45 mg.) in 0.1 ml. methyl laurate and lauric acid 45 mg. in 0.1 ml. methyl laurate decreased the color score to 4« The color score of the control in the light was 8. 51

Table 5

Color Scores for Heat Denatured Globin tJyohemichroine Irradiated in the Presence of Surface Active Agents and Acetone

Color score after 30 minutes

Treatment Pigment Acetone solution

Pigment only 9 3 red

1 ml. Brij-30 9 2 red

1 ml. oleic acid 4 - 0.1 ml. Brij-30 8 0

1 ml. Brij-30 9 2 red

1 ml. 2% Dreft 9 5 red

1 ml. 2% Dreft + 0.1 ml. oleic acid 7 5 brown

1 ml. 2^ Dreft 9 water 0 (no acetone)

Pigment only 9 water (no acetone)

It was desired to explore the function of surface action agents.

Samples were shaken with combinations of Brij-30, oleic acid and Dreft, using 0.54 moles of the purified hemichrome in 5 ml. of pH 6.20, 0.05

M. phosphate buffer and 5 ml. acetone. The color of the pigment was

scored. Color in the acetone was scored against the most highly colored

acetone solution arbitrarily scored 5*

It appeared frcsn Table 5 that Dreft was dispersing some of the protein while the fatty acid caused darkening of the dispersed red colored material in the light.

In viev/ of the fact that ambiguity existed in the work it was deemed necessary to check some of the earlier results using the myo- 52

hemichrane prepared by heating purified metmyoglobin. The earlier

results with fatty acids, amino acids and the effect of pH were

rechecked.

Possible components of meat that could conceivably affect color

loss were shaken in the light and in the dark with denatui’ed globin

myohemichroine prepared by heating purified metmyoglobin. In each

case samples were made up to a final volume of 5 ml. with water.

Iron, 100 p.p.m.; Fe (added as FeSO^); NaCl, 5% and the amino acids

(1 ml. of a 1% solution of each) glycine, tyrosine, tryptophan,

cysteine and methionine did not affect the rate of color loss in

the light or in the dark. The samples shaken in the light all scored

9 after 120 minutes while those shaken in the dark scored 9 or 10.

An earlier experiment with phosphate buffers at pH 7.20, 6.04 and

5.70 was repeated. New lots of 0.5 molar phosphate buffers were made

up to these pH values. The samples each contained O.3O micromoles of

heat denatured globin myohemichrome. The final volume in each case

was 5 ml.

The rate of color lose was the same for the samples in phosphate

buffers at different pH values and for the control sample in v/ater.

In 120 minutes in the light the color score was 8 while in the same

time samples shaken in the dark scored 9 .

It was also desirable to repeat some of the more critical expéri­

menta using fat derivatives and the purified myohemichrome. In each

case 0.30 micromoles of purified heat denatured globin myohemichrome

was used. In each case the total liquid volume was 5 ml. The samples were shaken vigorously with an illumination of 400 '.veston. 53

Table 6

The Effect of Fatty Materials on Discoloration of Denatured Globin I^ohemi.chrome in the Light and in the Dark

Color Score

Light Dark Treatment 60 min. 120 min. 60 min. 120 min.

Rancid fat (corn oil) 50 mg. 5 4 7 5

Oleic acid (recrystallized) 50 mg. 6 3 10 9

Methyl laurate 50 mg. 10 9 9 9

Methyl oleate 50 mg. 9 S 9 9

Lauric acid 50 mg. in 150 mg. Methyl laurate 7 5 9 8

Oleic acid 50 mg. in I50 mg. Methyl laurate 8 5 9 9

Pignent only (control) 10 9 10 10

Another experiment was carried out with the purified hemichrome and the results are given in Table 7.

In view of the fact that the amount of the nitric oxide deri­ vative in most of the samples was unknovm no conclusions can be drawn regarding the role of fat derivatives and reactions involving the loss of nitric oxide. It is however possible to draw scxne conclusions concerning the hemichrome. In light of later work, this was the major pigment in most of the samples. Free fatty acids in particular are capable of causing loss of color of the hanichrone. This may be due to splitting off of the heme group, possibly as free hematin.

Oxidized fat causes color loss. The loss of color in the presence of fatty acids is light induced and results in darkening. 54

Table 7

The Effect of Fatty Materials on Denatured Globin Myoheffilehrcsne Discoloration

Color Score after 60 minutes

Treatment Dark Light After addition of

Pigment only 9 8 9

0.1 ml. oleic acid 0.1 ml. methyl laurate 8 8

40 mg. lauric acid 0.1 ml. methyl laurate 8 4 9

0.1 ml. mi laurate 9 9 9

0.1 ml. ml oleate* 45 mg. lauric acid 9 8 9

50 mg. oxidized lard 8 4 9

53 mg. oxidized lard** 7 6 8

40 mg. lauric acid 5 3 7

*Peroxide value 95, smelled rancid **Some samples soon lost their color after the color improved on the addition of the Na2S20^.

The red color could be regenerated by Na2S20j^ in the few samples tried.

In several cases, however, a rapid loss of color followed an initial increase in redness. This light-induced darkening could be related to darkening of meat products in light.

Spectroscopy of the Pigment

It was desired to find out if information on the structure of the heated pigment could be obtained by spectroscopy. It was highly desir­ able to work with a solution and it was thought possible that gums might stabilize the heat denatured pigment to obtain a suitable dis­ persion. Nitric oxide myoglobin solutions were heated slowly with 55

water, gim arable 0.8%, gum damar 0.8% and gum ghatti 0.8%. With

water only, with gum damar and with gum arable a heavy red preeipi-

tate formed at 70°C. but only a small amount of precipitation took

place under the same conditions with gum ghatti. A solution of

nitric oxide myoglobin prepared by heat fractionation was ccmpared to

a sample in 2% gum ghatti which was heated to 80°C. Heating to 80°

would ordinarily precipitate the pigment in water.

Both of these solutions had nearly the same absorption spectra

with peaks at 546 and 578 millimicrons. These corresponded to peaks

for nitric oxide myoglobin. The results suggested that either the

gum prevented heat dénaturation or if the denatured pigment was

actually present it had absorption peaks similar to nitric oxide

myoglobin. Since the results were ambiguous, this approach was

abandoned.

The absorption spectrum of nitric oxide myoglobin has been re­

ported by Kiese and Kaeske (26). For this work, it was desirable to

determine the spectrum obtainable with the Beckman D.U. instrument.

Peaks were found at 545 and 577 millimicrons with minima at 505 and

564 millimicrons at pH 5.88 and this is essentially in agreement with

the report by Ginger and Schweigert (88).

Determination of the spectral reflectance of a layer of the heat

denatured pigment (prepared as described later for mancmetric experi­ ments) was unsatisfactory since the curves showed no distinct peaks and fading appeared to take place during preparation and observation of the samples. This method was also abandoned.

Another approach to the problem of obtaining the absorption 56

spectman of the denatured pigments was to suspend the heat denatured pigment in glycerol containing 0.2% Dreft. Dreft was used since such anionic detergents are known to solubilize and denature protein

(85). One suspension of the heat denatured pigment had a peak at

550 millimicrons with a gradual drop off of absorbance toward the red end of the spectrum. The curves obtained could not be interpreted at the time but they corresponded to what may have been mixtures of the hemichrome and the nitric oxide myohemochrome. This method was abandoned due to the turbidity encountered.

Finally to obtain spectral curves, a thin layer of the wet solid pigment was compressed for 30 minutes in a vise between glass plates made from standard microscope slides cut into 4 equal sized rectangles.

In each case, two of the glass rectangles were used for a blank. The two pieces of slide with the pigment between was in turn conprnssed between heavy glass blocks in order to prevent breakage of the thin glass during the pressing operation. Usually several attempts were necessary to obtain a sample without air holes. The pressed samples were usually nearly transparent.

A sample of lean tissue from purchased bacon showed no distinct peaks or troughs between 500 and 580 millimicrons. Absorbance dropped off at 580 millimicrons. Since no trough was observed at 500 milli­ microns the presence of metmyoglobin as an oxidation product was possible.

The heat denatured pigment, nitric oxide myohemochrome prepared as for the Warburg runs was compressed between glass plates. The curves obtained could be interpreted to be due to a mixture of 57

1.00

.9 0 -

.80-

.70-

U .60- m .50-

.30^

460 480 500 520 540 560 580 6 0 0 6 2 0 6 4 0 6 6 0 WAVELENGTH IN MILLIMICRONS

Figure 1

Absorbance Versus Wavelength for Denatured Globin Mj’ohemichrome and for Denatured Globin Nitric Oxide Myohemochrome After Irradiation

A. Denatured Globin Myohemichrome

B. Denatured Globin Nitric Oxide Myohaaochrome After Irradiation 505 Minutes in Warburg Flasks 58

.30"

5 .10-

jOO- 440 460 480 500 520 540 560 580 600 620 640 660 WAVELENGTH IN MILLIMICRONG

Figure 2

Absorbance Versus Wavelength for Pigment Samples Prepared as for the Manometrio Experiments 59

2.00-

1.80-

1.60-

1.40-

w 1.00-

.80-

6 0 -

«0 480 500 520 540 560 580 600 620 640 660 680 WAVELENGTH IN MILLIMICRONS

Figure 3

Absorbance Versus Wavelength for Pigment Samples Prepared as for the Manometrio Experiments (Curve 2) 60 denatured globin myohemichrome and the nitric oxide myohemochrome, assuming the nitric oxide myohemochrome has an absorption spectrum similar to nitric oxide myoglobin and the denatured globin myo­ hemichrome has peaks at 532 and 558 millimicrons as found for the heated metmyoglobin.

It was desired to obtain evidence that the heated pigment was the hemochrcme, but since it appeared that the heated nitric oxide derivative has an absorption spectrum similar to nitric oxide myoglobin, it was desirable to find out if the material left after loss of the nitric oxide was the myohemichrome.

The absorption spectrum of the myohemichrome prepared by heating purified metmyoglobin, was determined for a sample pressed between glass plates. This had a major peak at 532 millimicrons and a small peak at 558 millimicrons corresponding to those of the oxidized hemochrane studied by Drabkin and Anstin (91) and to the values of 530 and 558 millimicrons given by Lemberg and Legge (92) for denatured globin hemichrcme. A sample of nitric-oxide myohemochrcxne prepared as usual by addition of sodium nitrite and sodium hydrosulfite and irradiated in the Warburg apparatus for 505 minutes was centrifuged and washed three times with centrifugation. The pigment was compressed between glass plates and the absorption spectrum was determined. This had a spectrum nearly identical to that of the honichrcme except for possibly a very slightly higher absorption in region of 550 to 580 millimicrons. Although in this case only one curve was obtained, other curves in which the nitric oxide derivative was used showed the hemi­ chrcme peak at 532 millimicrons. 61

It is therefore likely that the nitric oxide derivative of the heated pigment is the hemochrome. In all cases where the spectrum of the solid pigment was observed, extinction values could not be obtained. It should be pointed out that this method leaves much to be desired but reliance can probably be placed on the positions of the absorption peaks.

Manometrio Experiments

For the most of the mancmetric experiments, a seven position

Warburg apparatus was used and was especially constructed for this work using a rectangular glass tank. The shaking rate was more rapid (180 to 200 strokes per minute with short strokes) than for an ordinary Warburg apparatus. This along with an added glass bead prevented the pigment frcsn settling.

The bottoms of conventional conical Waz'burg flasks were illumin­ ated by a system comprising a mirror set at a 45° angle to the hori­ zontal in the bath and a type AH-6 General Electric high pressure water jacketed mercury arc lamp. The light was restricted to the visible range with a Corning Glass filter #3389 of 3.0 mm. thickness, to cut off the ultra violet end of the spectrum at approximately 430 millimicrons, and an Ansco glass heat filter to remove the infra red.

The illumination was held constant by slightly shifting the lamp to sample distance to give the same reading (350) on a Weston photo­ graphic light meter held just above the bath over the sample positions.

The absolute light intensity was determined by the use of the Warburg,

Schocken (93) actinometer. Protoporphyrin, prepared fran blood hemo­ globin (94) was used as the sensitizer. For this determination each 62

wàrburg flask contained 10 mg. protoporphyrin, 200 mg. thiourea, and

5 ml. of pyridine. In this case the themo-baraneter contained 5 ml.

of pyridine.

The intensity used in most of the work including the experiments

involving nitrate and nitrite determination as well as seme of the

earlier runs (350 on a Weston meter) corresponded to 1.8 micrcmoles

of light quanta per minute per flask.

Preparation of Samples

Samples of denatured globin nitric oxide myohemochrome were pre­ pared by heating the metmyoglobin 15 minutes at 85°C. with a small quantity of sodium nitrite and sodium hydrosulfite. It was desired

to use a minimum quantity of the reagents in formation of the pigment in order to prevent the possibility of oxidation of the protein during preparation of the sample. In one experiment in which the quantity of sodium hydrosulfite were varied it was found that 1 cc. of

1% sodium hydrosulfite and 1.0 cc. of 0 .05^ sodium nitrite were sufficient to form maximum color on heating. The amount of nitrite added was doubled.

Approximately the same or slightly IcMver concentrations of sodium nitrite and sodium hydrosulfite relative to metmyoglobin were used in earlier Warburg experiments in which nitrite and nitrate were not deteimiined after irradiation of the sample.

In all of the experiments involving detemination of nitrite the samples were prepared by one of the two following methods both of which gave the same quantity of reagent per sample.

A quantity of metmyoglobin sufficient to give a final coneen- 63 tration of 0.4 micrcmoles per ml. was measured into a 100 ml. flask.

Sodium nitrite (10 ml. 1%) and sodium hydrosulfite (10 ml. Z%) was

added and the volume was made up with water to 100 ml. In sme

cases 1.0 ml. 0.05^ sodium nitrite and 1 ml. of 1^ sodium hydrosul­

fite were added to individual samples containing 2.00 micromoles of metmyoglobin before heating.

In order to study the oxygen uptake of the purified hanichrcme,

in a few cases the purified metmyoglobin was heated 15 minutes at

85° without added sodium nitrite or reducing agent. After heating for

15 minutes at 85°, the samples were held about 15 minutes at room

temperature. They were then centrifuged in a cold room and were washed with cold distilled water six times with centrifugation.

The precipitate in each case was quantitatively transferred to a

Warburg flask by mixing with a portion of 0.05 M pH 6.2 phosphate buffer sufficient to make 2 ml. of slurry. The slurry was removed to the Warburg flask with a 2 ml. pipette. A second approximately 2 ml. portion of buffer was added and this was removed with the same pipette. One ml. of 5*0^ NaCl was added to the centrifuge tube and the 2 ml. pipette was rinsed with this before the solution was trans­ ferred to the Warburg flasks. In this manner, it was possible to know the volume of material in the flask although the amount of phos­ phate buffer was not exactly known.

In some earlier work, before the elusive nature of the nitric oxide group was recognized, because of the subtle nature of the color change between that of the nitric oxide derivative and the myohani- chrcme when the pigments are in the pure form, the centrifugation and 64 washing was generally carried out at room temperature. This undoubt­ edly led to loss of a considerable amount of the nitric oxide.

Color scoring was carried out for part of the samples involving nitrate and nitrite determination, using the freshly prepared nitric oxide myohemochrome as a standard arbitrarily given a score of 10.

For the zero score, the myohemichrome was prepared by heating purified metmyoglobin. Color scores less than zero were used when the sample was visually judged to be below the hemichrome standard in redness. Both the zero score and 10 score standards were placed in

Warburg flasks for comparison with the irradiated samples. For this observation, the samples were quickly removed from the bath, ccmpared with the standards, and replaced. In early experiments, color was scored against a standard prepared in the same manner as the sample and the redness was judged using the standard as 10 and complete loss of redness as zero.

Color scoring was carried out with great difficulty since both pigments are red. The subtle difference between the two pigments could best be visually observed under tungsten lighting.

Some Warburg runs without benefit of nitrite and nitrate analysis were carried out with purified pigment under essentially the same conditions as used in the later e^qjeriments. In this case, oxygen uptake was observed in the light, in the dark, in air and in some cases in pure oxygen. In this case, the amount of myoglobin used for preparing samples, generally determined for each experiment as cyanometmyoglobin, was the same for each of the samples in a Warburg i"un but the amount varied between 1.8 and 3*13 micrcmoles for dif­ 65 ferent runs. In some experiments, oleic acid was added. It appears likely in the light of later experiments, that in most of these runs considerable loss of nitric oxide had occurred in seme of these sam­ ples before the start of the Warburg runs and in some cases it appears likely that less of the nitric oxide myohonochrome was in­ itially formed. Light intensity was generally between 200 and 400 as measured by the Weston meter. Except in the case where pH was the variable studied pH 6.0 and pH 6.2 buffers were used.

Warburg Experiments with Heat Fractionated Pigment

In the earliest Wferburg experiments, a five position Warburg apparatus was used, along with light supplied by an AH-6 General

Electric Mercury arc lamp and a type 3389 Corning glass filter.

Special flasks were constructed having a flat side. At first, shaking was carried out using iron armatures inbedded in glass which were shaken by magnets outside of the flasks. This systan of shaking proved unsatisfactory and the shaking was changed to shaking of the manometers. Since the light entered the side of the flasks and. during shaking the lighted area changed, metal shields were fitted to the flasks which had the same opening for each flask. Runs were carried out using pigment purified by heat fractionation of water extracts of beef. In this case, finely ground beef was mixed with 1 part water and was held overnight. The slurry was filtered and was heated to about 68°C. until some of the metmyoglobin began to pre­ cipitate as noted by the change in color of the precipitate. The material was quickly cooled and was filtered. The myoglobin in the supernatent liquid was determined as cyancmetmyoglobin (91). 66

Runs were carried out with this partially purified pigment to study the oxidation of the nitric oxide derivative and to try to obtain some idea whether or not a Van Slyke reaction was involved.

The nitric oxide derivative was prepared by heating the metmyoglobin solution with added sodium nitrite and sodium hydrosulfite and with subsequent washing of the pigment as described for the purified pigment.

In some of the early Marburg runs, results were very erratic. It was thought that this could possibly be due to bacteria. Necxnycin sulfate was added to prevent possible growth of bacteria during the runs. The oxygen uptake in the presence of 0.?0 mg. neomycin sulphate per sample was very high and amounted to $.0 and 5«5 micro­ moles in 433 minutes with KOH in the wells using pure oxygen. In

540 minutes, a sample with neomycin sulfate took up 69.0 microliters while one without necatiycin sulfite took up 47.0 microliters. Neomycin sulfate itself was possibly undergoing autoxidation. The use of this material was therefore abandoned. 67

Table 8

The Effect of Oxygen Pressure on Rate of Oxygen Uptake

pOp Microliters Oxygen Uptake in 310 Run m.m. Hg. Light Minutes

A 32 m.m. light 14

A 32 dark 3

A 148 light 23

A 148 dark 9

B 148 light 26

B 148 dark 3

B 742 light 58

B 742 dark 32

In place of neomycin sulfate samples were made 0.001 M. in sodium fluoride. This did not appear to effect the oxygen uptake and was used in a few Warburg runs, but it was later found that bacteria were probably not causing difficulty and the use of a bacteriastatic agent was abandoned entirely.

Oxygen uptake was observed at 3 pressures and for samples in the light, in the dark, and ior samples with and without KOH. It is not known whether the different oxygen uptake rates observed at the different oxygen pressures were due to oxidation of the globin or whether the effect was associated with other extraneous protein.

Although this method, using partially purified heat fractionated pig­ ment, saved some time in working out the many difficulties encountered with the Warburg equipment, and yielded information suggesting the possibility of a Van Slyke reaction, it was finally abandoned in favor 68

w 804

5 504 40 J

-I 30 4 204 10 H

0 100 200 300 400 600 600 TIME IN MINUTES

Figure 4

Rate of Net Gas Uptake for Partially Purified Denatured Globin Nitric Oxide Myohemochrome

A. Light, Air, KOH (Wells)

B. Light, Air, KOH (Wells) 69

70-

60 “

50-

30-

= 10-

50 100 ISO 200 250 300 350 400 450 500 550 600 TIME IN MINUTES

Figure 5

Rate of Oxygen Uptake for Partially Purified Denatured Globin Nitric Oxide tÿ-ohemochrome In Pure Oxygen and In Air

A. Light, Pure O2 , KOH (Wells)

B. Dark, Pure O2 , KOH (Wells)

G. Light, Air, KOH (Wells)

D. Dark, Air, KOH (Wells) 70 of the use of samples prepared from purified myoglobin.

Figures 4 and 5 shows the results of two of these runs and in­ dicates the possibility of a Van Slyke reaction due to the initial shape of the rate curve.

In separate experiments using a conventional Warburg bath with

100 watt lamps for the irradiation, the irradiation of heat dena­ tured samples as well as some samples of non-heat denatured nitric oxide myoglobin was carried out. The heat denatured nitric oxide myohonochrome in each flask in this case was prepared from 4*3 micro­ moles of purified metmyoglobin. Samples were irradiated 225 minutes in the light and in the dark with nitrogen freed of traces of oxygen by passing it through alkaline pyrogallol. In all cases KOH was used in the flask wells. Control samples in air were irradiated in the light and in the dark.

Non-denatured nitric oxide myoglobin prepared by heat frac­ tionation gave off a gas not taken up by KOH. In one experiment in the light 14 microliters of a gas was given off in N2 compared to

13 microliters of oxygen taken up in the air in 53 minutes. In another experiment in the light, I6 microliters of a gas was given off in nitrogen compared to 14 microliters oxygen uptake in air in 8? minutes. In the latter experiment 5 microliters of gas was given off in N2 in the dark.

Gas Uptake in the Presence of Sodium Pyruvate

Since a peroxide mechanism has been proposed for the oxidation of myoglobin to metmyoglobin it was desired to find out if hydrogen peroxide is formed in the oxidation. 71

An attempt was made to demonstrate the presence or absence of peroxides by the addition of sodium pyruvate, which reacts with hydro­ gen peroxide to produce CO2 . Sodium pyruvate was added in two of the

Warburg runs in which nitrate and nitrite were determined. The results are given in Table 9. The failure to find nitrite in three cases and the lower than normal nitrite value in the fourth case as well as the low nitrate values suggest that the pyruvate may be react­ ing with nitric oxide in these experiments. However, due to the large excess of pyruvate this should not effect any reaction of pyruvate with peroxides. The pH was at 6.2, a value close to the pK of the bicarbonate ion. Thus if CO2 was produced sane but not all CO2 would probably be liberated as a gas. Results relative to CO2 production can therefore be considered to be qualitative only.

Table 9

Oxidation of Denatured Globin Nitric Oxide Myohemochrome in the Presence of Sodium Pyruvate*

Micromoles Micromoles nitrite O2 Uptake (No KOH) in the supernatent Treatment Pyruvate No Pyruvate Pyruvate No Pyruvate

A. light** .22 .36 .33 .28

A. dark .22 .18 .006 —

B. light .94 .76 .006 .30

B. dark .0 .25 .006 .13

*For each 5.0 ml. sample 1.0 ml. 3*33^ sodium pyruvate was added **A irradiated 364 minutes, B irradiated 508 minutes

In these and earlier runs on the average there appeared to be no increase in CO2 liberated in the presence of sodium pyruvate. This indicates that hydrogen peroxide is probably not produced. 72

Since it appeared possible from the above results that sodium pyruvate may act to trap nitric oxide lost from the pigment, it was thought possible that pyruvate might effect the rate of color loss.

There was also a possibility that if H2O2 is produced during the oxidation that pyruvate might protect color.

Duplicate samples of bologna were soaked in water and in 1^ sodium pyruvate solution. Pairs in duplicate were packaged in cellophane and also in polyethylene vacuum packages. The color was compared with the controls treated with water. These were held under tungsten lighting at about 68°C. No difference in the rate of color loss between the sodium pyruvate treated and control samples was noted after 1, 2, 10 and 24 hours.

Results of Warburg Experiments with Purified Pigment in IVhich Nitrate and Nitrite Were Not Determined

A break in the oxygen uptake curves was noted in pure oxygen at about 200 minutes. This behavior was characterized by a period of slightly accelerating rate up to about 200 minutes, a drop in rate and finally return to a normal rate curve. This behavior is seen in

Figure 6. In pure oxygen in one experiment in 512 minutes 62 micro­ moles of O2 was taken up in the dark without KOH and 1.38 micromoles was taken up with KOH present. In the light 2.06 micromoles was taken up with KOH and 1.43 without KOH. This indicated a gas which can be taken up by KOH is produced, both in the light and in the dark.

For exploratory purposes the effect of pH on oxygen uptake in air, the light, with and without KOH was observed. Phosphate buffers

(0.04 M.) of pH values 7.10, 6.02, and 5.32 were used. With KOH in the wells using 2.00 micromoles of the nitric oxide myohemochrome made 73

UJ Z .6 -

.2 -

100 200 3 0 0 4 0 0 50 0 TIME INMINUT MINUTESES

Figure 6

Rate of Net Gas Uptake for Denatured Globin Nitric Oxide Myohemochrome

A. Light, Air, KOH (Wells)

B. Dark, Air, KOH (Wells)

C. Light, Air, No KOH (Wells)

D. Dark, Air, No KOH (Wells) 74 frcm purified metmyoglobin (sane hemichrome present), the oxygen uptake values in 335 minutes were 1.03, .89, and .94 micronoles respectively.

With no KOH the respective values were .90, .94, and .94 micromoles.

This suggests that pH has little effect on the rate of oxygen uptake or rate of CO2 production.

There was a rather wide difference in observed rate of fading under the same conditions. Part of this difference was probably due to differences in the relative amounts of the nitric oxide myohanochrone and the myohemichrone present at the start of the experiments and part of the difference could be due to differences in judgement of color since the difference in color between the nitric oxide derivative and the hemichrome is a very subtle one.

The net gas uptake value for the myohemichrome prepared by heating purified metmyoglobin with KOH in the wells in 296 minutes, was O.4O,

0 .62, 0 .52, 0.0 and 0.13 microliters respectively (ave.= 0.33 micro­ liters) with no KOH in the wells the average net gas uptake was O.O6 microliters in 296 minutes. This compares with an average net gas up­ take of 0.88 microliters for the nitric oxide derivative irradiated in air in the light in the same time.

The Effect of Oleic Acid on Oxygen Uptake

In seme of the early experiments, the pigment appeared to be supris- ingly stable ccmpared to what would be expected with meat. Since oleic acid appeared to be the only material that would cause color loss it was desirable to study the oxygen uptake of the pigment in the presence of oleic acid. It was thought possible that loss of color might be due to peroxides formed in the oxidation of the oleic acid. Haurowitz had 75

shown that linoleic acid is oxidized in the presence of heanin. It was

also considered possible that the fatty acids might interact with the

protein in close enough association that light energy might be trans­

ferred to the double bond in the oleic acid and make it possible for

formation of peroxides to readily occur. It was therefore desirable

to find out if oxygen uptake curves were modified by the presence of

oleic acid and whether oxygen uptake curves typical of fat autoxidation

occurred.

With the Warburg samples the shaking was considerably less vig­

orous than with the samples in the qualitative runs using test tubes.

A good dispersion of the fat was never obtained. Samples were shaken

in the light, in the dark and with and without KOH in the wells. Oleic

acid generally caused increased loss of red color with darkening but

the rate was never nearly as rapid as for the samples shaken in test

tubes. Since the belief in the role of fatty acids as the major

factor persisted with the author, attempts were made to obtain satis­

factory dispersions by adding surface active agents such as Tween-80,

but it was still not possible to get a satisfactory dispersion. Part

of the qualitative experiments described elsewhere were attempts to

obtain a suitable systan of pigment, fatty acid and surface active

agent for this purpose. In scane cases, it was desired to observe the

effect of extended oxidation. In this case, it was necessary to fill

the manometers with nitrogen at the end of a day’s run and hold the

samples overnight in the dark at 4°C. The flasks were filled with

oxygen or air the next day and the run was continued. Usually no unusual break in the rate curves were noted. In pure oxygen, oxygen 76 uptake values obtained in one experiment are given in Table 10. The slightly higher oxygen uptake noted here suggests that the possibility that the oleic acid might have taken up some oxygen. In another experiment, oxidation in the presence of methyl oleate was observed., All samples contained methyl oleate. The curves are shown in Figure ?. Where results are available for a direct ccgnparison during the same run for samples with and without oleic acid, they are given in Table 11.

Table 10

The Effect of Oleic Acid on the Oxygen Uptake of the Heat Denatui-ed. Pigment

Micrcxnoles Oxygen Uptake Treatment 485 minutes

Dark 0.58

Light 1.40

Light KOH 3.00

Light Oleic Acid 10 2.20

Light KOH Oleic Acid 10 3.60

Dark KOH 2.30 77

ui

< 80-

60-

100 200 300 400 TIME IN MINUTES

Figure 7

Rate of Oxygen Uptake of Denatured Globin Nitric Oxide Myohanochrome In the Presence of Methyl Oleate In Pure Oxygen

A. Light, KOH (Wells)

B. Light, No KOH

G. Dark, KOH

D. Light, No KOH

3. Light, KOH

F. Dark, No KOH 78

Table 11

Effect of Oleic Acid on O2 Uptake

Code Oleic Acid No Oleic Acid Special Treat- No KOH KOH No KOH KOH ment

7-2A 1.20 1.20 O2 dark 1.96 2.28 light

11-2 1.95 1.70 Air light 1.08 1.07 light

2A-2 3.60 3.00 02 light 2.20 1.40 light 1.58 2.8 1.46 2.35 light

In one run, the oxygen uptake was very high. In 334 minutes with

KOH and no oleic acid the uptake was 6.75 micromoles while with oleic acid it was 9.00 micromoles. In such a case, it was likely that something was wrong with the thermobar ometer.

In seme cases, the oxygen uptake was slightly lower in the presence of fatty acid than in their absence. This could be due to mechanical protection of some of the pigment against oxidation, by the fatty acid.

After recognition of the fact that the pigment in the early runs was largely the myohemichrone, an experiment was carried out with oleic acid in all flasks using two samples of the nitric oxide myo- hemochrcme and 4 samples of the myohemichrome prepared and handled in the same manner as for the runs in which nitrate and nitrite were determined. The results are given in Figure 8 and indicate that in the presence of oleic acid with the hemichrcsne a gas is given off.

It appears possible that CO2 was liberated due to the presence of the acid group of the fatty acid. In several other cases libera­ tion of a small amount of CO2 was noted in the presence of oleic acid. 79

404

304 A 204 B 104 c p £

26 0 300 TIME IN MINUTES

Figure 8

Rate of Oxygen Uptake of Denatured Globin Nitric Oxide Myohsnoohrome In the Light and In the Dark

A. Light, NO Mb (Denatured) KOH

B. Light, NO Mb (Denatured)

C. Light, Met Mb (Denatured) KOH

D. Light, Met Mb (Denatured)

E. Dark, Met Mb (Denatured) KOH

F. Dark, Met Mb (Denatured) 80

In all, 44 Warburg samples contained oleic acid. In one experi­ ment two samples out of 6 containing oleic acid showed oxygen uptake curves of the type that might be expected in fat autoxidation.

It appears likely that the pigment (largely the myohemichrome) did not cause fat autoxidation. Further work with larger quantities of fatty acid or methyl oleate would however be desirable.

Nitrate and Nitrite Production

Early in the work it was thought that available methods for determining the products of the reaction involving color loss would not be sensitive enough to be useful. It was later found that nitrite and nitrate could be estimated even though the amounts in the sample fell in the low range of the analytical methods available.

Since color stability may possibly be related to reformation of the pigment, it was desired to find out what happened to the nitric oxide lost from the pigment. Since the pigment was washed free of soluble material at the start, if nitrite or nitrate is formed then they could be determined in the solution after irradiation.

When nitrate and nitrite were to be determined, the suspensions of pigpent from the flasks were removed to 15 ml. centrifuge tubes with a pipette. The flasks were rinsed with small portions of water using the same pipette. The washings were ccmbined with the suspen­ sion. The suspension was quickly centrifuged and was washed twice with approximately 10 ml. aliquots of cold water.

This supernatant liquid was made up to a volume of 50 ml. with water. Aliquots of 15 ml. each were taken for analysis for nitrite and 30 ml. aliquots were taken for the nitrate analysis. 81

Nitrite expressed as NO2 in all cases was determined by the method of Shinn (97).

One ml. of $0% HCl was added to the 15 ml. sample in the 50 ml. flask. After three minutes 5 ml. of a 0,2% sulfanilamide solution was added. After 2 minutes 1 ml. of 0.1% (l-naphthyl) ethylene dia­ mine dihydrochloride was added. The solution was made up to 50 ml. with water. The absorption spectrum using a standard containing

0.025 mg. of NO2 was determined and had a peak at 542 millimicrons.

This wave length was used in all determinations using the Beckmann model D.U. spectrophotometer. Standards were prepared from reagent grade sodium nitrite.

The brucine sulfuric acid method of Mettette, Brodsky, and

Palmer (98), was used for determination of nitrate.

Either nitrate or nitrite was determined in the KOH but not both, due to the low concentration present.

A small amount (O.l ml. of 1%) sodium hydroxide solution was added to each sample for nitrate determination. The samples were then evaporated just to dryness on a steam bath. Several ml. of water were added to wash down the sides of the tubes. The solution was then evaporated just to dryness. Then 1 ml. 0.5% ammonium sulfamate solution was added to prevent interference by nitrite. One ml. of water was added to give the 2 ml. of sample required in the nitrate determination. For development of color 5 ml. of a solution of a brucine sulfuric acid reagent containing 200 mg. of brucine per

200 ml. of concentrated reagent grade sulfuric acid was added. The tubes were allowed to stand exactly five minutes. Then 5 ml. of 82

water was added and the solution was thoroughly mixed. The material

was cooled in a water bath at room temperature for ten minutes.

Absorbance was read at 440 i^ttusing the Beckmann model D. U. spectro­

photometer and a water blank. The amount of nitrate as NO3 was

obtained from a standard curve prepared for each lot of brucine sul­

furic acid reagent. Reagent grade potassium nitrate was used for

preparation of the standards. In five of the Warburg runs

irradiation was carried out for approximately 350 minutes and nitrate

and nitrite were determined. All of the Warburg experiments involving

nitrite and nitrate determinations and most of the earlier experiments

were carried out at 0.3°G.

It was desirable to follow the course of nitrate and nitrite

production, loss of color, and oxygen uptake. For this purpose sam­

ples were irradiated frcm 31 minutes to 434 minutes each, with ob­

servation of oxygen uptake. At the end of the irradiation period,

the slurry was removed and nitrate and nitrite were determined.

Nitrite only was determined in the KOH.

Two runs were carried out in which nitrate and nitrite were

determined to test the replication of results. In one run, three

samples were observed without KOH in the well. In 350 minutes, the

oxygen uptake values were .67, .58, and .63 micromoles respectively.

The nitrite recovered was .32, .28, and .22 micronoles respectively while the respective nitrate values were .49, .53, and .65 micromoles.

In a similar experiment with KOH in the wells, after irradiation for 332 minutes, the respective values for oxygen uptake were 1.11,

1.11, 1.25 micromoles, while values for nitrite were .37, .35, and 83

.35 micromoles, and nitrate values were .56, .68, and .76 micrcmoles respectively.

The reaction between H2O2 and nitrous acid has been studied in a recent paper (98). The possibility that an excess of nitrate over nitrite could be due to peroxides by the reaction NO2 + H2O2 = NO^ +■

exists but in this case continued destruction of nitrite and production of nitrate would be expected rather than a break in the using up of nitrite.

Results of Warburg Experiments in VJhich Nitrate and Nitrite Were Determined

As Table 12 shows, two major products of the oxidation of de­ natured globin nitric oxide hemochrcsae were the nitrite and nitrate ions. Since the starting amount of pigment was 2.00 micromoles and since the results represented 5 different runs at different times, it appeared possible that nitrite was also lost as seme other product. 84

Table 12

Nitrate and Nitrite Produced and Oxygen Taken Up in the Presence of Light with KOH in the Wells

Micro­ Micro­ moles Micro­ moles Nitrite moles Micro­ Micro­ Oxygen Minutes in Nitrate moles moles Uptake Total Superna­ Superna­ Nitrite Nitrate at Time Irradiation tant tant Wells Wells T Time T

0.10 0.59 0.20 1.00 362

0.19 0.70 0.21 1.00 362

0.19 0.45 0.16 1.07 362

0.37 0.56 0.15 1.11 332

0.35 0.68 0.16 1.11 332

0.33 0.76 0.10 1.25 332

0.24 0.;# 0.02 0.76 350

0.22 0.72 0.16 0.87 350

0.16 0.77 0.14 1.00 434

0.13 0.60 0.11 0.63 183

0.12 & J 8 0.07 0.36 110

0.07 0.20 0.05 0.26 45

Average

0.21 0.58 0.11 0.19 0.88 296

The sample was prepared from 2.00 micromoles of metmyoglobin in each case

It is evident from these results that nitrate or nitrite found in the KOH represents loss of NO as a gas, either in the form of NO or the f o m of NO2 . 85

Table 13

Nitrate and Nitrite Produced and Oxygen Taken Up in the Dark with KOH in the Wells^

Micro­ Micro­ Micro­ moles moles moles Micro­ Oxygen Minutes Nitrite Nitrate moles Uptake Total Superna­ Superna­ Nitrite at Time Irradiation tant tant Wells T Time T

0.080 0.44 0.03 0.44 35 0

0.190 0.40 0.09 0.6? 340

0.074 0.40 0.11 0.54 508

Average

0.11 0.41 0.08 0.55 400

*The sample was prepared frcm 2.00 micromoles of metmyoglobin in each case

Under the specific conditions of these experiments, based on the starting amount of metmyoglobin, the average quantum yield for the reaction was estimated to be 0.0016, or based on the amount of nitrate and nitrite recovered the quantum yield was estimated to be

0.0027.

As Table 13 shows, loss of nitric oxide with the formation of nitrite and nitrate occurred in the dark as well as in the light.

Figure 9 shows that fading occurred in the dark as well as in the light. 86

9-1

8 -

7 -

5 - 4-

2 -

TIME IN MINUTES

Figure 9

Visually Observed Rate of Color Loss In the Light (A) and In the Dark (B) 87

-10

cc O

.6 - ac

O .4- O O O .2 - - 2

50 100 150 200 250 300 400 TIME IN MINUTES

Figure 10

Rate of Color Loss and Rate of Oxygen Uptake of Denatured Globin Nitric Oxide i^yohemochrome In the Light, In Air

A. Visual Color Score

B. KOH in Wells

C. No KOH in Wells 88

.80—

.60-

.00 100 2 00 0 50030 TIME IN M I N U

Figure 11

Rate of Nitrate and Nitrite Production from Denatured Globin Nitric Oxide Myohemochrome

A. Nitrate, KOH (in Wells)

B. Nitrate, No KOH (in Wells)

C. Nitrite, No KOH (in Wells)

D. Nitrite, KOH (in Wells)

E. Nitrite in the KOH 89

Table 14

Nitrate and Nitrite Produced and Oxygen Taken Up in the Presence of Light vdthout KOH in the Wells Minutes Micronoles Micronoles Ratio of Total Nitrite in Nitrate in Nitrite O2 Uptake Irradiation Supernatant Supernatant Nitrate Time T Time

0.34 0.56 0.61 0.80 362

0.21 0.56 0.37 0.76 362

0.20 0.42 0.48 0.72 362

0.32 0.49 0.65 0.67 350

0.28 0 . 5 3 0.53 0.58 350

0.46 0.60 0.77 0 .94 332

0.22 0.65 0.34 0.63 350

0.39 0.60 0.64 0.58 350

0.25 0.74 0.34 0.63 434

0.21 0.68 0.31 0.36 183

0.17 0.44 0.39 0.16 110

0.13 0.26 0.50 0.10 45

Average

0.27 0 . 5 4 0.50 0.50 298

The sample was prepared from 2.00 micronoles of metmyoglobin in each case

In Figure 10 the color score and the oxygen uptake curves are given for the same Warburg run as that of Figure 11 showing the rate of nitrite and nitrate production.

Together these two curves showed that at about 200 minutes under the conditions used, a change occurred in the character of the oxi- 90 dation in which nitrate production decreased, rate of color loss in­ creased slightly, gas uptake rate decreased, and rate of production of CO2 increased somewhat.

For samples irradiated 25O minutes or longer, the average amount of nitrite and nitrate recovered, was estimated to be 60% based on the metmyoglobin used in preparing the sample with a maximum value of

65^.

The ratio of nitrite to nitrate for 13 samples without KOH in the wells was O.5O, Std. Dev. t 0.15 with a maximum value of 0.77* DISCUSSION

There are several possible mechanisms that can be proposed to account for the results found in these experiments, any one of which could be related to practical questions on meat discoloration.

Color stability may be related to reformation of the nitric oxide derivative by reducing materials. The loss of nitrite could beccane a limiting factor in color protection. Thus the question, what happens to the nitric oxide, becomes of practical importance.

Possible explanations include, first, possible participation of nitrite in the oxidation of the iron with formation of NO. In the presence of oxygen and water NO2 and subsequently nitrite and nitrate would be formed. The second possibility is that a cyclic reformation of the nitric oxide myohemochrome takes place in the presence of reducing activity associated with protein. Third]y, a Van Slyke reaction between protein free amino groups and nitric oxide may be involved.

The total recovered nitrite and nitrate was low, never over 65^ of the metmyoglobin used, thus some loss of nitric oxide may have occurred during the preparation of the sample. Part of the apparent nitric oxide loss may not have been due to loss of nitric oxide during preparation, but may be due to a lower affinity of the denatured globin myohemochrome for nitric oxide than is the case for myoglobin.

Lonberg and Legge (99) mention that the affinity of denatured globin honochrome for carbon monoxide is about one-half that of hemoglobin for capbon monoxide.

The sharp break in the curve of nitrate production, the decline

91 92

in color score, along with the change in the slope of the O2 uptake

curve, and increased KOH soluble gas production may be associated

with changes in oxidative processes from those predcminantly asso­

ciated with the nitric oxide derivative to those associated with

the hemichrome.

One possibility is that even with thoroughly washed solid pig­

ment seme reducing activity remains associated with the protein.

This could result in the regeneration of the nitric oxide derivative.

In this case oxygen uptake would be expected to continue at a high

rate until all of the reducing activity is used up, then the oxygen

uptake rate should drop off rather sharply. This scheme could well

account for the fact that the ratio of nitrite to nitrate is less

than one. A second possibility is that nitrite itself may be

directly concerned in oxidation of the heme iron. In this case the

resulting nitric oxide reacting with oxygen and water would be ex­

pected to give equimolar quantities of nitrite and nitrate. Nitrite

acts as a fairly strong oxidizing agent by the following half

reaction; e -j- 4- HNO2 = NO + HgO o. r . potential = -+ 0.95. The

oxidation reduction potential for the denatured globin hem.ochrome

hemichr one system is low (-0.098 volts at pH 7.06) (lOO).

Many papers have been devoted to the problem of methemoglobin

formation by oxidation of honoglobin by nitrite. The oxidizing

effect of nitrite is seen daily in meat packing operations as the

graying that takes place when meat is initially treated with curing materials containing nitrite.

Oxidation by nitrite would be in line with the rather low values 93

of Oxygen uptake observed. In this case only 3/4 moles of oxygen per

mole of nitric oxide lost would be required to account for the

products formed. If nitrite is oxidized to nitrate through another

pathway, a small amount of additional oxygen would be required.

In the light, the average oxygen uptake for 12 samples was 0.65

micromoles. The average of the recovered nitrate and nitrite was

0.81 micronoles. This is slightly less than the calculated value

of .93 micromoles of O2 required for production of products assuming

a starting amount of the nitric oxide myohemochrome equal to the sum

of the nitrate and nitrite recovered. The value is reasonable con­

sidering the experimental error. A mechanism based on cyclic

regeneration by reducing agents would have a theortetical oxygen

requirement somewhat higher unless a reaction between nitric oxide

and free amino groups was giving low obsei'ved oxygen uptake values.

The break in the curve of nitrate production before all of the

color is lost, however, is a rather strong argument against the

above possibility since conversion of nitrite to nitrate would be

expected to persist until all of the pigment is converted to the hemi­

chrome. This behavior is more in line with the possibility that a

cyclic regeneration of the nitric oxide derivation takes place in the

presence of reducing activity associated with the protein. In this

case, it would be e^cpected that excess nitrite production would

occur until all of the reducing groups are used, a sanewhat higher O2

uptake would be expected and the cured color of the materials would be expected to persist.

The observations are in line with the possibility that preser- 94 vation of color depends on regeneration of color. In this case, the cured color would be expected to persist until one or more of the following conditions occurs;

1. All of the reducing activity is used up.

2. All of the nitrite is gone.

3. The hemichrome loses its ability to reform the cured meat

pigment.

A possibility of a reaction between nitric oxide and free amino groups cannot be ignored since this could account for many of the observations. At a pH value of 6.2 and ivith a nitrite concentration of the order of 4 parts per million a Van Slyke reaction would probably not occur to a measurable extent. If such a reaction occurs, it must be the result of free nitric oxide produced directly from the nitric oxide myohemochrome. Such a reaction is suggested by obser­ vation that a gas was given off when samples were shaken in a nitrogen atmosphere. It has been shov/n that the rate of this reaction depends on the concentration of the free acid (101). The possibility also exists that the observed break in the curve for nitrate production could be due to oxidation of sulfhydryl groups by nitrite. If this is the case, this could be of practical significance both to the question of curing and of color preservation. If such a reaction occurs in curing, it means that that part of the nitrite used in curing could be destroyed by oxidation of sulfnydryl groups. SUMMARY AND CONCLUSIONS

Sûme indirect evidence has been presented indicating that the heated pigment is denatured globin nitric oxide myohemochrome.

A quantitative method for the study of the chemistry of the fading reaction has been presented. This method includes the use of washed solid heat denatured pigment in suspension and the determina­ tion of nitrite and nitrate appearing in solution after experimental treatment. Heretofore, the only tools available were visual color scoring and reflectance spectrophotometry neither of which were capable of yielding quantitative information about the reaction.

The mancmetric technique alone was found to be inadequate for the quantitative study of the fading reaction since oxidative pro­ cess associated both with reactions involving nitric oxide loss and oxidation of the protein occurred simultaneously. In all mancmetric experiments, some myohanichrome was probably present at the start of experiments.

In the oxidation of denatured globin nitric oxide myohemochrcme in the presence of light and air or in air alone part of the nitric oxide is further oxidized to give the nitrite and nitrate ions.

A gas which is absorbable by concentrated potassium hydroxide solutions and which is probably CO2 is produced in the oxidation of the pigment in light and air.

Oxygen absorption as well as gas production takes place both in the light and in the dark, but both take place at a lower rate in the dark.

Oxygen uptake values observed were much lower than those observed

95 96 by George and Stratmann for the oxidation of oxymyoglobin to met- myoglobin. Consequently the same oxidizable groups therefore may not

be involved.

Some evidence has been presented that hydrogen peroxide forma­

tion is not involved in reactions associated with the fading of the purified pigment.

Denatured globin myoheraichrome shaken in the presence of free

fatty acids undergoes a darkening not observed in the light.

Several possible pathways have been suggested for the fate of nitric oxide lost from denatured globin nitric oxide myohemochrome which could possibly be of practical significance to the problem of cured meat discoloration. BIBLIOGRAPHY

(1) Gamgee, A. Textbook of Physiology, E. A. Schafer, Ed. VI, 1 ed. pp. 185-260, V. J. Pentland Pub. (1898).

(2 ) Haldane, J. The Red Color of Salted Meat. Journal of Hygiene 1: 115-121 (1901).

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I, Hcnvard Ned Draudt, was born in Franklin County, Ohio,

January U, 1921.

liy undergraduate training was received at The Ohio State

University, from which I received the degree Bachelor of Science

in Food Technology in 1949. In 1950 I received the degree Master

of Science in Agricultural Biochemistry. In 1950 I entered the

field of industrial research with Kingan Inc., of Indianapolis,

Indiana. In 1952 I returned to The Ohio State University to work

toward the Ph.D. Degree in the field of Agricultural Biochemistry.

In 1955 I returned to industrial research work at the Kingan, Inc.,

Indianapolis laboratory.

105