BIOCHEMICAL CHARACTERISTICS OF CUCURBITACEAE SALT-SOLUBLE PROTEINS.
Item Type text; Dissertation-Reproduction (electronic)
Authors AL-KANHAL, MOHAMAD AHMAD.
Publisher The University of Arizona.
Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Download date 04/10/2021 12:40:04
Link to Item http://hdl.handle.net/10150/186471 INFORMATION TO USERS
This reproduction was made from a copy of a document sent to us for microfilming. While the most advanced technology has been used to photograph and reproduce this document, the quality of the reproduction is heavily dependent upon the quality of the material submitted.
The following explanation of techniques is provided to help clarify markings or notations which may appear on this reproduction.
I. The sign or "target" for pages apparently lacking from the document photographed is "Missing Page(s)". If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting through an image and duplicating adjacent pages to assure complete continuity.
2. When an image on the film is obliterated with a round black mark, it is an indication of either blurred copy because of movement during exposure, duplicate copy, or copyrighted materials that should not have been filmed. For blurred pages, a good image of the page can be found in the adjacent frame. If copyrighted materials were deleted, a target note will appear listing the pages in the adjacen t frame.
3. When a map, drawing or chart, etc., is part of the material being photographed, a definite method of "sectioning" the material has been followed. It is customary to begin filming at the upper left hand corner of a large sheet and to continue from left to right in equal sections with small overlaps. If necessary, sectioning is continued again-beginning below the first row and continuing on until complete.
4. For illustrations that cannot be satisfactorily reproduced by xerographic means, photographic prints can be purchased at additional cost and inserted into your xerographic copy. These prints are available upon request from the Dissertations Customer Services Department.
5. Some pages in any document may havc indistinct print. In all cases the best available copy has been filmed.
Uni~ Microfilms International 300 N. Zeeb Road Ann Arbor, MI48106
8319723
AI·Kanhal, Mohamad Ahmad
BIOCHEMICAL CHARACTERISTICS OF CUCURBITACEAE SALT·SOLUBLE PROTEINS
The University of Arizona PH.D. 1983
University Microfilms International 300 N. Zeeb Road. Ann Arbor, MI48106
PLEASE NOTE:
In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark_,/_.
1. Glossy photographs or pages _ V"/'
2. Colored illustrations, paper or print ~/
3. Photographs with dark background __
4. Illustrations are poor copy __
5. Pages with black marks, not original copy __
6. Print shows through as there is text on both sides of page __
7. Indistinct, broken or small print on several pages 1//
8. Print exceeds margin requirements __
9. Tightly bound copy with print lost in spine __
10. Computer printout pages with indistinct print __
11. Page(s) lacking when material received, and not available from school or author.
12. Page(s) seem to be missing in numbering only as text follows.
13. Two pages numbered . Text follows.
14. Curling and wrinkled pages __ 15. Other______
University Microfilms International
BIOCHEMICAL CHARACTERISTICS OF
CUCURBITACEAE SALT-SOLUBLE
STORAGE PROTEINS
by
Mohamad Ahmad Al-Kanhal
A Dissertation Submitted to the Faculty of the
COMMITTEE ON AGRICULTURAL BIOCHEMISTRY AND NUTRITION (GRADUATE)
In Partial Fulfillment of The Requirements for the Degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
The University of Arizona
198 3 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE
As members of the Final Examination Committee, we certify that we have read the dissertation prepared by Mohamad Ahmad Al ~Kanhal entitled Biochemical characteristics of Cucurbitaceae salt..-.soluble
storage proteins
and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy
Date ~ < /1,/qf'3 Date 0 7 Da"!!7J {'1 / J ?
Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College.
I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfillment of requirements for an advanced detree·at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.
Brief quotations from this dissertation are allowable without special permission, provided that accurate ack knowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.
SIGNED: DEDICATION
TO NORAH
iii ACKNOWLEDGMENTS
I would like to express my gratitude to all the people who have helped me during phases of this study. In particular, I want to thank professor J. W. Berry, my major professor, for his helpful suggestions and patience throughout this study. Thanks are also due to professors
W. F. McCaughey, B. L. Reid, R. L. Price and J. W. Stull for serving on my doctoral committee.
I am very grateful to Dr. R. E. Allen, for the use of equipment in his laboratory. Also, thanks are due to Mr. A. Watkins, for his assistance during the two dimensional analysis.
Special thanks are due to all of the wonderful people in our laboratory who made my task very enjoyable.
My deep gratitude is expressed to all of my family members in Saudi Arabia, for their emotional support and constant encouragement.
My special thanks go to my wife Monirah and to my lovely daughter, Norah, who introduced happiness to my life.
iv TABLE OF CONTENTS
Page
LIST OF ILLUSTRATIONS viii · '/
LIST OF TABLES '/ . ix
ABSTRACT .. I • xi
INTRODUCTION 1
LITERATURE REVIEW 3 • I
Seed Proteins 3 • 1 Seed Oil 12 • 1 Starch 13 Other Products 14
MATERIALS AND METHODS 16
Seeds Source 1 16 Proximate Analysis 16 Moisture . . 1 16 Lipid Extraction 17 Crude Protein 18 Acid Detergent Fiber 20 Ash .... 1 ••• 21 Isolation of Albumin and Globulins 22 Defatting the Samples 22 Albumin and Globulins Separation I 23 Lowry Method 1 • • • • 24 Standard Curv, . . . . 24 Albumin Sampl, Analysis 26 Salt Extract Analysis 27 Electrophorettc Ahalysis of Globulin an4 Albumin Fractions . 27 Detergent Gel System . . . 27 Staining and Oestaining of the Gel Column I •• • • • • 30
v vi
TABLE OF CONTENTS--Continued
Page
Molecular Weight Determination of Albumin and Globulin by Gel Filtrations ...... 31 Two-Dimensional Electrophoresis of the Salt Extracts 33 First Dimension 33 Equilibration of the Gels 37 Freezing and Storage of the Gels ...... 37 Staining and Destaining the Gels ...... 37 Measurement of pH Gradient 37 Second Dimension . . . . 38 Loading the Isoelectric Focusing Gels onto the Slab Gels 41 Electrophoresis in the Second Dimension ...... 42 Staining and Destaining the Slab Gels ...... 42 Drying the Destained Slab Gels 43 Amino Acid Analysis Globulin Fractions 43
RESULTS 46
Isolation of Albumin and Globulins ...... 49 Characterization of the Salt Soluble Protein Extracts 50 Isoelectric Focusing . . 50 Two Dimensional Analysis 60 Characterization of Albumin and Globulin in Fractions . 60 SDS-Polyacrylamide Gel Electrophoresis . . . 60 Molecular Weight Determination by Gel Filtration 61 Amino Acid Analysis ...... 67 vii
TABLE OF CONTENTS--Continued
Page
DISCUSSION . 70
Isoelectric Focusing 70 SDS-Polyacrylamide Gel Electrophoresis . . . . 72 Globulin Fractions . 72 Albumin Fractions 72 Molecular Weight Determination 73 Globulin Fractions 73 Albumin Fractions 74 Amino Acids Analyses 75
SUMMARY AND CONCLUSION 77
APPENDIX A 79
REFERENCES 104 LIST OF ILLUSTRATIONS
Figure Page
1. Standard Curve for Protein Determination by Lowry Method . . . . 25
2. Calibration Curve; Logarithmic Plot of Molecular Weight versus Distribution Coefficient (Kav) for Chromatography over Sephacryl S-300 32
3. O'Farrell Isoelectric Focusing Gels of Salt-Soluble Proteins 48
4. Electropherogram Component Pattern of C. digitata Salt-Soluble Proteins after Isoelectric Focusing in Disc Gel as Described by O'Farrell 53
5. Electropherogram Component Pattern of C. foetidissima Salt-Soluble Proteins after Isoelectric Focusing in Disc Gel as Described by O'Farrell 54
6. Electropherogram Component Pattern of C. sororia Salt-Soluble Proteins after Isoelectric Focusing in Disc Gel as Described by O'Farrell 56
7. Electropherogram Component Pattern of C. palmeri Salt-Soluble Proteins after Isoelectric Focusing in Disc Gel as Described by O'Farrell 57
8. Electropherogram Component Pattern of C. moschata Salt-Soluble Proteins after Isoelectric Focusing in Disc Gel as Described by O'Farrell 58
9. Electropherogram Component Pattern of A. undulata Salt-Soluble Proteins after Isoelectric Focusing in Disc Gel as Described by O'Farrell 59
viii LIST OF ILLUSTRATIONS--Continued
Figu~e Page
10. Savoy SDS-Gel Electrophoresis of Globulin Fractions . . . . . 62
11. Savoy SDS-Gel Electrophoresis of Albumin Fractions ...... 63
ix LIST OF TABLES
Table Page
1. Proximate Composition of Seed Embryos ...... 47
2. Recovery Data for Salt-Soluble Proteins ...... 51
3. Recovery Data for Albumin and Globulin Fractions . . . . 52
4. Molecular Weight Determination of Globulin Fractions by Gel Filtration on Sephacryl 8-300 . 64
5. Molecular Weight Determination of Albumin Fractions by Gel Filtration on Sephacryl S-300 66
6. Amino Acid Content of Globulin Fractions Expressed as Percent Protein ...... 68
7. Amino Acid Content of Albumin Fractions Expressed as Percent of Protein ...... 69
x ABSTRACT
Salt-soluble protiens of Apodanthera undulata,
Cucurbita digitata, f. foetidissima, f. moschata, f. palmeri and ~. sororia, were isolated from the decorticated seeds.
The isoelectric focusing pattern of ~. undulata was divided into two groups, the first falling between pH
4.43 to 5.48 and consisting of six distinct peaks. The pattern of C. digitata consisted of three groups of six, two and three peaks falling in the pH ranges of 5.19 to
6.14, 6.48 to 7.00 and 7.14 to 7.89, respectively. The pattern for ~. foetidissima was divided into three groups of four, two and three peaks which fell in the pH ranges
4.82 to 5.82, 6.38 to 6.86 and 7.00 to 8.00, respectively.
The patterns of C. palmeri and f. sororia were virtually identical, with two groups of eight and five peaks which fell in the pH range 4.54 to 5.72 and 6.24 to 7.77, res pectively. The f. moschata pattern showed three groups of seven, seven and five peaks in the pH range 5.00 to
5.91, 6.00 to 7.00 and 7.24 to 8.29, respectively.
On electrophoresis the globulins of all five
Cucurbita species displayed six distinct bands with the same relative mobilities, while the globulin pattern of
xi xii
~. undulata was quite different. There was no similarity among the patterns of the Cucurbita albumins, and none resembled that of A. undulata. Two-dimensional analysis of the salt-soluble extracts showed similarity among the five Cucurbita species and differences from ~. undulata with respect to the relative mobilities of the resolved bands.
Molecular weight determination by gel filtration, resulted in resolution of each globulin fraction into four peaks except that of ~. moschata, which was resolved into five peaks. Molecular weight values ranged from 12,000 to
660,000. Each albumin fraction was resolved into three peaks except that of .~. undulata which possessed four peaks.
The molecular weight values ranged from 11,000 to 446,000. INTRODUCTION
Food production appears to be trailing the increase in world population. This is causing scientists to explore new frontiers for new and non-conventional sources of food. A large part of the world is arid and semi-arid where the usual crops cannot be produced econo mically. This makes the problem of food supply complicated in this part of the world. However, there are plant families and species which require low amounts of moisture and nutrients and which may have promise as food sources.
The use of cucurbits as food items has been documented in early civilization. They are potential sources of seed protein, seed oil, root starch and other products. Certain wild xerophytic cucurbits produce oil and protein in the seed and possess large starch bearing storage roots. In order for these plants to be more productive, vigorous research and development in many aspects of agri cultural sciences are required.
This study is concerned with the biochemical features of the salt-soluble seed proteins of certain xerophytic and mesophytic cucurbits, because the salt soluble proteins, albumin and globulin, make up the major
I 2 part of the storage protein body (Alekseeva, 1958;
Castanada-Agullo, 1948).
The objectives of this study were to isolate and purify the major storage proteins, albumins and globulins, from these different cucurbit species, study their properties utilizing different biochemical techniques, and establish any relations that may exist among the species studied. LITERATURE REVIEW
There are twenty-seven species in the genus
Cucurbita which are known by various common names such as squashes, pumpkins, gourds or marrows (Whitaker and
Bemis, 1975).
Species in this genus are a very promising source of food, because they require comparatively moderate amounts of moisture and nutrients for good growth
(Whitaker, 1968). Many other members of the family
Cucurbitaceae such as cucumbers and melons, are already recognized food sources. The use of cucurbits as food items has been documented in early civilizations; maize, beans and squashes were major foods of the ancient Aztec, Inca and Maya Indians. Several wild cucurbits, most notably Cucurbita foetidissima, f. digitata and Apodanthera undulata are potential food crops, especially in arid and semi-arid regions of the world. Possible products of these cucurbits include seed protein, seed oil, root starch and perhaps cucurbitacins.
Seed Protein
The characterization of seed proteins of xerophytic cucurbits and the relationship of those proteins to their
3 4 me sophy tic counterparts was of particular interest in this dissertation.
The general approach for isolation and fraction ation of proteins starts with enriching the protein source.
This step includes the separation of the undesirable components of the sample, e.g., separating the seed coat from the embryo, defatting the sample and liberating the protein from the cellular material by blending and centri fugation. The next step is the extraction with either aqueous solvents such as sodium chloride or ammonium sulfate, or the extraction with organic solvents and dilute acids and dilute alkalies. Dialysis is also an important step in protein enrichment and fractionation.
The intermediate purification procedure includes the utilization of ion-exchange chromatography, gel filtra tion and affinity chromatography. The final stage of protein purification requires the use of polyacrylamide electrophoresis and isoelectric focusing.
Osborne (1892) described a method of globulin isolation from squash seeds, and other investigators have used similar methods with application to a number of cucur bit seeds. He extracted squash seed meal three times with 20% sodium chloride solution. The clear extract was saturated with amonium sulphate. The precipitate was dissolved in 10% sodium chloride and then submitted to dialysis. A crystalline globulin was separated by 5 filtration. Jones et al. (1923) isolated the globulin frac tion from cantaloupe seeds by extracting the seed meal with
5% sodium chloride solution and then treated the salt extract by one of the following methods. In the first, the salt ex tract was dialyzed against chilled running water for ten to fifteen days. The second method involved dilution of the salt extract with ten volumes of distilled water. The third method required acidification of the extract with acetic acid fol lowed immediately by dilution with ten volumes of distilled water. The fourth method depended on making the extract 40% saturated with ammonium sulfate. In all cases the precipi tate was removed by filtration, redissolved by addition of water and the globulin was precipitated by dialysis.
Vickery et al. (1941) modified the procedures of Osborne
(1892) and Krishnan et al. (1939) for preparation of large amounts of globulin. They omitted the fat extraction step and the separation of the testa from the seeds. They also intro duced a heat coagulation step designed to remove the albumin fraction. The seeds were ground in a meat grinder then extracted wi th warm (30 - 350 C) 10% sodium chloride solution. Filter paper clippings were added and incorporated until a mass was produced.
The mass was pressed to produce an emulsion which was heated in a hot water bath to 750 C and allowed to stand overnight at roan tEmperature. The turbid aqueous phase was siphoned fran beneath the layer of fat emulsion and protein coagulum, filtered and washed with 10% sodium chloride solution. The emulsion was 6
The emulsion was centrifuged, filtered, warmed to 60oC, diluted with four volumes of water, also at 60oC, and
allowed to stand overnight in a cold room. The supernatant
fluid was removed and the globulin was collected on a hard paper in a Buchner funnel.
Investigators have reported the effect of the solvent, pH, temperature and ionic strength on the compo sition and physico-chemical properties of the extracted proteins (Wang et al., 1976; Betschart, 1974; Lawhon et al., 1974).
The amino acid composition of seed globulins from several species of Cucurbitaceae has been determined
(Jones et al., 1923; Smith et al., 1946). Smith and Green
(1947) used the amino acid composition to establish minimum molecular weights. They found values of 55,000 for globulins from cucumber and watermelon, and 58,000 for those
from pumpkin and squash. The same researchers reported that globulins from different genera could be distinguished on the basis of differences in the amino acid composition
(Smith et al., 1946).
The physico-chemical properties of cucurbit globu lins also have been explored. Byerrum et al. (1950) studied the electrophoretic mobility of the globulin from ~. pepo at pH 4 and described it as essentially homogeneous at that pH. Fuerst et al. (1954) found that squash seed globulin in acetate buffers contained only a single electrophoretic 7 component at low pH, while at pH 4.7 it contained two sharply defined components. Their results also showed four defined fractions of this globulin with sedimentation coefficients of 3.0S, 7.1S, 9.4S and 12.1S. The presence and proportion of these fractions were affected by time, pH and concentration of the proteins. Mourgue et al. (1968) also provided information on the physico-chemical proper ties of the seed globulins of ~. maxima, showing that the globulin fraction consisted of three distinct components by use of ultracentrifugation and chromatography on DEAE
Sephadex columns.
In a detailed study, PichI (1976) isolated globulins from six different species of the family Cucurbitaceae. His results showed no significant differences in the nitrogen content of the globulins isolated. The mean value of the nitrogen content was 18.55%. The most abundant amino acids were arginine and glutamic acid. On the average, the amount of arginine was three times greater than that of glutamic acid. The contents of histidine, proline, serine and tyrosine showed relatively high variability within the group of species compared. Ultracentrifugation measure ments revealed the presence of three fractions in all the species studied except Momordica charantia. The presence and proportion of these fractions were affected by concen tration of the proteins, pH and time. Gel filtration studies on Sephadex G-200 showed no substantial difference 8
dimerization dimerization pH > 4.5 pH = ? +6.48 (?)
3.08 >6.48 11.08 >158
pH < 4.5 ~______~T_r_i_m_e_r_l_'z __ a_t_i_O_n __ (_?_.~)~
in the elution properties of the globulins. In all cases they were eluted as a single peak under these experimental conditions: G-200 column (2.1 x 65 cm); sample applied was
2 ml containing 5 mg of protein in 2 M NaCl; sample was eluted with 2M NaCl adjusted with Tris to pH 8. The molecular weights varied within the range of 218,000 -
256,000.
Evident differences were observed in electrophoretic properties of the globulin subunits on 5% acrylamide with
8M urea. In contrast, the differences in the number of fractions, formed after oxidative splitting, were not so pronounced as in the case of electrophoresis in 8M urea.
PichI concluded that this type of analysis "may offer a prospective tool in employing the above mode of analysis as one of ~he chemotaxonomic criteria for determination of at leRst generic pertinence within a given family or tribus".
The diversity of globulin and albumin components in cucurbit seeds is clear. Experimental conditions for 9 isolation, fractionation and determination of physical properties vary considerably, leading to apparently contradictory results. Selected examples from species other than cucurbits are presented below for comparison.
Soybean storage proteins have been characterized by sucrose gradient-sedimentation and by polyacrylamide gel electrophoresis (Hill et al., 1974). They reported
three classes of proteins with sedimentation coefficients of 2.2S, 7.5S and 10.88
Barker et al. (1976) employed several extraction and fractionation procedures to isolate the major storage proteins of the seeds of Phaseolus vulgaris. They found three proteins which were soluble at pH 4.7, and one
insoluble at that pH. The characteristic subunits of the three soluble proteins as they appeared on SDS-gel electro phoresis had molecular weights of 50,000, and 47,000,
32,000 and 23,000 respectively; those of the pH 4.7 insoluble fraction had molecular weights of 6,000 and 20,000.
Youle and Huang (1976 and 1978) isolated albumin and globulin from castor bean and showed that the albumin fraction constitutes approximately 40% of the protein bodies. This fraction had a sedimentation value of 2S and could be resolved into several proteins with molecular weights between 50,000 and 60,000. Youle and Huang (1979) subsequently separated cottonseed protein into three 10 approximately equal fractions with sedimentation values of
2S, 5S and 9S. The 5S and 9S proteins were typical globulin storage proteins and had similar amino acid composition.
The 2S proteins were albumins and also storage proteins as judged by their amino acid compositions and relative abun dance in the seed. The 2S storage albumin proteins were shown to be identical with the cottonseed allergens.
The globulin and the albumin of Lupinis albus have been isolated using distilled water at pH 5.0 and 7.0, and salt extraction (1M NaCl). The albumin was then separated from the globulin during dialysis (Cerletti et al., 1978).
They found that the globulin was the major component
(83.5 ~ 1.0% of the protein). A gel filtration study on
Ultragel AcA34 showed that the globulin fraction contains at least seven distinct molecular species, which differ in molecular weight, subunit size, distribution and ionization behavior as shown by electrophoresis and ion exchange chromatography. Albumins, on the other hand, form only a minor part of the proteins extracted (7.2 ~ 1.2%), but at least five different species appear to be present. However,
Jouber (1955) reported only one albumin component in blue lupine seed.
Salt-soluble proteins from three varieties of sunflower seeds, which contain about 80% of the total seed nitrogen, have been fractionated by using buffered 10% NaCl, 11
gel filtration on Sephadex G-50, dialysis, and gel
filtration on Sephadex G-200 (Baudet et al., 1977). They
confirmed the occurrence of three groups of proteins,
calling them light albumin (20% of extracted protein), heavy
albumin (5-10%) and globulin (70-80%).
The first group was isolated on Sephadex G-50,
while the other two were separated from each other by
dialysis. The molecular weight of each group was determined
using Sephadex G-200. Light albumin proved to be homogeneous
with a molecular weight of 14,000 and an amino acid composi
tion showing high amounts of methionine, cystine, arginine
and glutamine. Heavy albumin was shown to have a molecular weight of 48,000 and a very different amino acid composition with a high level of lysine. Globulin was ·composed of at
least four fractions with molecular weights of 13,000,
25,000, 43,000 and 95,000. The first two globulin fractions
appeared to be subunits of the last two.
Rahma and Rao (1979) studied sunflower meal protein
using disc electrophoresis, gel filtration and ultracentri
fugation. The electrophoretic pattern showed three major bands of low mobility and two to three bands of high mobility. The sedimentation velocity pattern of the meal
proteins in 1.OM NaCl showed three peaks whose S20,w values were 11.OS, 7.0S and 2.0S. Gel filtration showed three major peaks on Sepharose 6B-IOO. On the other hand, 12
Sabir et al. (1973) obtained five fractions from extracts of sunflower meal using Sephadex G-200.
Seed Oil
Unsaturated fatty acids predominate in cucurbit seed oils. In some species about one-third of this unsaturation is in the form of conjugated triene (Jacks et al., 1972). They also showed that a "typical" cucurbit oil contains 28.5% oleic acid, 47.3% linoleic acid, 14.2% palmitic acid and 8.4% stearic acid. The analysis of 37 samples of cucurbit seed showed that the decorticated cucurbit seeds contain 49.5 ~ 2.3% oil and 35.0 + 2.4% by weight (Earle and Jones, 1962).
The fatty acid composition indicates that certain cucurbit seeds are a potential source of edible oil. Pumpkin seed oil, for instance, is known to be more than 99% digested by rats. The results of feeding studies with mice conducted by Bemis et al. (1977) and by Khoury et al. (1982) with chicks showed that buffalo gourd seed oil is nutritionally comparable with commercial oils. Although the amount of conjugated fatty acids is variable, certain cucurbit seed oils are likely to develop rancidity by oxidative degradation. Some cucurbit seed oils (e.g., buffalo gourd) contain only a small amount
(2.3%) of the conjugated triene (Vasconcellos et al., 1980).
This level drops to 0.48% after processing (Vasconcellos 13 et al., 1981), and the oxidative stability of the processed oil was acceptable even in the absence of antioxidants (peroxide value (pv) = 100 after 4.9 hr).
Starch
Cucurbit seeds are clearly an important source of high quality protein and oil. In addition, Berry et al.
(1975) reported that potentially commercial amounts of starch can be isolated from the roots of buffalo gourd.
A seasonal variation occurs in the starch content of the root from 18% (dry weight basis) in May during the initia tion of vine growth and fruiting to a peak of 52% in
August, which is maintained until growth is initiated again in May (Berry et al., 1978).
The digestibility of raw buffalo gourd starch was found to be 41.1%, which is between that of potato starch
(26.5%) and corn starch (96.5%) or tapioca starch (97.2%).
Cooking of buffalo gourd starch raised its digestibility to 95.6% (Dreher et al., 1981).
The rheological properties of buffalo gourd root starch were found to be quite different from those of typical root or cereal starches. It shows viscosity stability with a higher initial pasting temperature, a peak viscosity at 900 C intermediate between that of potato and cOrn starches, and only a slight increase in viscosity on cooling (Dreher, 1979). 14
Other Products
A potential use of the buffalo gourd root is the conversion of its starch to alcohol by fermentation. It has been found comparable to corn meal as a fermentation sub strate (Gathman and Bemis, in press).
Many members of the Cucurbitaceae possess an extreme bitterness in the fleshy part of the fruit (Whitaker et al., 1962) and in the roots (Berry et al., 1978) due to the presence of triterpenoid glycosides (cucurbitacins).
Buffalo gourd roots cannot be eaten unprocessed because of the cucurbitacins present. Fortunately, most cucurbitacins can be readily extracted with water during the starch isolation.
The role of bitter cucurbits as insect attractants has been reported by many investigators (Chambliss and
Jones, 1966a and 1966b·; Sharma and Hall, 1971 and 1973).
The potential use of cucurbitacins in medicine or research as laxatives, antitumor drugs and antihelminthics has been reported (Sharma, manuscript in preparation).
However, their use is limited by their toxicity to mammals.
The vine growth of the cucurbits could be used as fuel, either for direct combustion or as a cellulose source for conversion to ethanol. The vines contain 28% fiber
(dry weight basis), predominantly cellulose (Berry et al.,
1976). Assuming 50% conversion of cellulose to sugars 15 and 50% conversion of sugars to ethanol, a yield of 125 L
(33 gal.) of alcohol per acre would be obtained (Gathman and Bemis, in press). MATERIALS AND METHODS
Seeds Source
The following six species of the family
Cucurbitaceae were used in this study: Apodanthera
undulata (A.P.), Cucurbita digitata (C.D.), f. foetidissima (C.F.), f. moschata (C.M.), f. palmeri (C.P.), and C. sororia (C.S.). All of the seed samples were obtained by the courtesy of Dr. William P. Bemis from the
University of Arizona Collection.
Proximate Analysis
Moisture (Lyne, 1976)
Accurately weigh in duplicate between 2 - 3 gm of ground seed embryo into a tared dish. Dry in oven overnight at 1200 C. Allow samples to cool in desiccator and weigh.
Calculation: moisture weight loss, gm X 100 % initial wet weight, gm
16 17 Lipid Extraction, (Schoch, 1964)
1. Apparatus:
a. Vacuum oven
b. Goldfisch extraction apparatus with cooling
2. Material:
a. Thimbles
b. Goldfisch beakers
c. Thimble holders
3. Procedure:
Accurately weigh in duplicate about 2.0 gm of 10 mesh sample and quantitatively transfer to thimbles. Attach thimbles in holders. Then attach to the snaps in the condensing apparatus so that the bulb-like portion of the holder is approximately one half-inch from the snap. Add
30 ml of hexane to the Goldfisch beaker and attach beaker to condensing apparatus using metal ring. Start cooling system for both refrigerator and pump. Adjust heating apparatus to about 1 - 2 inches below the bottom of the beaker. Adjust temperature control to "high" and turn on switch at the right side of the apparatus. Extract for at least 8 hr and cool.
Lower heating unit~ and remove Goldfisch beakers. Evaporate solvent on hot plate in the hood but do not boil dry. When solvent is completely evaporated, place beakers in desicca tor, cool, weigh. 18 Calculation: % crude lipid lipid weight, gm X 100 dry sample, gm
Crude Protein
1. Apparatus:
a. Kjeldahl digester unit connected to
aspirator in the hood.
b. Kjeldahl distillation apparatus
2. Material and Reagents:
a. 30 ml Kjeldahl flasks
b. Cigarette paper
c. Stopwatch
d. Microburet
e. 2% boric acid solution (20 gmjL)
f. 40% sodium hydroxide (40 gm NaOH + 60 g water)
g. 0.01 N hydrochloric acid (1 ml conc HCljL)
h. Catalyst: 0.05 gm Hengar's selenium
0.15 gm copper sulfate (anhydrous)
i. Digestion solution:
concentrated sulfuric acid plus phosphoric
acid mixture 3:1 (v:v)
j. Indicator (modified methyl red):
0.125 gm methyl red
0.0825 gm methylene blue
100 ml ethanol 19
3. Procedure:
(In duplicate) Dry defatted meal in a vacuum oven overnight at 60oC. Lightly grind material in a mortar to pass a 150 mesh screen. Dry 150 mesh material in a vacuum oven overnight at 60oC. Tear a piece of cigarette paper and accurately weigh approximately 50 mg of sample. Fold sample in paper and place in a 30 ml Kjeldahl flask. Add about
1/2 of a small scoop spatula of catalyst and 5 ml of digestion solution. Place the flasks on the digester under a hood. The digester is set at low temperature until the danger of frothing is over; then increase heat. When completely digested, the sample is water clear or light blue in color. Run a blank using cigarette paper. Remove digested sample from digester and place in a wire basket until cooled. Transfer solution to a 25 ml volumetric flask and make up to volume when cool. After thoroughly flushing the distillation equipment, fill outer jacket with water to just above the heating coil. Use a 50 ml Erlenmeyer flask, add 10 ml of 2% boric acid solution from a buret, add 3 drops of indicator and attach to apparatus with condenser tip immersed in liquid, Withdraw 5 ml sample with a volumetric pipet and empty pipet into Kjeldahl apparatus. Add 10 ml
40% NaOH with a graduated cylinder and rinse with a small amount of deionized water. Close off apparatus and run water in the outside jacket of condenser column. Turn on 20 heating element and let the sample boil. When the first drop of condensate falls from the cold finger into the boric acid solution, start a stopwatch and let distilla- tion proceed for 4.5 minutes. Then lower the flask to clear the apparatus of the distillate, and let it run for another 30 seconds. Turn off the apparatus. Titrate the green boric acid sample with 0.01 N HCI until there is a distinct color change back to blue or purple. Record the volume of titrant used in ml to two decimal places.
Calculation:
(titrant used (ml))(conversion factor)(dilution) crude protein % dry sample weight (mg)
Conversion factor
(N HCI)(0.014 gm nitrogen)(6.25 gm protein)(IOOO mg)(IOO) (1 mg weight) (1 gm nitrogen)
Acid Detergent Fiber (Van Soes~ 1963)
1. Apparatus:
a. Vari-heating unit (Precision Scientific Co.)
2. Materials and Reagents:
a. 600 ml Berzelius beakers
b. 250 ml centrifuge bottles
c. 2% hexadecyltrimethylammonium (HDTA) bromide
dissolved in 1 N sulfuric acid.
d. Decahydronapthalene 21 3. Procedure
(In dupl ica te ) Accurately weigh 2 gm of dry sample
(or equivalent weight of wet material) into 600 ml Berzelius beakers. Add 100 ml of HDTA bromide solution to 2-6 ml of decahydronaphthalene (antifoamant). Place in heating unit with condenser. Heat to boiling and reflux for 60 min from the onset of boiling.
Filter the acid detergent fiber using light suction on a dry, tared sintered glass crucible (tall form, coarse porosity, 40 or 50 mm in diameter). Wash with a stream of hot deionized water (90 - 100oC). Wash adhering particles down from sides of crucible and wash twice with cold acetone or until washings appear colorless. Suck crucible free of acetone and dry in a forced air oven at 1000C for two hr.
Cool in desiccator and weigh. The acid detergent fiber value is determined by subtraction of the dry crucible weight.
Ash (Smit~ 1964)
1. Apparatus:
a. Muffle Furnace (Wheelco Model No. 293)
b. Fiseher burner
2. Material and Reagents:
a. 100 ml capacity silica dishes
b. crucible tongs 22
3. Procedure:
(In duplicate) Accurately weigh approximately 5 gm of sample into a dry tared silica dish. Heat dish and con- tents carefully over an open flame until sample is com- pletely charred. The partially decomposed sample is then heated in a muffle furnace at 5250 C until the residue is free from carbon; overnight is usually sufficient. If ignition of carbon is slow, the process can be hastened by cooling the dish, moistening the residue with a few drops of water and reheating. After completion, the dish and ash are cooled in a desiccator prior to weighing on the analytical balance.
Calculation:
ash weight, gm x 100 % ~sh sample weight, gm
Isolation of Albumins and Globulins
Defatting the Samples
The cold fat extraction procedure described by Savoy
(1976) was employed to avoid heat denaturation of proteins.
Procedure:
The dehulled embryos were ground using a blender. To
20 gm of the ground material add 100 ml of cold acetone (90~) 23 containing ammonium hydroxide (50 ]11/100 ml). Collect the sample by sedimentation, 20,000 g, for 30 min at 4 o C.
Repeat this process three times. Quick freeze the sample then 1ypholize and store at -20oC.
Albumin and Globulin Separation
The pr8cedure described by PichI (1976) was employed with some modification. The defatted seed meal was extracted for 3 hr with stirring in 10% NaC1 added in the ratio of 1:40 at room temperature. The slurry was allowed to settle and the supernatant was filtered through Whatman
No.1 paper with a layer of cellulose powder. The filtrate was dialysed for eight days, at first against running water
(4 hr) and then against deionized distilled water at 4 0 C
(eight changes). The resultant precipitate (globulin fraction) was collected by centrifugation at 5000 g for 15 min at 2oC. The supernatant which contains the albumin fraction was transferred to a glass pan and freeze dried, o then stored at -20 C. The globulin fraction was washed with distilled water and collected again by centrifugation under the same condition as above (repeated three times).
The globulin was finally suspended in a minimum volume of distilled water, lypholized and stored at -20oC. 24
Protein determination in the separated fractions
(albumin and globulin) and in the salt extract.
The Kjeldahl method was used for protein determi
nation in the globulin fractions. The Lowry method
(Lowry et al., 1951) was used for protein determina
tion in the salt extract and in the albumin fractions
in order to conserve the albumin samples.
Lowry Method
Reagent:
a. 2.0% solution of NaCo in 0.1 N NaOH 3
b. 0.5% solution of CuS04 .5H20 in 1.0% sodium or potassium tartrate.
c. Alkaline-copper solution - 50 ml of
solution a and 1 ml of solution b (discard
after one day).
d. Folin and Ciocalteru 1 s phenol reagent -
2 N (from Sigma Chemical Co.) diluted
1:1 with distilled water.
Standard Curve (Figure 1)
Crystalline bovine serum albumin (Sigma Chemical
Co.) was used to prepare the working solutions; 25 mg was dissolved in 100 ml of 0.85% NaCl solution (250 ~g/ml). 1.8
1.6
1.4
1.2 w u Z 1.0 Figure 1. Standard Curve for Protein Determination by Lowry Method. tv CJl 26 Procedure: 1. Pipet an aliquot of protein solution into a colorimeter tube to provide 25 - 250 11 g of protein per tube. 2. Add water to a volume of 1.0 ml (prepare blank with 1 ml of water). 3. Add 5 ml of Reagent c, alkaline-copper solution 4. Mix well and allow to stand at least 10 min. 5. Add 0.5 ml of Folin reagent (Reagent d) rapidly and mix immediately. 6. Allow to stand for 30 min. 7. Read at 750 nm. 8. The standard curve is not linear and must be plotted. Albumin Sample Analysis 1. Weigh accurately about 5.0 mg of the isolated albumins. 2. Dissolve the protein in 5 ml of 0.85% NaCl solution. 3. Dilute 1.0 ml of the protein solution to 5.0 ml by adding 4.0 ml of the saline solution. 4. Use a 0.4 ml aliquot of each diluted protein solution to measure the protein content of each albumin fraction according to the above procedure. 27 Salt Extract Analysis 1. Dilute 1.0 ml of each salt extract to 100 ml by adding 99 ml of 0.85% NaCl solution. 2. Follow the above Lowry procedure using 0.4 ml aliquot of each diluted salt extract. Electrophoretic Analysis of Globulin and Albumin Fractions The purified preparations of albumin and globulin of each species were analyzed electrophoretically using SDS- polyacrylamide gels. The electrophoretic analysis was used primarily to study the structural similarities of various protein fractions. Detergent Gel System Detergent SDS-electrophoresis was carried out in 10% acrylamide gels utilizing the basic systems of Davis (1964), Ornstein (1964) and Shapiro et al. (1967) as described and further refined by Weber and Osborn (1969), Grula and Savoy (1971) and Savoy (personal communication, 1981). Reagent: Polyacrylamide gel forming reagents were obtained as a kit from the Eastman Company. 28 Procedure: Stock solution A contained 7.8 gm NaH P0 .H 0, 2 4 2 38.6 gm Na HP0 .7H 0 and 2.0 gm SDS per liter and was stored 2 4 2 at room temperature in a dark bottle. Stock Solution B contained 11.1 gm acrylamide and 400 mg N,N' - methylene- bis~crylamide (BIS) per 50 ml; insoluble materials were always removed from the solutions by filtration through Whatman No.1 filter paper and stored under refrigeration in a dark bottle. Both solutions were prepared fresh every two weeks if not depleted. The glass tubes used to prepare the gel column were 125 mm long with an inner diameter of 5.0 mm. Before use, the tubes were cleaned with acid cleaning solution (Chromerge in concentrated sulfuric acid), rinsed in distilled water (three times), soaked in Photo-Flo cleaning solution (one ~rop/IOO ml H20), rinsed again (three times) and oven dried (lOOoC for 30 min). One end was covered with a double layer of Parafilm then positioned on the Bio-Rad Universal gel preparation rack. For electro- phoresis of a set of gels (12 tubes), 15 ml of stock solution A and 13.5 ml of stock solution B were mixed. Then 30 WI of N,N,N' ,N' -tetramethylenediamine (TEMED) were added. The mixture was degassed for 5 min and 1.5 ml of freshly prepared ammonium persulfate solution (8 mg/ml H20) was added, the solution was mixed thoroughly and immediately placed into the tubes using a syringe. A few drops of distilled water 29 were carefully layered on top of the gel columns so as not to disturb the acrylamide surface, and this was completed before the acrylamide polymerized (within 10 - 15 min after the final mixing). Columns were kept at room temperature overnight to allow for complete polyermization before the electrophoresis was attempted. Two hrs prior to electro phoresis, the water was replaced by the running buffer. Just before sample application, this buffer and the Parafilm were removed. Protein samples were solubilized (3 mg/ml) in screw-capped tubes at 900 C for 15 min in solvent buffer (pH 7.1) containing 78.0 mg NaH P0 .H 0, 386.0 mg Na HPO.7H 0, 2 4 2 2 2 1.0 gm SDS and 5 ml glycerol in distilled water (99 ml total volume). Immediately before use, 1 ml 2-mercaptoethanol was added to the 99 ml of solvent (100 ml total volume). Any insoluble materials were removed from the solubilized sample by centrifugation at 1500 g. The samples were applied (40 ~l) to the tops of the gels using an automatic pipet, and the remainder of each tube was carefully filled with running buffer (stock solution A diluted 1:1 with distilled water). Buffer chambers were filled with this diluted solution, and electrophoresis was performed at room temperature with the anode located in the lower chamber (Bio-Rad model 151 tube gel electrophoresis cell). To enhance resolution, an amperage of 1 rna/tube was used for the first 30 minutes followed by a final 30 running amperage of 5 rna/tube. Bromophenol blue (0.1% solution) served as the tracking dye (3 ~l/tube). After electrophoresis (12 hours) the gel columns were removed from the tubes by gently rin~ing both ends of the gel-glass boundary of the gel tube with a 2 inch no. 22 needle supplied with a gentle stream of water. If the gel did not break free, a 10 ml syringe was connected to the electrophoresis tube via a short piece of Tygon tubing, and the gel was slowly forced out by pressure on the syringe. Finally, the freed gel was placed on a clean flat surface and a needle was used to puncture through the gel right at the front of the tracking dye. Staining and Destaining of the Gel Columns The gel was placed dye front down in a screw-capped tube. The tube was filled with 0.025% Coomassie blue in an aqueous solution containing 25% isopropanol and 10% acetic acid and allowed to stand overnight. Staining solution was replaced with 25% isopropanol, 10% acetic acid for 5 hrs. This solvent was replaced with a solution containing 5% methanol and 5% aceiic acid and allowed to stand about 8 hours. If necessary, the latter solvent was changed until blue protein bands became visible against a relatively clear gel background. The gel columns were stored in 7% acetic acid until scanned at 580 nm using Gilford gel scann8r model 31 2520 with slit plate (0.1 x 2.36) connected to Beckman spectrophotometer model 2400. Molecular Weight Determination of Albumins and Globulins by Gel Filtration Gel filtration experiments were performed in a K 26/70 (2.6 cm x 70 cm) column from Pharmacia Fine Chemicals. Four hundred ml of the settled pre-swollen Sephacryl S-300 superfine was suspended in the eluent buffer, Tris/HCl buffer solution (0.1 M, pH 8.0) containing 0.5 M NaCl and 0.02% sodium azide. Then the column was packed according to the procedure described in the handbook (Gel Filtration Theory and Practice, Pharmacia Fine Chemicals) and according to the instructions included with Sephacryl S-300 pre-swollen gel. The same buffer used in equilibrating the column was also used in the elution of both standard proteins and samples. About 20 mg of a sample protein were dissolved in the elution buffer and applied to the column using the sample applicator, four way valve (LV-4) and a peristaltic pump (Gilson Company) with a flow rate of 24 ml/hr. Eluent was monitored at 280 nm with an Isco Model UA-5 multi-wave length absorbance monitor (Instrumentation Specialties Company). Elution volume was recorded by a Linear Instru- ments Corporation recorder Model 141. A part of this experiment was monitored by an LKB monitor Uodel 2138 0.6 0.51- 1. CHYMOTRVPSONIGEN A "- 2. 0 VALBUMIN 3. ALDOLASE 4. FERRITIN 5. THYROGLOBULIN aT ~ KAV 0.3 0.2 0.1 4 5 6 LOG MW Figure 2. Calibration Curve; Logarithmic Plot of Molecular Weight versus Distribution Coefficient (Kav) for Chromatography over 8ephacryl 8-300. c.u tv 33 Uvicord S. and LKB peristaltic pump model 2132 microperpex and recorded with an LKB recorder model 2210. Standard solutions (20 mg/2 ml) containing ovalbumin (43,000) and aldolase (158,000) in the fi~st run and chymotrypsinogen A (25,000), ferritin (440,000) and thyroglobulin (669,000) in the second run were loaded onto the column and the elution volumes (Ve) obtained. The void volume (Vo) was obtained as the volume required for elution of blue dextran (4 mg/2 ml eluent buffer; MW 2,000,000). A calibration curve (Figure 2) was constructed using the linear relationship between Kav and log MW, where: Ve - Vo Kav Vt - Vo Two-Dimensional Electrophoresis of the Salt Extracts In this experiment proteins were separated according to their isoelectric point by isoelectric focusing in the first dimension and according to their molecular weights by SDS electrophoresis in the second dimension. First Dimension The isoelectric focusing in disc gels was carried out utilizing the procedure deBcribed by O'Farrell (1975). 34 Solutions: % in the final solution I. 7.1 gm acrylamide 28.38 (w/v) acrylamide 0.4 gm Bis-acrylamide 1.62 (w/v) Bis-acrylamide dilute with water to 25 ml Keep refrigerated in amber container II. 8 M urea 4.8 gm/IO ml Final III. Equilibration Solution Volume (ml) Concen.tration 0.5 M Tris-HCl, pH 6.8 10.00 50 mM 10% SDS 20.00 2% 2-mercaptoethanol 5.00 5% water 65.00 100.00 Final IV. SDS-Freezing Buffer Volume (ml) Conc~tion 0.5 M Tris-HCl, pH 6.8 12.4 62.5 mM glycerol 10.0 10% 2-mercaptoethanol 5.0 5% 10% SDS 23.0 2.3% water 49.6 100.00 35 Final V. Voltnne (ml) Concen:tration 10% SDS 2.0 2% 2-mercaptoethanol 0.88 8.8% 3/10 Ampholines (LKB) 0.88 8.8% NP-40 0.35 3.5% water 5.89 10.0 Sample Preparation: Dilute each salt extract to 1:10 with distilled water Then add 0.5 ml of each diluted protein solution to 1 ml of Solution V. To 80 ~l of the final solution of each protein in a plastic vial add 80 mg of solid urea and let it dissolve at room temperature (avoid heating). Procedure: The isoelectric gels were made in glass tubes (12.5 x 0.5 cm in diameter) soaked in chromic acid, rinsed in distilled water, soaked in alcoholic KOH solution (1.0 liter of 95% ethanol + 120 ml H 0 containing 120 gm KOH), then 2 rinsed extensively and air dried. The bottom of each tube was sealed with tW0 layers of Parafilm. To make 10 ml of gel mixture (4% acrylamide), the following were combined: 5.5 gm solid urea in a 125 ml sidearm flask, 0.2 ml Nonidet p40 (Np-40), 3.8 ml distilled water, 1.33 ml of acrylamide stock solution (I) and 0.5 ml of 3/10 Ampholines. The flask 36 was swirled until the urea was completely dissolved (without heating), 7 WI of TEMED was added and the mixture was degassed for 5 min; then 50 WI of 10% ammonium persulfate (fresrily prepared) was added. Immediately after the addition of the persulfate, the solution was loaded into the gel tubes using a syringe with a long narrow gauge hypodermic needle. The tubes were filled to 0.5 cm from the top, and the gels were overlayed with solution II. After 3 hrs the overlay was removed, the Parafilm was also removed, and the ends of the tubes were covered with dialysis membrane held in place by 3 mm section of Tygon tubing. The lower reser voir of the tube electrophoresis cell (Bio-Rad's Model 151) was filled with 0.01 M H P0 . The gels then were placed in 3 4 the electrophoresis apparatus, and the samples were loaded and overlaid with 25 WI of solution II. The tubes and upper reservoir were filled with 0.02 N NaOH (extensively degassed). The gels were run at 200 volts for 14 hrs, then at 400 volts for one hr and finally at 800 volts for one hr. The gels were removed from the tubes by connecting the gel tube to a 10 ml syringe via a short piece of Tygon tubing, and the gel was slowly forced out by pressure on the syringe. The gels were stained, or equilibrated and immediately loaded on the second dimension gel, or equilibrated and stored frozen at -200 C. 37 Equilibration of the Gels The gels were suspended in equilibration solution at room temperature for 30 min with mild agitation. The gels were then suspended in new solution for 30 min at 800 C with mild agitation. Freezing and Storage of the Gels After equilibration each gel was placed in 5 ml of SDS-freezing buffer (IV) in a stoppered tube (25 ml) and the tube placed on its side in a freezer (-20oC) Staining and Destaining the Gels The unequilibrated gels were stained in 0.25% Fast Green in 10% acetic acid for 2 hrs. Destaining was then carried out in a diffusion destainer for at least 48 hrs in a mixture of 10% acetic acid and 30% methanol. The gels were scanned at 635 nm using the gel scanner described previously. Measurement of pH Gradient The unstained isoelectric focusing gels were cut into 5 mm sections which were placed in vials containing 2 ml of degassed distilled water. These vials were capped and shaken for 10 min, and the pH was then measured with a pH meter. 38 Second Dimension The second dimension was performed on PROTEAN Dual 16 cm Slab Cell (Bio-Rad) utilizing the discontinuous SDS gel system described by Laemmli (1970). Solutions: 1. Stock solutions: a. Acrylamide:Bis 30:0.8 300 gm acrylamide 3 gm bis-acrylamide Make up to 1000 ml with water. Filter and keep cold in the absence of light. b. 1.5 M Tris-Cl, pH 8.8 18.15 gm Tris 20 ml water Adjust pH 8.8 with HCl Make up to 100 ml with water c. 0.5 M Tris-Cl, pH 6.8 3 gm Tris 20 ml water Adjust to pH 6.8 with HCl Make up to 50 ml with water 39 2. Separating Gel (10% acrylamide). The following amounts are sufficient to pour 2 slabs 1.5 mm thick: Volume (ml) Acrylamide:Bis 30:0.8 25.0 1.5 M Tris-HCl, pH 8.8 18.75 10% SDS 0.75 TEMED 37.51-11 water 29.75 1% ammonium persulfate 0.75 3. Spacer Gel (5%) Volume for 2 slabs (ml) Acrylamide:Bis 30:0.8 2.5 0.5 M Tris-HCl, pH 6.8 2.76 10% SDS 0.15 TEMED 0.008 water 8.6 10% ammonium persulfate 0.16 4. Electrode Buffer To make 4 liters of the electrode buffer, mix the following: 40 12.1 gm Tris (0.025 M) 57.6 gm glycine (0.192 M) 40 ml of 10% SDS (0.1%) Make up to four liters with water. 5. Staining Solutions: Final Concentration 0.3 gm Coomassie Blue 0.033% 450 ml methanol 50% 378 ml water 63 ml glacial acetic acid 7% 6. Destaining Solutions: Final Concentration a. 300 ml methanol 30% 100 ml acetic acid 10% 600 ml distilled water Final Concentration b. 100 ml methanol 5% 150 ml acetic acid 7.5% 1750 ml water 41 7. Slab Gel Backs Treatment: Boil the backs in 5% sodium bicarbonate and 50 mm EDTA for 5 min. Rinse with distilled water until the water is clear. Store in distilled water at room temperature. Procedure: The 1.5 mm width gel sandwiches were assembled using two of the one-piece 16 em clamps. The assembled sand- wiches were securely locked to the casting stand with the two Delvin cams per sandwich. The separating gel was poured into each sandwich up to 1.0 em from the top using a small plastic funnel connected to a plastic automatic pipet tip. A few drops of butanol were laid on top of the gel. After the gel was cast, the spacer gel was poured and a few drops of butanol were layered on top of it. After the spacer gel was cast, the upper buffer chamber was slipped over the sandwiches in the casting stand and locked to the sandwiches with the cams which were transfered from the casting stand to holes in the buffer chambers. Loading the Isoelectric Focusing Gels onto the Slab Gels The cylindrical gel was placed on a piece of Parafilm, straightened, and placed close and parallel to one edge of 42 the Parafilm. Meltedagarose solution (1% in equilibration buffer) was added to fill slab to notch on glass plate (minimum amount), then the Parafilm was used to transfer the equilibrated cylindrical gel into· the notch, which was then covered with melted agarose. Electrophoresis in the Second Dimension About twenty min were allowed for the agarose to set, then the entire upper chamber and the sandwich assembly were lifted from the casting stand and placed in the .lower buffer chamber for operation. Electrode buffer was added and the air bubbles were removed from the bottom of the gel by a stream of running buffer from a Pasteur pipet which was bent at the end and attached to a 10 ml syringe. A small well was made on the agarose gel and 15 ~l of 0.1% bromo- phenol blue was added as a tracking dye. Gels were run at 30 rna/slab constant current until the dye front reached the bottom of the gel. Running time was about 8 hours. Staining and Destaining of the Slab Gels After the current was turned off, the glass plates were pried apart by applying finger pressure on the spacers against the upper glass plate. The slab gels were stained for 20 min with mild shaking, then destained with mild shaking in several changes of destaining solution. 43 Drying the Destained Slab Gels The drying of the gels was carried out on Gel slab dryer model 224 (Bio-Rad). The detained gels were immersed in an aqueous solution containing 1% glycerol and 10% acetic acid for 40 min. A pretreated cellophane membrane backing sheet (treated in the backing sheet solution) was laid on the stainless steel support of the dryer, and stretched smooth by passing a hand over the sheet. The gel laid over the sheet and was covered with another pretreated cellophane sheet. The air bubbles were removed. The Mylar sheet was placed, smooth side down, over the gel, covered with the rubber sealing sheet. Then the vacuum was connected and the heat was turned on for 3 hrs. Amino Acid Analysis of Albumin and Globulin Fractions The ion exchange analysis of amino acids was carried out with a Beckman model 121 automatic amino acid analyzer. Reagents: 1. sodium thioglycollate 2. 6N Hel 3 sodium citrate buffer, pH 2.2 4. thiodiglycol 44 Dissolve 19.6 gm sodium citrate dihydrate using approximately 400 ml of deionized water in a liter mixing cylinder. Add 16.5 ml of concentrated HCl and 5.0 ml of thiodiglycol. Dilute to within a few ml of a liter with deionized water and mix. Adjust pH to 2.2 using HCl or NaOH as needed. Dilute to a liter with deionized water and mix. Procedure: 1. Weigh duplicate 50 mg samples into 250 ml round bottom flasks. Add approximately 100 mg sodium thioglycollate to one sample in each set. 2. Add approximately 25 ml of 6N HCl to each flask and cover by inverting a small beaker over each. 3. Autoclave for 16-18 hrs at 1210 C (15 psi) 4. Evaporate the sample to dryness on a rotary evaporator. 5. Dissolve the samples with 1 ml sodium citrate buffer (pH 2.2) for each estimated % protein in the sample. 6. Filter the samples using Whatman #2 paper and collect the filtrate in vials (samples must be clear). 45 7. Adjust pH to 2.2 if necessary. 8. Allow the samples to stand overnight in the refrigerator before submitting them for analysis. Refilter or draw off a clear sample from each vial if necessary. Amino acids as percent of protein were determined by comparison with standards. RESULTS The first step in this investigation was theproxi mate analysis of the seed of the six species (Table 1). Each exhibited an analytical profile characteristic of cucurbits. The decorticated seeds contained (by weight) about 50% oil and 65% protein (defatted embryo). The ash content (4%) and the fiber content (8%), were similar to those of other oil seeds. Three materials were prepared for protein characterization from each cucurbit species. They were: a) a salt-soluble protein extract; b) a globulin fraction; and c) an albumin fraction. The globulin and albumin fractions were components of and isolated from the salt soluble protein extract. The extracts of all species were characterized by the following sequence of biochemical methods. 1. Isoelectric focusing . Proteins were separated into groups according to their isoelectric point. Staining was employed to visualize individual components (Figure 3). 46 Table 1 . Proximate Composition of Seed Embryos (Dry·Weight Basis). Species % of Moisture % of Fat % of Protein % of Ash % of Fiber APODANTHERA UNDULATA 4.55 51.30 . 74.25 3.82 9.33 CUCURBITA DIGITATA 4.72 49.77 76.93 4.52 7.18 CUCURBITA FOETIDISSIMA 4.61 48.92 63.73 5.16 8.48 CUCURBITA MOSCHATA 4.22 46.28 64.41 4.62 9.15 CUCURBITA PALMERI 4.51 45.70 61.71 6.22 7.52 CUCURBITA SORORIA 3.74 50.80 61.45 .4.58 12.23 Fat, protein, ash and fiber determined on dry weight basis; protein, ash and fiber determined on fat-free basis. ~ .....:] 48 F A B c D E Figure 3. O'Farrell Isoelectric Focusing Gels of Salt Soluble Proteins of A: A. undulata, B: C. digitata , C: C. foetidi~sima, D: C. mosEhata, E: C. palmeri~ F: C. sororia. 49 2. Two dimensional analysis Isoelectric focusing was applied to fresh samples to separate proteins into groups without staining. Those groups were then separated on the basis of molecular weight by the application of SDS polyacrylamide gel electrophoresis, a procedure which denatures protein and may produce protomers. Staining was used for visualization. The globulin and albumin fractions were characterized by the following sequence of methods: 1. SDS-polyacrylamide gel electrophoresis Separation was made on the basis of molecular weight of denatured proteins, possibly as pro tomeric units. Staining was used for visualization. 2. Gel filtration (molecular sieve separation) Separation was made on the basis of molecular weight without denaturation. Components were detected by spectrophotometry. 3. Amino acid analysis. Isolation of Albumins and Globulins The salt-soluble proteins, albumins and globulins, usually constitute 80 - 95% of the family Cucurbitaceae 50 seed proteins (Alekseeva, 1958 and Castaneda-AguIlo, 1948). One purpose of this study was to isolate pure albumin and globulin fractions with no regard for the yield recovered. Therefore, cold lipid extraction, and the use of a cellu- lose layer for filtration and dialysis for a long period, were factors which definitely reduced the amount of protein recovered as shown in Tables 2 and 3. Also, the small amounts of albumin recovered relative to globulin can be seen in Table 3. Characterization of the Salt-Soluble Protein Extracts Isoelectric Focusing The isoelectric'patterns were arbitrarily divided into groups according to the pH range of the isoelectric points. The patterns of ~. digitata and ~. foetidi$sima were very similar (Figures 4 and 5). Both patterns could be broken into three groups. For C. digitata, the majority of the proteins fell in the first group between pH 5.19 to 6.14 and consisted of six distinct bands. Similarly, the first group ~. foetidissima proteins consisted of four bands and fell in the pH range 4.82 to 5.82. The second group consisted of two almost identical peaks between pH 6.58 to 7.00 for ~. digitata and pH 6.36 to 6.86 for ~. foetidissima. The third group consisted of three almost identical peaks Table 2. Recovery Data for Salt Soluble Proteins. Weight Weight of Total Volume Protein in Total of the Defatted of the Salt the Salt Protein Species Sample (gm) Sample (gm) Extract (m!) Extract (m!) (gm) APODANTHERA UNDULATA 20.00 7.50 .60 27.19' 1.631 CUCURBITA DIGITATA 47.00 33.6 206.4 16.15 3.330 CUCURBITA FOETIDISSIMA 16.00' 10.97 85.0 . 15.70 1.335 I CUCURBITA MOSCHATA 20.00 10.27 58.90', 19.84 1.150 CUCURBITA PALMERI· : 9.30 5.43 35.0 20.73 '0.726 CUCURBITA SORORIA 20.00 8.95 70.0 14.90 1.043 CJ1. f-l Table 3. Recovery Data for Albumin and Globulin Fractions. Weight of Weight of Albumin + Species Albumin (gm) Golbulin (gm) Globulin (gm) APODANTHERA UNDULATA 0.123 1.142 1.2650 CUCURBITA DIGITATA 0.177 2.53 2.7070 CUCURBITA FOETIDISSIMA 0.1374 1.0817 1.2191 CUCURBITA MOSCHATA 0.055 0.475 0.530 CUCURBITA PALMERI 0.048 .0.3074 0.3554 CUCURBITA SORORIA 0.0744 0.2026 0.2170 CJl ~ 53 + I I I 9.0 ao 7.0 6.0 5.0 4.0 3.5 pH Figure 4. Electropherogram Component Pattern of C. digitata Salt-Soluble Proteins after Isoelectric Focusing in Disc Gel as Described by O'Farrell (1975). 54 + I I 9.0 ao 7.0 6.0 5.0 4.03.S pH Figure 5. Electropherogram Component Pattern of C. foetidissima Salt-Soluble Proteins after Isoelectric Focusing in' Disc Gel as Described by O'Farrell (1975). 55 pH 7.14 to 7.89 for C. digitata and 7.00 to 8.00 for C. foetidissima. The patterns of C. sororia and ~. palmeri salt soluble proteins were also very similar (Figures 6 and 7). They were divided into three groups; the major group consis ted of eight bands and fell between pH 4.54 to 5.72 for the two species. The second group consisted of five bands and fell between pH 6.24 to 7.77. Both patterns of these two species showed a third group of light bands in the very acidic region. The isoelectric pattern of ~. moschata salt-soluble proteins also could be broken into three groups (Figure 8). The major group consisted of seven bands; these bands fell between pH 5.00 to 5.91. The second group also was made up of seven bands within the pH range 6.00 to 7.00. The third group which contained five bands was within the pH range 7.24 - 8.29. The pattern of A. undulata could be broken into two groups. One group fell between pH 4.43 - 5.48 and con sisted of six distinct bands. The major part of the salt soluble proteins of this species, as judged by the peak heights, fell in this group. The other group fell between pH 5.62 to 6.48 and was made up of four bands (Figure 9). The isoelectric focusing pattern of !. undulata was clearly different from the patterns of the five Cucurbita species studied. 56 + I I 9.0 ao 7.0 6.0 5.0 4.0 3.5 pH Figure 6. Electropherogram Component Pattern of C. sororia Salt-Soluble Proteins after rsoelectric Focusing in Disc Gel as Described by O'Farrell (1975). 57 I I 9.0 ao 7.0 6.0 5.0 4.0 3.5 pH Figure 7. Electropherogram Component Pattern of C. palmeri Salt-Soluble Proteins after Isoelectric Focusing in Disc Gel as Described by O'Farrell (1975). 58 + rA.-.-....' .. ~~r~--- I I 9.0 ao 7.0 6.0 5.0 4.0 3.5 pH Figure 8. Electropherogram Component Pattern of C. moschata Salt-Soluble Proteins after rsoelectric Focusing in Disc Gel as Described by O'Farrell (1975). 59 ,t -I' III I I t I ! I 'II I I I ! + ~- I I 9.0 ao 7.0 6.0 5.0 4.0 3.5 pH Figure 9. Electropherogram Component Pattern of A. undulata Salt-Soluble Proteins after Isoelectric Focusing in Disc Gel as Described by O'Farrell (1975). 60 Two Dimensional Analysis The salt-soluble protein extracts that were focused in gel columns were analyzed by SDS-polyacrylamide gel electrophoresis in the second dimension to further- resolve, according to their molecular weights, the subunits of the proteins that were separated according to their charge in the first dimension. All five Cucurbit species studied had the same general protein patterns (Appendix 2-6). The relative mobilities were the same for the bands detected which indicates approximately equal molecular weights. The number of bands resolved was the same (5). The staining technique was not sufficiently sensitive to detect trace quantities of protein which were also undoubtedly present. The two dimensional pattern of A. undulata salt-soluble proteins was clearly different from those of Cucurbi ta species, (Appendix 1). The bands resolved had higher relative mobilities. which means lower molecular weights, and the horizontal distances between the bands were also different. These horizontal distances had originated in the isoelectric focusing gel columns. Characterization of Albumin and Globulin Fractions SDS-Polyacrylamide Gel Electrophoresis Electrophoresis in the 2-mercaptoethanol SDS-PAGE system of the globulins of the six species studied and subsequent examination of the data revealed that all 61 five Cucurbita species had six distinct bands with the same relative mobility, which implies similar molecular weights. The globulin pattern of !. undulata was clearly different. Band number 2 was absent, and the pattern had a very distinct band, number 4, which was absent in the Cucurbita patterns (Figure 10). The electropherogram of the gel columns (Appendix 6to 12) revealed the minor components in addition to the major ones. Direct examination of the gel columns (Figure 11) and the comparison of the electropherogram (Appendtx 13 - 18) of the albumin patterns obtained from the six species studied revealed no close similarities either in the number or kinds of bands present. The maximum number of subunits (25) was detected in C. foetidissima albumin, while the minimum number (19) was detected in that of ~. palmeri. In general, the electropherogram albumin patterns of the five cucurbit species could not be distinguished from that of A. undulata. Molecular Weight Determination by Gel Filtration The gel filtration patterns of the globulins consisted of four major peaks, except that of ~. moschata which contained five peaks. Molecular weights ranged from 12,000 to 660,000 (Table 4). The fraction 12,000 or'13,OOO 62 A B c D E F Rm 0.0 1 2 0.5 3 4 5 6 7 1.0 Figure 10. Savoy SDS-Gel Electrophoresis of Globulin Fractions. A: C. digitata, B: C. foetidissima, C: C. moschata,-D: A. undulata,-E: C. palmeri, F: C. sororia. 63 A B c D F Figure 11. Savoy SDS-Gel Electrophoresis of Albumin Fractions. A: C. digitata, B: C. foetidissima, C: C. moschata,-D: A. undulata,-E: C. palme ri, F: C. sororia. Table 4 . Molecular Weight Determination of Globulin Fractions by Gel Filtration on Sephacryl 8-300. Molecular Species Peak No. l 1 0.062 525,000 2 0.187 204,000 CUCU.RBITA DIGITATA 3 0.495 19,000 4 0.544 13,000 1 0.062 524,000 2 0.136 309,000 CUCURBITA FOETIDISSIMA I 3 0.258 95,000 4 0.544 13,000 1 0.124 339,000 2 0.272 107,000 CUCURBITA MOSCHATA 3 0.384 45,000 4 0.544 13,000 5 0.561 12,000 1 0.037 660,000 380,000 CUCURBITA PALMERI 2 0.111 3 0.210 174,000 4 0.557 12,000 1 0.074 501,000 81,000 CUCURBITA SORORIA 2 0.309 3 0.433 30,000 4 0.557 12,000 (j) ~ 65 was present in all species; otherwise there was no similarity either in the molecular weight or in the relative proportion of each fraction. Figures (Appendix 19 to24) show that all four fractions of A. undulata globulin were present in almost equal amounts as shown by their absorbance intensities. Fraction number 2 (204,000) in ~. digitata globulin was the major fraction, while the number 3 fractions (95,000), (45,000) and (174,000) were major components in ~. foetidissima, ~. moschata and ~. palmeri globulins, respectively. Fraction number 1 (501,000) was the major fraction of ~. sororia globulin. The gel filtration patterns of the albumin fractions consisted of three major peaks except those of A. undulata and C. sororia which contained four peaks. The molecular weight values ranged from 11,000 to 446,000 (Table 5) and were substantially lower than those of globu lins. Fraction numbers 2 and 3 (19,000) and (11,000), respectively; were the major fractions in C. digitata albumin (Appendix 19 to 24). Fraction number 3 (15,000) was the major fraction in ~. foetidissima albumin, and fraction number 1 (229,000) was present in substantial amount. The three fractions of C. moschata albumin were present in almost equal amounts. Fraction number 2 (114,000) was the major fraction in ~. palmeri albumin, and fraction number 1 (304,000) and fraction number 3 (14,000) were present in equal amounts. Table 5. Molecular Weight Determination of Albumin Fractions by Gel Filtration on 8ephacryl 8-300. Molecular KAV Species Peak No. Weights 1 0.186 209.000 APODANTHERA UNDULATA 2 0.309 80,000 3 0.433- 30.000 4 0.532 14,000 1 0.322 145.000 CUCURBITA DlGITATA 2 0.495 19,000 3 0.557 11,000 1 0.173 229,000 CUCURBITA FOETIDISSIMA 2 0.334 66,000 3 0.520 15,000 1 0.161 257,000 CUCURBITA MOSCHATA 2 0.297 87,000 3 0.444 27,000 1 0.136 309,000 CUCURBITA PALMERI 2 0.260 114,000 3 0.532 14,000 1 0.087 446,000 2 0.198 186,000 CUCURBITA SORORIA 3 0.421 33,000 4 0.544 13,000 The C. sororia albumin profile showed that fraction number 3 (33,000) was the major fraction; the others were present in almost equal amounts. Amino Acid Analysis The globulin fractions of all species showed similar amino acid profiles (Table 6). Arginine, glutamic acid and aspartic acid were the most abundant amino acids. Cysteine was the least abundant and the contents of threonine, methionine and tyrosine were comparatively low. The albumin fractions of all species also showed similar amino acid profiles (Table 7). Arginine and glutamic acid contents were high, while those of isoleucine, leucine, tryptophan, phenylalanine and aspartic acid were relatively low. The content of cysteine was higher than that in globulins except for C. digitata and C. foetidissima. Table 6'. Amino Acid Content of Globulin Fractions Expressed as Percent Protein. Speclss Ly. HI. Arg Asp Thr Ser Glu Pro Gly AlII CVI VIII Mat liB Leu Tyr Pho APODANTHERA UNDULATA 3.27 2.64 16.829.37 2.68 4.61 17.91 2.83 4.66 4.79 1.16 6.24 2.86 3.94 7.S8 2.70 6.87 CUCURBITA DlGlTATA 3.68 2.23 17.83 9.62 2.61 4.85 20.46 2.64 4.84 4.46 0.34 6.16 2.66 4.24 7.47 2.92 6.28 CUCURBITA FOETIDISSIMA 3.03 2.04 17.47 8~q5 2.33 4.39 19.72 2.61 6.16 4.18 0.68 4.78 2.38 3.94 7.02 2.73 6.74 CUCURBITA MOSCHATA 3.49 2.34 18.769.97 2.74 6.10 21.60 2.66 4.83 4.71 0.39 6.50 2.76 4.27 8.12 3.07 6.74 CUCURBITA PALMERI 3.43 2.29 18.00 9.78 2.69 S.01 20.69 2.66 4.98 4.63 0.00 6.34 2.64 4.11 7.80 3.06 6.73 CUCURBITA SORORIA 3.59 2.29 18.92 10.03 2.73 6.20 22.19 2.62 6.26 4.66 0.11 6.43 2.86 4.22 8.07 3.07 6.88 (j) OJ Table 7. Amino Acid Content of Albumin Fractions Expressed as Percent of Protein. Species LVI HI, Arg Asp Thr Ser Glu Pro GlV Ala eVa Vel Mot lie Leu Tyr PhD APODANTHERA UNDULATA 4.35 1.94 14.56 7.02 1.87 3.44 19.21 2.93 7.17 3.46 3.37 2.71 * 2.47 6.88 1.92 2.07 CUCURBITA DIGITATA 5.97 2.09 11.12 5.96 2.35 3.72 15.32 2.50 11.05 3.17 0.33 3.37 1.03 2.30 4.41 1.97 1.64 CUCURBITA FOETIDISSIMA 4.83 1.70 9.49 4.85 2.05 2.96 12.29 2.06 8.05 2.88 0.41 2.55 1.12 1.89 3.38 1.67 1.31 CUCURBITA MOSCHATA 6.29 1.98 14.84 8.66 3.00 4.03 20.73 3.42 ~p.77 0.49 2.38 0.46 1.87 3.24 6.59 2.41' 2.68 CUCUR81TA PALMERI 8.03 2.62 20.529.81 3.37 5.64 25.79 3.41 14.14 5.50 4.01 4.67 2.62 3.32 7.85 3.17 2.95 CUCURBITASORORIA 6.64 2.08 14.63 7.76 2.90 4.40 19.28 2.92 11.62 4.29 2.13 3.71 1.84 2.71 6.24 2.68 2.27 * Not determined. m (.l) DISCUSSION Savoy had suggested in 1976 the existence of a universal standard protein electrophoretic pattern for all peanut genotypes based on analysis of defatted seed meal. In cucurbits, salt-soluble protein makes up 80% or more of the total storage protein. Thus, it is a reasonable assumption that there is a universal standard protein pattern for Cucurbita storage protein, which could be found in the salt-soluble extract. This study involved analyzing the salt-soluble proteins by several biochemical techniques to learn whether there is support for the existence of a universal standard protein pattern for Cucurbita species. Isoelectric Focusing Initially the isoelectric focusing patterns of the salt-soluble.proteins in the salt extract as a whole were established. Since isoelectric focusing is not a molecular sieving medium, the patterns provided a good picture of the charge homogeneity of the proteins from each species. The patterns were arbitrarily divided into groups according to the pH range into which they fell. The close 70 71 similarity of the isoelectric focusing patterns of the salt- ~oluble proteins of ~. digitata and~. foetidissima sup l~rts the numerical taxonomy reported by Bemis et al. (1970). The phenogram of numerical taxonomy relationships showed a correlation coefficient of + 0.1 for these two species. Similar correlation was apparent in the patterns of ~. palmeri and ~. sororia; the phenogram in that instance showed a correlation coefficient of + 0.3 for those species. The pattern of C. moschata salt-soluble proteins resembled those of all the species studied except A. undulata, but it was closer to the patterns of ~. palmeri and C. sororia than those for ~. digitata and ~. foetidissima. This observation is also in agreement with the numerical taxonomy. The similarity of the ~. moschata proteins pattern to those of 'the other four Cucurbita species supports the suggestion of Whitaker et al. (1975) that C. moschata "occupies a key role as the putatiYe ancestor of the genus". A'consideration of the number of bands and their pH range indicates that the isoelectric focusing patterns of the salt-soluble proteins of Cucurbita species are similar, but all differ from the pattern of ~. undulata. The patterns of the Cucurbita species were divided generally into three groups of bands within the pH range 4.54 to 8.29, while the pattern of ~. undulata was broken into two groups of bands within the range 4.43 to 6.48. 72 SDS-Polyacrylamide Gel Electrophoresis Globulin Fractions The electrophoretic patterns that all five Cucurbita species exhibited agree generally with other investigators' findings. PichI (1976) reported similar electrophoretic behavior of the globulin polypeptides from five cucurbit species using SDS-gel after cleavage with performic acid. Byerrum et,al. (1950) and Fuerst et al. (1954) studied the electrophoretic mobility in acetate buffer, pH 4 and 3.9, of f. pepo and f. maxima, respectively. They reported that the globulin was essentially homogenous at those pH values. The globulin pattern of C. foetidissima was slightly different from the other four species of Cucurbita in the fact that band number 3 was less concentrated and appeared to have split into two smaller bands. This slight change was probably a result of a very short period of germination to which this seed sample had been subjected, which would have caused a rapid mobilization and catabolism of the globulin storage proteins (Youle et al., 1979). Albumin Fractions No electrophoretic pattern common to all five Cucurbita species was found which distinguished them from A. undulata. 73 A possible explanation of the absence of a general albumin pattern is the lesser abundance of albumin in the storage proteins. Also, some investigators have not recognized albumin as storage protein but have considered it as a mixture of enzymes and other metabolic proteins (Altshul et al., 1966; Boulter et al., 1971; Ashton, 1976). Others have described it as atypical seed storage protein because of its water solubility and low molecular weight (Youle et al., 1979). Molecular Weight Determination Globulin Fraction All the globulin fractions studied were eluted from the gel filtration column in four peaks except those of C. moschata. The fact that ~. moschata globulins were eluted in five peaks can be explained if peaks number 1 and number 2 were a single protein which had been dissociated under the experimental conditions. Alternatively, it could be a result of evolutionary changes, because ~. moschata is considered the putative ancestor of the genus (Whitaker et al., 1975). The difference in the number and molecular weight of the fractions resolved from the globulin of each specie is not surprising since many investigators have reported conflicting results. Mourgue et al. (1968) found three components in the globulin isolated from C. maxima, 74 with a major fraction of molecular weight 340,000. PichI (1976) reported only one fraction of globulin eluted from Sephadex G-200 for all six cucurbit species he studied. Blagrove et al. (1980) found two fractions of globulins from the four species they studied, the molecular weight of the major and minor component being 325,000 and 630,000, respectively. Baudet et al. (1977) reported at least four different fractions of globulins from sunflower seeds with molecular weights of 13,000, 25,000, 43,000 and 95,000. Albumin fraction Except for the presence of the lower molecular weight fractions, 11,000 - 14,000, there was no similarity in the gel filtration patterns of albumins studied either in molecular weight or in the relative proportion of each fraction. The same conclusion that has been drawn from the albumin electrophoretic patterns can be drawn from the albumin gel filtration patterns; neither displayed any similarity among the five Cucurbita species. The reason may be that albumins are less abundant and may be better 'studied as enzymes and other metabolic proteins than as storage proteins. The albumin fraction in storage proteins has not received as much attention as the globulin fraction. However, there have been reports about the physico-chemical properties of seed albumins from different plant families 75 and species. Baudet et al. (1977) isolated three albumin fractions from sunflower seeds. Their molecular weights in the native form were 13,000, 48,000 and 50,000. Youle et al. (1978 and 1979) isolated albumins from castor bean and found the molecular weight of the denatured albumin to be around 11,700. Amino Acids Analysis The globulin and albumin fractions of all six species studied showed amino acid profiles characteristic of storage proteins (Boulter et al., 1971; Derbyshire et al., 1976). The relative amounts of the most abundant amino acids were similar to those of other seed globulins (PichI, 1976; Youle et al., 1978). In general, the amino acid profiles which the globulin and albumin fractions exhibited were analogous to the globulin and albumin profiles of other plant families (Boulter et al., 1971). This makes difficult a distinction among families or species on the basis of amino acid profile. The results of isoelectric focusing and two dimen sional analysis of the salt-soluble proteins, and of the electrophoresis of globulins reveal that a standard salt soluble protein isoelectric focusing pattern and a globulin electrophoretic pattern probably exist for all Cucurbita species. 76 Neither the gel filtration patterns of the globulins and albumins nor the amino acid profiles of these two fractions from the five Cucurbita species studied resemble each other. Moreover, the fact that they are not distinct from those of A. undulata suggests that no general patterns based on gel filtration or amino acid profile exist for the globulins or the albumins of Cucurbita species. SUMMARY AND CONCLUSION Salt-soluble protein extracts and albumin and globulin fractions from A. undulata, ~. digitata, ~. foetidissima, ~. moschata, ~. palmeri and C. sororia were prepared from the decorticated seed meals using 2M NaCI buffer. Albumins were separated from globulins by dialysis and centrifugation. The physico-chemical properties of each material were studied. The isoelectric focusing pattern of each salt extract was established. Clear similarities were apparent among the patterns of the five Cucurbita species which were very different from that of !. undulata. The electrophoretic patterns of the Cucurbita globulin fractions were also very similar and clearly different from that of A. undulata. Two dimensional analysis of the salt extracts also revealed similarity among the five cucurbit species and a clear difference from that of A. undulata. No distinction among the albumin fractions could be made on the basis of the electrophoretic patterns; molecular weights determined by gel filtration did not successfully differentiate albumins or globulins. 77 78 The albumin and globulin fractions generally exhibited amino acid profiles characteristic of other storage proteins. Arginine, glutamic acid and aspartic acid were the most abundant amino acids. The isoelectric focusing patterns and electro phoretic patterns of the globulin fractions support the idea that a universal globulin pattern exists for Cucurbita species. Future study should involve more species and focus on globulin electrophoresis and the salt-soluble extract isoelectric focusing to confirm the suggested pattern. APPENDIX A 79 80 0-- -~------~------~~------ Figur Al. Two Dimensional Analy i ·of A. undula a Salt-Solub Prot ins. First Dim n. ion: I soeJe tric Focus:ng in Dis G l from Righ to L ft. Sncond Dimension : SDS -Polya rylamide Gradi nt G J El ctropbor sis from Top to Bo ttom . 81 Two Dimonsio1 al Analysis of C. digi ata Sa. L- Sol ub1e Prot - ins . Fir t Dimen -:Lon: I o 1 tri Fo u .·ing in Dis G 1 £rom Rj_gh t to L Ft. s ~ ond Dim - nsion: SDS-Polya rylamtde Gradie n t G 1 E1 trophor sjs from To j_J to Bottom . 82 0------~~--~------~------ Figur Aa Two Dim nsi ysis of C. foeti_dissima Salt- Solubl Prot ins . Fir.~ Dim nsion: Fo u . ing in Dis Gel from Right and Dimension : SDS-Pol. a rylamide G l Ele trophor ,s i s from Top to 83 0-- ·~------~------~------~~---- Figure A4. Tw o Dim nsion al An al si of C. mos ha a Salt-:--Solubl . Proteins . F · rst Dimension : I soele Lri ./o u sin g i n Di.) G l from Ri r~ht Lo L T t . S e o n d Dj_r,_·L nsion: SDS-Polya rylamide Gradi n t G l Ele trophor is from Top to l3 ttom. 84 Figure A5 . Two Dimensio1: a 1 Ana lys j_. o ·r C. palm ri Sal t - -Solubl Prot in . :Firs Ditnnsion : I oeJGctri Fo using in Disc G 1 from Ri~hL to L I . S ond L ·m =>n ,·i 1 : SDS - Polya rylami.d e Gradi nt Gel El trophor sis from Top o Bo tom. 85 0 11 12 Figur A6. Two Dimc nsj_o -: al An aly ._ is of g. soyq_ria Salt- Soluble Proteins . First Dime nsion : Iso l ctri · Fo u sing in Disc Ge l Jrom .u.igbt to L ft. S cond Dj_m n ion : SDS - Polya rylamide Gradient G l El trophro s i s from Top to Bottom . 86 1 ~ + Figure A7. E1ectropherogram Component of A. undu1ata Globulin Fraction after sDS-Polyacry1amide Electrophoresis as Described by Savoy (1981). 87 + Figure A8. Electropherogram Component of C. digitata Globulin Fraction after sDS-Polyacrylamide Electrophoresis as Described by Savoy (1981). 88 1D ~ I + l Figure A9. Electropherogram Component of C. foetidissima Globulin Fraction after sDS-Polyacrylamide Electrophoresis as Described by Savoy (1981). 89 1.0 as I I . ~ FigureAlO. E1ectropherogram Component of C. moschata Globulin Fraction after sDS-Polyacry1amide Electrophoresis as Described by Savoy (1981). 90 1D + Figure All Electropherogram Component of C. palmeri Globulin Fraction after sDS-Polyacrylamide Electrophoresis as Described by Savoy (1981). 91 1D J + Figure A12. Electropherogram Component of C. sororia Globulin Fraction after sDS-Polyacrylamide Electrophoresis as Described by Savoy (1981). 92 ~o Q9 :~ t pJ i ,J \ i Figure Al3. E1ectropherogram Component Pattern of A. undu1ata Albumin Fraction after SDS-Po1yacry1amideE1ectrophoresis as Described by Savoy (1981). 93 1D 09 08 07 OS uw Z =~ as ~m C OA ) + Figure Al~ Electropherogram Component Pattern of C. digitata Albumin Fraction after SDS-PolyacrylamireElectrophoresis as Described by Savoy (1981). 94 o.a 0.7 o.s + Figure A15. Electropherogram Component Pattern of C. foetidissima Albumin Fraction after SDS-PolyacrylamideElectrophoresis as Described by Savoy (1981). 95 1.0 Q.9 . 0.8 u z'" :5 DC ~m < + Figure Al6. E1ectropherogram Component Pattern of C. moschata Albumin Fraction after SDS-Po1yacrY1amideE1ectrophoresis as Described by Savoy (1981). 1D + Figure A17. Electropherogram Component Pattern of f. palmeri Albumin Fraction aft r SDS-PolyacrylamideElectrophores1s7 as Described by Savoy (1981). 97 1D + Figure Al& Electropherogram Component Pattern of C. sororia Albumin Fraction after SDS-PolyacrylamideElectrophoresis as Described by Savoy (1981). 98 1.0 0.9 0.8 W 0.7 ~ 0.6 ALBUMIN Ci3 0.5 ~ 0.4 en CD 0.3 « 02 0.1 o 3 4 5 6 7 8 9 10 II 12 TIME (hrs) 1.0 0.9 0.8 W 0.7 GLOBULIN ~ 0.6 Ci3 0.5 0:: o 0.4 en CD 0.3 « 02 0.1 o 3 4 5 6 7 8 9 10 II 12 TIME (hrs) Figure Ale. Molecular Weight Chromatography of A. undulata Albumin and Globulin Fractions from Gel Filtration on Sephacryl S-300. 99 1.0 0.9 0.8 W 0.7 ~ 0.6 ALBUMIN ~ 0.5 0: 0 0.4 en CD 0.3 2 I I I I I I I I I I 3 4 5 6 7 8 9 10 II 12 13 14 15 16 TIME (hrs) 1.0 0.9 0.8 W 0.1 ~ 0.6 ~ 0.5 GLOBULIN ~ 0.4 ~ 0.3 <{ 02 0.1 O~------~ 3 4 5 6 1 8 9 10 II 12 TIME (hrs) Figure A2Q. Molecular Weight Chromatography of C. digitata Albumin and Globulin Fractions from Gel Filtration on ~ephacryl 8-300. 100 1.0 0.9 W 0.1 U o.S Z <{ o.S ALBUMIN CD a:: 0 0.4 ~Q.3 <{ 0.2 0.1 0 1 1 1 I I I I I 1 I .2 3 4 S S 1 8 9 10 II 12 13 14 IS IS TIME (hrs) 1.0 GLOBULIN I· ! I I I I I I I 4 5 6 7 8 9 10 \I 12 13 14 15 16' TIME (hrs) FigureA21. Molecular Weight Chromatography of C. foetidissima and Globulin Fractions from Gel Filtration on 8ephacryl 8-300. 101 1.0 0.9 O.B W 0.7 ~ O.S ~ 0.5 ~ 0.4 ALBUMIN (f) (D 0.3 « 0.2 0.1 ~ o N\ I I I I I I I I 3 4 5 6 7 8 9 10 II 12 TIME (hrs) 1.0 0.9 O.B W 0.7 ~ O.S GLOBULIN ~ 0.5 ~ 0.4 ~ 0.3 « 0.2 0.1 o 4 5 6 7 8 9 10 II 12 TIME (hrs) Figure A22. Molecular Weight Chromatography of C. moschata and Globulin Fractions from Gel Filtration on Sephacryl S-300. 102 1.0 0.9 O.B W 0.7 ~ 0.6 ALBUMIN <:[ (l) 0.5 ~ 0.4 en (l) 0.3 « 02 0.1 oL------~ 3 4 5 6 7 B 9 10 II 12 TIME (hrs) 1.0 0.9 O.B W 0.7 ~ 0.6 ~ 0.5 GLOBULIN ~ 0.4 ~ 0.3 « 02 O~·------J 3 4 5 6 7 B 9 10 II 12 TIME (hrs) Figure A23. Molecular Weight Chromatography of f. palmeri and Globulin Fractions from Gel Filtration on Sephacryl S-300. 103 1.0 0.9 0.8 W 0.7 ALBUMIN ~ 0.6 ~ o.S ~0,4 ~Q.3 1.0 W 01 U 0.6 Z I I I I I I I I 5 6 7 8 9 10 II 12 13 14 IS 16 TIME (hrs) Figure A24. Molecular Weight Chromatography of C. sororia and Globulin Fractions from Gel Filtration on 8ephacry1 8-300. LITERATURE CITED Alekseeva, M. V. (1958). The seed proteins of Cucurbita. Trudy Pokhim. Prirod. Soedin., Kishinev. Gos .. Univ:103-108. Chern. Abstr. 54:1667f. 1960. Alekseeva, M. V. (1961). A comparative study of nitrogen containing substances in the seeds of some representatives of the gourd family. Trudy Pokhim Prirod. Soedin., Kishinev. Gos. Univ:37-43. Chern. Abstr. 58:10514d. 1963. Altshul, A. M., Yatsu, L. Y., Ory, R. L., and Engleman, E. M. (1966). Seed proteins. Ann. Rev. Plant Physiology. 17:113. Ashton, F. M. (1976). Mobilization of storage proteins of seeds. Ann. Rev. Plant Physiology. 27:95. Barker, R. D., Derbyshire, E., Yarwood, A., and Boulter, D. (1976). 'Purification and characterization of the major storage proteins of Phaseolus vulgaris seeds, and their intracellular and cotyledonary distri bution. Phytochemistry 15:751. Bandel, J., and Mosse, J. (1977). Fractionation of sun flower seed proteins. J. Am. Oil Chern. Soc. 54: 82A. Bemis, W. P., Rhodes, A. M., Whit~ker, T. W., and Carmer, S. C. (1970). Numerical taxonomy applied to Cucurbita relationships. Amer. J. Bot. 57:404. Bemis, W. P., Berry, J. W., and Weber, C. W. (1977). Breeding, domestication, and utilization of the buffalo gourd. In Non-conventional Proteins and Foods: Proceedings of NSF grantee-users conference, October 18-20, 1972. Berry, J. W., Bemis, W. P., Weber, C. W., and Philip, T. (1975). Cucurbit root starches: Isolation and some properties of starches from Cucurbita foetidissima HBK and Cucurbita digitata Gray. J. Agr. Food Chern. 23(4):825. 104 105 Berry, J. W., Weber, C. W., Dreher, M. L., and Bemis, W. P. (1976). Chemical composition of buffalo gourd, a potential food source. J. Food Sci. 41:465. Berry, J. W., Scheerens, J. C., and Bemis, W. P. (1978). Buffalo gourd roots: Chemical composition and seasonal changes in starch content. J. Agr. Food Chern. 26:354. Betschart, A. A. (1974). Nitrogen solubility of a·lfalfa protein concentrate as influenced by various factors. J. Food Sci. 39:1110. Blagrove, R. J., and Lilley, G. G. (1980). Characteriza tion of cucurbitin from various species of the Cucurbitaceae. Eur. J. Biochem. 103:577. Boulter, D. and Derbyshire, E. (1971). Taxonomic aspects of the structure of legume proteins. In J. B. Harborne, D. Boulter and B. L. Truner, eds. Chemotaxonomy of the Leguminosae. Academic Press New York. pp. 285-308. Byerrum. R. V., Brown, S. A., and Ball, C. D. (1950). The action of electrolytes on oxalacetic decarboxylase from Cucurbita seeds. Arch. Biochemistry. 26:442. Castaneda-Agullo, M., and Salazar, V. P. (1948). Investiga tion on crystallized globulins of pumpkin seeds. Anales escuela Nacl. Cienc. BioI. 5:65. Chern. Abst. 43:5450i. 1949. Cerletti, P., Fumagalla, A., and Venturin, D. (1978). Protein composition of seeds of Lupinus albus. J. Food Sci. 43(5):1409. Chambliss, O. L., and Jones, C. M. (1966a). Cucurbitacins: Specific insect attractants in Cucurbitaceae. Science 153:1392. Chambliss, O. L. and Jones, C. M. (1966b). Chemical and genetic basis for insect resistance in cucurbits. J. Am. Soc. Hort. Sci. 89:394. Davis, B. J. (1964). Disc. Electrophoresis-II. Method and Application to Human serum proteins. Ann. NY Acad. Sci., 121:404. 106 Derbyshire, E., Wright, D. J., and Boulter, D. (1976). Legumin and vicilin storage proteins of legume seeds. Phytochemistry 15:3. Dreher, M. L. (1979). Ph.D. dissertaion. Univ. of Arizona. Dreher, M. L., Weber, C. W., Bemis, W. P., and Berry, J. W. (1981). Nutritional evaluation of buffalo gourd root starch. Nut. Rep. Int. 23:1-8. Earle, F. R., and Jones, Q. (1962). Analysis of seed samples from 113 plant families. Econ. Bot. 16: 221. Fuerst, C. R., McCalla, A. G., and Colvin, J. R. (1954). Electrophoretic and sedimentation characterization of crystalline squash seed globulin. Arch. Bio chern. Biophys. 49:207. Gathman, A. C., and Bemis, W. P. The history, biology and chemistry of the buffalo gourd. (manus'cript in print). Grula, E. A., and Savoy, C. F. (1971). A detergent polyacrylamide gel system for electrophoretic resolution of membrane and wall proteins. Biochem. Biophys. Res. Commun. 43:325. Hill, J. E., and Briedenbach, R. W. (1974). Protein of soy bean seeds. I. Isolation and characterization of the major components. Plant Physiol. 53:742. Jacks, T. J., Hensarling, T. P., and Yatsu, L. Y. (1972). Cucurbit seed. I. Characteristics and use of oils and protein. A review. Econ. Bot. 26:135. Jones, D. B., and Gersdorff, C. F. (1923). Proteins of the cantaloupe seed, Cucumis melo. J .. BioI. Chern. 56:79. ---- Joubert, F., (1955). The proteins of Lupin seed (Lupinus luteus). Bioch. Biophs. Acta. 17:446. Khoury, N. N., Dagher, S., and Sawaya, W. (1982). Chemi cal and physical characteristics, fatty acid composition and toxicity of buffalo gourd oil J. Food Tech. 17:19. 107 Kirshnan, P. S., and Krishnaswamy, T. K. (1939). CLVII. Proteins and other nitrogenous constituents of watermelon seeds, Citrullus vulgaris, Biochemical J. 33:1284. . Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T . Nature 227:680. 4 Lawhon, J. T., and Carter, C. M. (1971). Effect of processing method and pH of precipitation on the yields and functional prop~rties of protein isolates from glandless cotton seed. J. Food Sci. 36:372. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the folin phenol reagent. J. BioI. Chern. 193:265. Lyne, F. A. (1976). Determination of starch in various products. Examination and analysis of starch and starch products. J. A. Radley, ed. Applied Science Publishers, Ltd. London. 167 p. Mourgue, M., Baret, R., Renai, J., and Savary, J. (1968). Sur les euglobulines des graines de courge (Cucur bita maxima duch). C. R. Soc. BioI. 162:1128. O'Farrell, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. J. BioI. Chern. 250: 4007. Ornstein, L. (1964). Disc-electrophoresis. I. Background and theory. Ann. NY Acad. Sci. 121: 321. Osborne, T. B. (1892). Crystallized vegetable proteins. American Chemical J. 14:662. PichI, I. (1976). Seed globulins of various species of Cucurbitaceae. Phytochemistry 15:717. Rahma, E. H., and Narasingarao, M. S. (1979). Characteri zation of sunflower proteins. J. Food Sci. 44(2):579. 108 Sabir, ·M. A., Sosulski, F. W., and MacKenzie, S. L. (1973). Gel chromatography of sunflower proteins. J. Agr. Food Chern. 21:988. Savoy, C. F. (1976). Peanut (Arachis hypogael). Seed protein characterization and genotype sample classification using polyacrylamide gel electro phoresis. Biochem. Biophys. Res. Comm. 68(3'): 886. Schoch, T.· J. (1964) . Fa tty substances in starch. Methods in Carbohydrate chern. Roy L. Whistler, ed. Academic Press, New York and London IV:56. Shapiro, A. L., Vinuela, E., and Maizel, J. V. (1967). Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. Biophys. Res. Commun. 28:815. Sharma, G. C., and Hall, C. V. (1971). Influence of cucurbitacins, sugars, and fatty acids on cucurbit ·susceptibility to spotted cucumber beetle. J. Am. Soc. Hort. Sci. 96:675. Sharma, G. C., and Hall, C. V. (1973). Relative attrac tance of spotted cucumber beetle to fruits of fifteen species of Cucurbitaceae. Env. Ent. 2: 154. Smith, R. J. (1964). Determination of ash. Methods in Carbohydrate Chern. Roy L. Whistler, ed. Academic Press, New York and London. IV:41. Smith, E. L., and Greene, R. D. (1947). Further studies on the amino acid composition of seed globulins. J. BioI. Chern. 167:833. Smith, E. L., and Greene, R. D. (1948). The isoleucine content of seed globulins and S-lactoglobulin. J. BioI. Chern. 72:111. Smith, E. L., Greene, R. D., and Bartner, E. (1946). Amino acid composition of seed globulin. J. BioI. Chern. 164:159. 109 Van Soest, P. J. (1963). Use of detergents in the analysis of fibrous feeds. II. A rapid method for the determination of fiber and lignin. J. Assn. Off. Agr. Chern. 46:829. Vasconcellos, J. A., Berry, J. W., and Weber, C. W. (1980). The properties of Cucurbita foetidissima seed oil. J. Am. Oil Chern. Soc. 57:310. Vasconcellos, J. A., Bemis, W. P., Berry, J. W., and Weber, C. W. (1981). The buffalo gourd, Cucurbita foetidissima HBK. as a source of edible oil. In New Sources of Fats and Oils, Am. Oil Chern. Soc. monograph #9. Vickery, H. B., Smith, E. L. Hubbell, R. B., and Nolan, L. S. (1941). Cucurbit seed globulins. I. Amino acid composition and preliminary tests of nutritive value. J. BioI. Chern. 140:613. Wang, J., and Kinsella, J. E. (1976). Functional properties of novel proteins: Alfalfa leaf proteins. J. Food Sci. 41:286. Weber, K., and Osborn, M. (1969). The reliability of mclecular weight determination by dodecyl sulfate-polyacrylamide gel electrophoresis. J. BioI. Chern. 244:4406. Whitaker, T. W. (1968). Ecological aspects of the cultivated Cucurbita. Hort. Science. 3(1):9. Whitaker, T. W., and Bemis, W. P. (1975). Origin and evolution of the cultivated Cucurbita. Bulletin of the Torrey Botanical Club 102(106):362. Whitaker, T. W., and Davis, G. N. (1962). Cucurbits Botany, Cultivation, Utilization. Interscience Publisher, Inc. NY Youle, R. J., and Huang, A. H. (1976). Protein bodies from the endosperm of castor bean. Plant Phys. 58:703. Youle, R. J., and Huang, A. H. (1978). Albumin storage proteins in the protein bodies of castor bean. Plant Phys. 61:13. Youle, R. J., Huang, A. H. (1979). Albumin storage protein and allergens in cotton seeds. J. Agr. Food Chern. 27(3):500.