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Chemical, rheological and ultrastructural properties of a major alkali-soluble protein of rapeseed Gill, Thomas Allan 1976

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I CHEMICAL, RHEOLOGICAL AND ULTRASTRUCTURAL PROPERTIES OF A MAJOR ALKALI-SOLUBLE PROTEIN OF RAPESEED by THOMAS ALLAN GILL B . S c , U n i v e r s i t y o f Guelph, 1970 M . S c , U n i v e r s i t y o f Guelph, 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF FOOD SCIENCE UNIVERSITY OF BRITISH COLUMBIA We a c c e p t t h i s t h e s i s as conforming t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September, 1976 (cP) Thomas A l l a n G i l l , 1976 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d tha t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depa rtment The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date OJ 5 ) /7 7l, ABSTRACT A 12S glycoprotein was i s o l a t e d from commercial rapeseed meal (Brassica campestris) and examined by chemical, microscopical, and rheological methods. The molecular weight of the protein was estimated to be 129,200 daltons by conventional sedimentation equilibrium u l t r a c e n t r i f u g a t i o n although the presence of higher molecular weight material was detected in the preparation. The 12S protein was found to be oligomeric, dissociating, into low molecular weight fragments in the presence of urea or sodium dodecyl sulfate. The protein aggregate was separated into subunits with apparent molecular weights of approximately 42,000, 37,600, 30,100, 17,400 and 12,200 by SDS gel e l e c t r o -phoresis. Electrophoretic patterns of non-reduced and reduced samples indicated the presence of intermolecular d i s u l f i d e bonds although the cystine content was low. The 12S protein contained 12.9% (w/w) carbohydrate and reacted strongly when oxidized and treated with S c h i f f reagent. PAS-treated SDS gels indicated that most of the carbohydrate was present i n one low molecular weight fragment. SDS immunoelectrophoretic analysis suggested that the glyco-peptide portion i s located on the surface of the complex. Although the i s o l a t e contained a high molecular weight contaminant (17S), immunoelectrophoretic analysis i i r e s u l t e d i n t h e f o r m a t i o n o f one h o m o g e n e o u s p a i r o f p r e c i p i t i n a r c s . T h i s w o u l d s u g g e s t t h a t t h e 12S p r o t e i n s e l f - a s s o c i a t e s t o f o r m a g g r e g a t e s o f h i g h e r m o l e c u l a r w e i g h t . I n a n a t t e m p t t o s e p a r a t e t h e 17S a n d 12S f r a c t i o n s b y g e l f i l t r a t i o n , a 3 3 . 9 S p r o t e i n was i s o l a t e d , p r e s u m a b l y t h e p r o d u c t o f a s e l f -a s s o c i a t i n g s y s t e m . SDS e l e c t r o p h o r e t i c p a t t e r n s o f t h e 3 3 . 9 S a n d 12S p r o t e i n s w e r e s i m i l a r . H i s t o c h e m i c a l s t u d i e s r e v e a l e d t h a t t h e 12S g l y c o -p r o t e i n was p r e s e n t i n some b u t n o t a l l o f t h e c e l l s o f t h e i n t a c t r a p e s e e d k e r n e l . S c h i f f - p o s i t i v e a l e u r o n e s w e r e d i s t r i b u t e d r a n d o m l y t h r o u g h o u t t h e k e r n e l s . T r a n s m i s s i o n e l e c t r o n m i c r o s c o p y o f n e g a t i v e l y - s t a i n e d s p e c i m e n s r e v e a l e d t h a t t h e p r o t e i n was m o r u l a - s h a p e d w i t h a maximum p a r t i c l e o d i a m e t e r o f 1 2 0 A . T h e 12S r a p e s e e d p r o t e i n f o r m e d g e l s when d i s p e r s i o n s o f t h i s m a t e r i a l w e r e h e a t e d . T h e r h e o l o g i c a l a n d u l t r a -s t r u c t u r a l e x a m i n a t i o n o f t h i s phenomenon r e v e a l e d t h a t g e l s t r u c t u r e d e p e n d e d u p o n pH a n d i o n i c s t r e n g t h b u t t o a l e s s e r d e g r e e o n l o w l e v e l s o f u r e a o r d i t h i o t h r e i t o l . i i i TABLE OF CONTENTS Page LIST OF TABLES v i i LIST OF FIGURES v i i i ACKNOWLEDGEMENTS X± I INTRODUCTION 1 II LITERATURE REVIEW 3 III EXPERIMENTAL 6 A. Microstructure and Ultrastructure of the Rapeseed Kernel (Brassica campestris) and Rapeseed Meal 6 1. Light microscopy 6 2. Electron microscopy 7 B. Recovery of the 12S Protein from Rapeseed Meal 7 1. A l k a l i extraction 7 2. Gel f i l t r a t i o n 8 C. Chemical Characterization of the 12S Rapeseed Globulin 1. Amino acid analysis 9 2. Determination of the component sugars of the 12S glycoprotein 9 a) Phenol-sulfuric acid method 10 b) S i a l i c acid determination 11 c) Gas chromatography 12 d) Hexosamine determination 16 3. Ultracentrifugation 16 a) Sedimentation v e l o c i t y 16 b) Sedimentation equilibrium 17 i v 4. D i s c g e l e l e c t r o p h o r e s i s 17 a) Davis method 17 b) E l e c t r o p h o r e s i s i n urea 18 c) D i s c immunoelectrophoresis and immunodiffusion 18 d) E l e c t r o p h o r e s i s i n the presence of sodium dodecyl s u l f a t e 22 e) SDS g e l immunoelectrophoresis 25 5. Gel f i l t r a t i o n on Sepharose 6B and examination of a high molecular weight component 2 6 D. M i c r o s t r u c t u r a l and U l t r a s t r u c t u r a l Examination of the 12S G l y c o p r o t e i n 2 7 1. L i g h t microscopy 2 7 2. Transmission e l e c t r o n microscopy 2 7 3. Scanning e l e c t r o n microscopy 2 ^ E. R h e o l o g i c a l P r o p e r t i e s of D i s p e r s i o n s and Thermally-Induced Gels df the 12S 30 G l y c o p r o t e i n 1. Sample p r e p a r a t i o n 3 <^ 3 7 2. R h e o l o g i c a l c h a r a c t e r i z a t i o n IV RESULTS AND DISCUSSION 3 3 A. M i c r o s t r u c t u r e and U l t r a s t r u c t u r e of the Rapeseed K e r n e l ( B r a s s i c a campestris) and Rapeseed Meal . B. Recovery of the 12S P r o t e i n from 3 Q Rapeseed Meal C. Chemical C h a r a c t e r i z a t i o n of the 12S Rapeseed G l o b u l i n 1. Amino a c i d composition ^ 2 2. Carbohydrate composition ^4 v -Page 3. U l t r a c e n t r i f u g a t i o n 52 4. D i s c g e l e l e c t r o p h o r e s i s 56 5. Gel f i l t r a t i o n on Sepharose 6B and examination of a high molecular weight component 68 D. M i c r o s t r u c t u r a l and U l t r a s t r u c t u r a l Examination o f the 12S G l y c o p r o t e i n 70 1. L i g h t microscopy 70 2. Transmission e l e c t r o n microscopy 73 3. Scanning e l e c t r o n microscopy. 7 7 E. R h e o l o g i c a l P r o p e r t i e s of D i s p e r s i o n s and Gels of the 12S G l y c o p r o t e i n 8 0 V SUMMARY AND CONCLUSIONS 1 0 4 REFERENCES 1 0 7 APPENDIX I 1 1 4 APPENDIX I I 1 1 6 APPENDIX I I I H 9 APPENDIX IV I 2 3 APPENDIX V 12 7 v i L I S T OF TABLES TABLE Page I MOLECULAR WEIGHT MARKERS FOR SDS ELECTROPHORESIS . 24 I I AMINO ACID COMPOSITION OF THE 12S GLYCO-PROTEIN FROM RAPESEED MEAL 43 I I I MONOSACCHARIDE AND AMINO ACID COMPOSITION OF THE 12S GLYCOPROTEIN FROM RAPESEED MEAL (B. CAMPESTRIS L. VAR. SPAN) 48 I V POWER-LAW PARAMETERS FOR STEADY SHEAR FLOW BEHAVIOR OF AQUEOUS 12S GLOBULIN DISPERSIONS AT 25°C AND pH 9.2 (EXPERIMENT I ) . 8 5 V POWER-LAW PARAMETERS FOR STEADY SHEAR FLOW BEHAVIOR OF 4.5% 12S GLOBULIN GELS AT 23°C (EXPERIMENT I I ) 9 2 v i i LIST OF FIGURES Figure Page I Hydrolysis tube prepared from a 16 x 125 mm culture tube I 3 II Light micrograph of a section of rapeseed stained by PAS method 34 III Light micrograph of rapeseed tissue stained with toluidene blue and showing the protein-r i c h aleurone grains 34 IV Light micrograph of a rapeseed kernel tissue c e l l r i c h i n PAS-positive aleurone grains 37 V Light micrograph of commercial rapeseed meal stained by the PAS procedure 37 VI Electron micrograph of rapeseed showing int e r n a l c e l l u l a r structure 38 VII Electron micrograph demonstrating the aleurone grain surrounded by a single membrane and l i p i d bodies 38 VIII Flow diagram for the preparation of 12S rapeseed i s o l a t e and concentrated dispersions .... 41 IX Elution p r o f i l e of the gel f i l t r a t i o n of crude rapeseed extracts on Sephadex G-100 in 0.1M borate buffer pH 9.2 41 X A t y p i c a l gas chromatogram of the a l d i t o l acetate derivatives of the neutral and amino sugars i n the 12S glycoprotein 46 XI Schlieren patterns of undissociated and dissociated 12S protein extracted from commercial rapeseed meal 53 XII Densitometric scan of a disc gel of the 12S glycoprotein is o l a t e d from commercial rapeseed meal 60 XIII Photograph of the disc immunoelectrophoresis pattern of the 12S rapeseed glycoprotein which was diffused against rooster anti-12S antiserum 6 0 XIV Densitometric scan of an SDS disc gel (12S glycoprotein) ' 63 v i i i Figure Page XV Densitometric scans of SDS gels which were run w i t h unreduced d i s s o c i a t e d 12S g l y c o p r o t e i n and 12S p r o t e i n which was reduced w i t h 0.15M 2-mercaptoethanol XVI Upper. A photograph of a S c h i f f - s t a i n e d . SDS g e l showing the PAS-positive fragment 67 Lower. A densitometric scan of a PAS-t r e a t e d g e l showing the p o s i t i o n of the carbohydrate-containing band 67 XVII The p r e c i p i t i n arcs formed by the PAS-p o s i t i v e fragment of the 12S p r o t e i n i n Ionagar No. 2 67 XVIII The e l u t i o n p r o f i l e of a concentrate of 12S rapeseed g l y c o p r o t e i n 69 XIX Densitometric scans of SDS gels loaded w i t h 12S and 33.9S rapeseed p r o t e i n f r a c t i o n s prepared by chromatography on Sepharose 6B ^ 9 . XX L i g h t micrograph of hydrated 12S rapeseed p r o t e i n i s o l a t e o x i d i z e d w i t h p e r i o d i c a c i d and s t a i n e d w i t h S c h i f f s reagent 71 XXI PAS-positive rapeseed p r o t e i n present as g l o b u l a r masses w i t h i n the water d r o p l e t 71 XXII L i g h t micrograph showing the water-protein i n t e r f a c e 72 XXIII E l e c t r o n micrograph of a p o s i t i v e l y -s t a i n e d s e c t i o n of hydrated i s o l a t e 74 XXIV E l e c t r o n micrograph of a p o s i t i v e l y -s t a i n e d s e c t i o n of i s o e l e c t r i c p r e c i p i t a t e of rapeseed p r o t e i n a l k a l i n e e x t r a c t 74 XXV E l e c t r o n micrograph of 12S rapeseed g l y c o p r o t e i n (negatively-stained) 76 XXVI A higher m a g n i f i c a t i o n of the 12S g l y c o p r o t e i n aggregate 76 XXVII Scanning e l e c t r o n micrographs of th e r m a l l y -induced, c r y o f r a c t u r e d g e l s formed from 4.5% d i s p e r s i o n s of 12S rapeseed g l y c o p r o t e i n 78 ix Figure Page X X V I I I XXIX XXX XXXI XXXII X X X I I I XXXIV Higher magnifications of gel structure formed from heated 4.5% rapeseed protein dispersions showing the e f f e c t of pH on the 3-dimensional ordering ' ^9 Rheograms of 12S protein i s o l a t e dispersions i n 0.01M borate buffer pH 9.2 8-* Rheograms ind i c a t i n g the e f f e c t s of heat and PCMB on v i s c o s i t y of 1% dispersions of 12S protein i s o l a t e at pH 9.2 ... 8 4 Rheograms of pH 9.2 12S protein gels (4.5%) measured under steady shear at 23°C demonstrating the effects of urea, d i t h i o t h r e i t o l , NaCl and ageing i n the cold ...... 8 7 Rheograms of 4.5% rapeseed protein gels measured under steady shear at 23°C demonstrating the effects of pH and reductive a l k y l a t i o n 91 Time p r o f i l e of a dynamic shear experiment on a v i s c o e l a s t i c material as measured with the Weissenberg Rheogoniometer 9 6 Dynamic shear storage moduli as a function of o s c i l l a t o r y frequency for 4.5% rapeseed protein gels at pH 9.2 and 23°C demonstrating the effects of urea, d i t h i o t h r e i t o l , NaCl and reductive a l k y l a t i o n .... 97 XXXV Dynamic shear loss moduli as a function of o s c i l l a t o r y frequency for 4.5% rapeseed protein gels at pH 9.2 and 23°C demonstrating the effects of urea, d i t h i o t h r e i t o l , NaCl and reductive a l k y l a t i o n 99 XXXVI Dynamic shear storage moduli as a function of o s c i l l a t o r y frequency for 4.5% rapeseed protein gels prepared at various pH le v e l s and measured at 23°C , 101 XXXVII Dynamic shear loss moduli as a function of o s c i l l a t o r y frequency for 4.5% rapeseed protein gels prepared at various pH levels and measured at 23°C , 102 x ACKNOWLEDGEMENTS I wish to express my gratitude to Dr. M.A. Tung for his aid and encouragement throughout the course of t h i s study. I would also l i k e to thank Mrs. Pam G i l l for her help in numerous a c t i v i t i e s associated with the electron microscope. I f e e l that I have been p a r t i c u l a r l y fortunate in choosing a graduate committee whose members have s a c r i f -iced not only t h e i r time but have also supplied me with numerous pieces of equipment. In p a r t i c u l a r , I would l i k e to thank Dr. R. Fitzsimmons for furnishing the laboratory animals used in the immunological study. Last but not le a s t , I would; l i k e . t o dedicate t h i s thesis to my wife Jeanette for her many s a c r i f i c e s during my years of graduate study and for her work i n preparing the manuscript. xi I INTRODUCTION Rapeseed (Brassica napus and Brassica campestris) i s the major oilseed crop grown i n Canada and thi s country now leads the world i n rapeseed exports (Downey et. aJL. , 1974) . While at the present time, the seed i s crushed for i t s high o i l content (up to 45% on dry basis), the current i n t e r e s t i n plant protein sources may make rapeseed a t t r a c t i v e for i t s protein as well since levels of 20% (dry basis N x 5.53) are common. U n t i l recently, the future for rapeseed as an inexpensive protein source for human n u t r i t i o n appeared doubtful due. to toxic factors present i n the protein. As a res u l t , the defatted meal which i s a by-product of the vegetable o i l industry has been used only at low levels as an animal meal supplement. The toxic factors, thiocyanates and isothiocyanates, are formed i n the meal by the enzymatic break-down of glucosinolates which are o r d i n a r i l y found i n the seeds. Several techniques suggested for the d e t o x i f i c a t i o n of the protein (Armstrong and Stanley, 1975; Woyewoda and Nakai, 1974; and Ohlson, 1973) as well as recent advances i n plant breeding (Agriculture Canada, 19 74), have made t h i s material more a t t r a c t i v e as a potential ingredient for food manufacture. In order to u t i l i z e a protein i s o l a t e or concentrate e f f e c t i v e l y , both physical and chemical characterization of the in d i v i d u a l proteins are necessary. Numerous studies of this nature have been reported i n the l i t e r a t u r e concerning 1 2 the constitutive proteins of soybean, for example, the most recent by Badley et a l . (1975). The rapeseed protein selected for t h i s study was the 12S glycoprotein described by Bhatty et a l . (1968), Finlayson et al. (1969), and Goding et a l . (1970). This protein, i s o l a t e d by previous investigation, was extracted from seed rather than commercial meal and constituted approximately 21% of the sal t - s o l u b l e nitrogen. The purpose of the present study was to develop a method for the i s o l a t i o n of the 12S globulin from meal i n s u f f i c i e n t quantities to permit chemical, physical, and u l t r a s t r u c t u r a l characterization which would provide an insight into possible functional q u a l i t i e s of t h i s oilseed globulin. I I LITERATURE REVIEW B h a t t y e t a l _ . ( 1 9 6 8 ) e x t r a c t e d t h e p r o t e i n s o f r a p e -s e e d w i t h 0 . 0 1 M s o d i u m p y r o p h o s p h a t e ( p H 7 . 0 ) a n d s e p a r a t e d t h e m i n t o 9 c h r o m a t o g r a p h i c a l l y d i f f e r e n t f r a c t i o n s . M o s t o f t h e t o t a l r a p e s e e d n i t r o g e n w a s f o u n d i n 2 f r a c t i o n s , a n e u t r a l h i g h m o l e c u l a r w e i g h t p r o t e i n a n d a b a s i c 1 . 7 S f r a c t i o n w i t h a m o l e c u l a r w e i g h t o f 1 3 , 8 0 0 . T h e l a r g e r p r o t e i n h a d a n o b s e r v e d s e d i m e n t a t i o n c o e f f i c i e n t o f 1 2 S i n 0 . 1 M b o r a t e b u f f e r ( p H 8 . 6 ) . T h e 1 2 S p r o t e i n w a s a l s o r e c o v e r e d i n a 10% N a C l e x t r a c t o f r a p e s e e d a n d r e p r e s e n t e d 21% o f t h e n i t r o g e n r e c o v e r e d . T h i s w a t e r - i n s o l u b l e p r o t e i n w a s c h r o m a t o g r a p h i c a l l y a n d e l e c t r o p h o r e t i c a l l y p u r e a t a l k a l i n e p H ' s b u t d i s s o c i a t e d i n t o s u b f r a c t i o n s a t p H v a l u e s b e l o w 3 . 5 a n d i n u r e a s o l u t i o n s . I n a d d i t i o n t o t h e 3S a n d 7S f r a g m e n t s w h i c h a p p e a r e d i n t h e u l t r a c e n t r i f u g e p a t t e r n s e v e n a t a l k a l i n e p H ' s , a 1 7 S c o m p o n e n t w a s d e t e c t e d i n t h e 1 2 S g l o b u l i n a n d w a s c o n s i d e r e d a c o n t a m i n a n t . I n a n o t h e r s t u d y , F i n l a y s o n e t a l . ( 1 9 6 9 ) r e p o r t e d d i f f e r e n c e s i n t h e a m i n o a c i d c o m p o s i t i o n s o f t h e 1 2 S p r o t e i n s i s o l a t e d f r o m d i f f e r e n t s p e c i e s a n d v a r i e t i e s o f r a p e s e e d . A l t h o u g h t h e m e t h o d o f r e c o v e r y w a s t h e s a m e a s t h a t r e p o r t e d b y B h a t t y e t a l . ( 1 9 6 8 ) , t h e 1 2 S i s o l a t e i n t h e s e c o n d s t u d y c o n t a i n e d a l m o s t 2% m o r e n i t r o g e n t h a n i n t h e f i r s t s t u d y a l t h o u g h n o a p p a r e n t r e a s o n f o r t h i s p h e n o m e n o n w a s g i v e n . T h e r e w e r e c o n s i d e r a b l e d i f f e r e n c e s i n c y s t i n e a n d m e t h i o n i n e 3 4 contents noted between species, and c u l t i v a r s within the same species. It was concluded that because obvious differences did occur i n structurally-important amino acids, the 12S proteins from the d i f f e r e n t species probably had d i f f e r e n t structures although similar sedimentation c o e f f i c i e n t s were observed. Further characterization of the 12S rapeseed globulin was reported by Goding et a_l. (1970) . A d i f f e r e n t technique for the i s o l a t i o n of the protein was outlined i n t h e i r study. The new procedure involved the extraction of protein i n a b a l l m i l l using 0.IM borate buffer (pH 9.2) containing IM sodium chloride. The insoluble residue and l i p i d were separated from the soluble material by centrifugation and the 12S protein enriched by high speed centrifugation at 110,000 x g. F i n a l p u r i f i c a t i o n was c a r r i e d out by gel chromatography on Sephadex G-100. The 12S protein was dissociated and chromatographed at pH 2.8 i n 2M urea. Four fractions were col l e c t e d from dissociated 12S proteins prepared from B. napus L. var. Target and B. campestris L. var. Echo. Cationic gel electrophoresis i n 6M urea resulted i n the separation of 7 components from the dissociated complex. It was concluded that cystine was present intramolecularly rather than intermolecularly since mercaptoethanol did not e f f e c t electrophoretic or chromatographic separation. The major component of the 12S complex, a 2.7S protein, was composed of 2 polypeptide chains and contained most of the carbohydrate. 5 The 12S glycoprotein complex contained 0.15 - 0.20% galactosamine and 1 - 1.5% glucose and arabinose. Similar studies have been reported on other members of the genus Brassica. MacKenzie and Blakely (1972) reported the recovery of the 12S globulins from B. juncea, B. nigra and B. h i r t a . Besides p u r i f i c a t i o n procedures described by previous authors, the 12S f r a c t i o n was chromatographed on Sephadex G-200 (superfine). Species differences i n amino acid composition of both the 12S and 1.8S proteins were analyzed by a multivariate s t a t i s t i c a l technique. MacKenzie (1975) reported that the impurities separated from the 12S protein in the 1972 study had amino acid compositions which were indistinguishable from the major component. The author suggested that such "impurities" actually represented products of a se l f - a s s o c i a t i n g system. According to MacKenzie, mercaptoethanol did e f f e c t the d i s s o c i a t i o n of the 12S complex isola t e d from B. juncea although no e f f e c t was observed at levels lower than 0.1M. The dissociated, reduced 12S protein of B. juncea was separated into f i v e a c i d i c , f i v e basic and one neutral component by preparative i s o e l e c t r i c focussing. The basic components were present i n much greater amounts than the a c i d i c components although amino acid analyses of the basic fractions indicated that aspartic and glutamic acids were present i n greater abundance than basic amino acids, suggesting that the former acids were presumably i n the amide forms of asparagine and glutamine. I l l EXPERIMENTAL A. Microstructure and Ultrastructure of the Rapeseed Kernel (Brassica campestris) and Rapeseed Meal. 1. Light microscopy The objective of t h i s study was to elucidate the microstructure of the rapeseed kernel, to locate the protein-r i c h components and to observe the e f f e c t s of commercial solvent extraction and drying on the meal. Intact seeds and meal were fixed for 1 h i n 2.5% glutaraldehyde i n Sorensen's phosphate buffer (Sorensen, 1909) pH 7.0. The seeds were s p l i t while immersed in the f i x a t i v e . Samples were dehydrated in a series of ethanol solutions (30, 50, 70, 95, 100%), each change l a s t i n g for 10 min. After 2 or 3 changes of 100% ethanol, samples were i n f i l t r a t e d and embedded in Spurr 1s epon (Spurr, 1969). Thick (1 ym) sections were cut on a Reichert Om U3 ultramicrotome and heat fixed to glass s l i d e s . Slides were placed i n saturated ethanolic-KOH for 20 min to dissolve the embedding material and rinsed exhaustively, f i r s t with tap water and then with d i s t i l l e d water. Slides were stained with S c h i f f ' s reagent (Sheehan and Hrapchak, 1973) for 15 min in order to examine any periodic acid S c h i f f (PAS) p o s i t i v e material. The s l i d e s were rinsed three times i n 2% sodium metabisulfite, each rinse l a s t i n g for about 2 min, and then counterstaihed with hematoxylin. Thick sections of seeds were 6 7 also stained for proteinaceous material with toluidene blue for approximately 1 min and then rinsed with tap water. Prepared s l i d e s receiving the above treatments were examined using a Wild M20 microscope equipped with a 35 mm camera system. 2. Electron microscopy The preparative methods for electron microscopical study of u l t r a t h i n sections of seed kernels were i d e n t i c a l to those described for l i g h t microscopy except that glutaraldehyde f i x a t i o n was followed by a 60 min f i x a t i o n with 1% OsO^ i n pH 7.0 phosphate buffer. S i l v e r sections were cut with a Reichert Om U3 ultramicrotome using freshly broken glass knives. Sections were mounted on uncoated 300 mesh copper grids and stained with uranyl acetate (Watson, 1958) and lead c i t r a t e (Reynolds, 1963). Micrographs were taken on an AEI Corinth 275 transmission electron microscope operating at a 60 kV accelerating voltage. B. Recovery of the 12S Protein from Rapeseed Meal. 1. A l k a l i extraction Commercial defatted rapeseed meal (Brassica campestris) was extracted for 12 h at 4°C with 0.1M borate buffer pH 9.2 (1 part meal: 9 parts b u f f e r ) . Extraction was ca r r i e d out on a Fisher Thermix magnetic s t i r r e r with the speed adjusted 8 such that the sl u r r y was moving slowly enough to prevent foam formation on the surface. The crude extract was centrifuged at 20,000 x g for 30 min at 4°C i n a Sor v a l l RC2-B refrige r a t e d centrifuge. The supernatant was f i l t e r e d through Whatman number 1 f i l t e r paper and applied to a Sephadex G-T00 column. 2. Gel f i l t r a t i o n F i f t y ml aliquots of crude extract were applied to an 82 x 5 cm Sephadex G-100 column and eluted with 0.1M borate buffer pH 9.2 at 2 ml/min. The f i r s t peak contained the 12S fr a c t i o n and was dialyzed against water at 4°C for 72 h. The brown color c h a r a c t e r i s t i c of alkaline extracts of rape-seed was removed completely af t e r t h i s time, leaving a white water-insoluble dispersion in the d i a l y s i s bag. The dispersion was then either pervaporated to a desired concentration for rheological characterization or cryogenically frozen and ly o p h i l i z e d . The void volume of the column was determined with Blue Dextran 2,000 (Pharmacia Fine Chemicals). Elution patterns were observed as absorbance at 280 nm. Protein contents of meal, crude extracts and 12S fractions were determined by the rapid micro Kjeldahl method of Concon and Soltess (1973), (%N x 6.25) . The 12S f r a c t i o n was i d e n t i f i e d as such i n 0.1M borate buffer pH 9.2 by sedimentation v e l o c i t y u l t r a c e n t r i f u g a t i o n 9 as described i n Section III.3.a. C. Chemical Characterization of the 12S Rapeseed Globulin. 1. Amino acid analysis Samples of freeze-dried 12S i s o l a t e were derivatized with 4-vinylpyridine by a method described by Cavins et a l . (1972). The authors found that the S - 3 - ( 4 - p y r i d y l e t h y l ) - L -cysteine formed from the sel e c t i v e a l k y l a t i o n of cystine and cysteine residues, was stable under the conditions' of acid hydrolysis. The alkylated protein was hydrolyzed with p-toluenesulfonic acid i n glass ampoules which were heat sealed under a vacuum of 20 to 3 0 ym Hg. Samples were hydrolyzed for 24, 25, 26, 27, 28 and 36 h at 110°C. Hydrolysis i n p-toluenesulfonic acid was suggested by Liu and Chang (1971) as a means of protecting tryptophan from hydrolytic destruction. Samples and standards were analyzed on a Hitachi Model KLA-3B amino acid analyzer u t i l i z i n g a single column elution system. Details of the sample preparation are described i n Appendix I. 2. Determination of the component sugars of the  12S glycoprotein It would be reasonable to expect that a large amount of carbohydrate chemically bound to protein molecules would 10 have a pronouced e f f e c t on the physical behavior of the macro-molecules. Glycoproteins generally exhibit lower p a r t i a l s p e c i f i c volumes than proteins containing no carbohydrate (Gibbons, 1966). The anomalous behavior of glycoproteins i n sodium dodecyl sulfate electrophoresis becomes more pronounced as the carbohydrate content for a given glycoprotein increases (Segrest and Jackson, 1972). It i s believed that carbohydrate side chains tend to radiate outward from the amino acid back-bone i n some glycoproteins (Morawiecki, 1964 and Winzler, 1969). As a r e s u l t of i t s hydrophilic nature, the carbohydrate portion of the macromolecular complex i s often responsible for the immunological i d e n t i t y of the structure. Such molecules would be expected to show a r e l a t i v e l y high degree of protein-protein i n t e r a c t i o n i n solution as well as apparent v i s c o s i t i e s which are highly shear dependent (non-Newtonian). A detailed analysis of the sugar components of the 12S glyco-protein was undertaken i n order to aid i n the explanation of chemical and physical properties. a) Phenol-sulfuric acid method. I n i t i a l l y , an approximate carbohydrate content was determined by the phenol-sulfuric acid method of Dubois et a l . (1956). Since the i d e n t i f i c a t i o n of the carbohydrate component was not carried out at t h i s time, the content was estimated from a standard curve for glucose. i i b) S i a l i c acid determination. A preliminary examination of electrophoretic mobility i n urea led to the suspicion that an acid carbohydrate may be present on the surface of the 12S complex occluding a basic amino acid backbone. The presence of s i a l i c acid was suspected because the undissociated complex migrated toward the anode, however, the dissociated fragments appeared to be neutral or basic i n nature. S i a l i c acid content was determined by the method of Warren (1959). Acid hydrolysis was carried out i n 16 x 125 mm screw cap test tubes. Samples of the 12S protein and of bovine thyroglobulin (Sigma) weighing between 10 and 20 mg were dissolved i n 10 ml 0.IN HpSO^. Hydrolysis was carried out for 1 h at 8 0°C i n a water bath. Tubes were cooled and 0.2 ml of each sample were mixed with 0.1 ml of solution containing 0.2M sodium metaperiodate i n 9M phosphoric acid. After 20 min at ambient temperature, 1 ml of sodium arsenite solution (10% sodium arsenite i n a solution of 0.5M sodium sulfate and 0.IN H2SO^) was added and the tube shaken u n t i l the yellow color disappeared. Three ml of a th i o -b a r b i t u r i c acid solution (0.6% th i o b a r b i t u r i c acid i n 0.5M sodium sulfate) were added and the sample tubes placed i n a b o i l i n g water bath for 15 min. During t h i s time, the chromogen developed (a complex between 3-formyl pyruvic acid produced from s i a l i c acid oxidation and t h i o b a r b i t u r i c acid) i n samples containing s i a l i c acid. Tubes were cooled and 4.3 ml cyclo-hexanone were added and shaken to extract the chromogen. The 12 tubes were then centrifuged at 1,000 x g for 2 min and the solvent layer read at 549 nm i n a Beckman DB spectrophotometer. A standard curve was prepared for standards containing between 0 and 20 yg s i a l i c acid (Sigma). c) Gas chromatography. Analyses for t o t a l neutral and amino sugars were carried out using a modification of the procedure described by Porter (1975). The major advantages to gas-liquid chromatographic analysis of the sugars as t h e i r a l d i t o l acetate derivatives were that both neutral and amino sugars could be analyzed simultaneously and that multiple peaks for each sugar, as a r e s u l t of anomerization and ring isomerization, were avoided. The method described by Porter u t i l i z e d an hydrolysis procedure i n which the sugars were liberated from the protein i n the presence of a cation exchange r e s i n and the amino sugars released from the re s i n through a nitrous acid deamination reaction. Because several modifications of the procedure were incorporated, d e t a i l s of the technique are described. i) Hydrolysis. Samples weighing between 2 and 3 mg were placed i n hydrolysis tubes consisting of 16 x 125 mm screw cap culture tubes which had been drawn out to approximately 20 mm i n length (Figure I ) . A 3 mm hole was d r i l l e d i n the center of each cap and the cardboard cap. l i n e r s replaced with s i l i c o n rubber septa. To each v i a l was added 100 y l of a 13 FIGURE I: Hydrolysis tube prepared from a 16 x 125 mm culture tube. 40% (w/v) suspension of AG 50W-X2 (H form) 200-400 mesh ion exchange res i n (Bio-Rad Laboratories) i n 0.02N HC1, followed by 100 y l d i s t i l l e d H20. The v i a l s were placed i n a 100 ±2°C oven for 40 h. In order to assess completeness of hydrolysis, two samples were hydrolyzed for an extra 12 h. In i n i t i a l runs, 0.4 ymoles of myoinositol were added to each v i a l i n a 50 y l i n j e c t i o n as an internal standard. I t soon became apparent that i n o s i t o l was also present i n the sample, there-fore, xylose was substituted as an i n t e r n a l standard in sub-14 sequent runs. i i ) Derivatization. The samples and standards were deaminated with the addition of 20 ul of a freshly prepared 5.5M solution of NaN02. After intermittent vortexing for a period of 30 min, 200 u l of a 40% aqueous suspension of AG 50W-X2 (H + form) res i n were added and intermittent vortexing continued for 30 min. The liberated sugars were then separated from the protein components by quantitatively transferring the samples to ion exchange columns prepared from Pasteur pipets plugged with glass wool and f i l l e d with 0.4 ml of a 40% aqueous suspension of AG 50W-X2. The pipets were arranged such that each pipet would drain into a second pipet f i l l e d with 1 ml of a 20% aqueous suspension of 200-400 mesh AG 1-X8 (Cl form prepared from formate form). Hydrolysis tubes were rinsed three times with 0.4 ml d i s t i l l e d water and transferred to the columns. The columns were then washed with 0.8 ml of 50% methanol in water and the effluents collected i n test tubes. The sugar solutions were dried under a stream of nitrogen and reduction carried out by the addition of 100 u l d i s t i l l e d water and 100 y l 0.22M NaBH^. After 1 h, excess borohydride was decomposed with the addition of 40 y l g l a c i a l acetic acid and the samples dried under nitrogen. Borate was v o l a t i l i z e d with the addition of four 200 y l portions of methanol-concentrated HC1 (1,000:1) with concen-t r a t i o n to dryness af t e r each addition. Acetylation of the sugars was carried out with the addition of 100 y l pyridine 15 and 100 y l acetic anhydride. The tubes were sealed with rubber stoppers wrapped i n Teflon tape, placed i n a 100°C oven for 15 min, vortexed, and heated for an additional 15 min. i i i ) Analysis. Portions (5 yl) of the samples and standards were chromatographed on a Tracor MT220 gas chromatograph equipped with 1/4 i n x 6 f t dual s i l a n i z e d glass columns packed with 3% ECNSS-M on 100/120 mesh Gas Chrom Q (Applied Science Laboratories) as suggested by Laine et aJL. (1972) . The i n j e c t i o n port temperature was 230°C and the flame i o n i -zation detector was 280°C. Oven temperature was programmed from 150°C to 190°C at 1°C min - 1. The nitrogen c a r r i e r gas was adjusted to 4 0 ml min 1 and a l l samples were applied v i a on-column i n j e c t i o n . A l d i t o l acetate derivatives were i d e n t i f i e d by retention times as compared to standards prepared from reagent grade sugars. The fact that xylose eluted at exactly the same time as glucosamine precluded the use of xylose as an int e r n a l standard. Standard curves r e l a t i n g the peak areas to sugar concentrations were prepared and samples were chromatographed on the same day as the standards. Standards were analyzed i n quadruplicate and three concentrations of standard mixtures run i n duplicate, both before and aft e r samples had been run. Peak areas were integrated with an electronic planimeter (Numonics Corp.). 16 d) Hexosamine determination. Since glucosamine could not be resolved from xylose by gas chromatography, and since no other amino sugars were detected (galactosamine and mannosamine were resolvable on the 3% ECNSS-M column), the colorimetric method of Elson and Morgan (1933) was used to detect glucosamine. Hydrolysis of the glycoprotein was carried out as described for gas chroma-tography and separation of the amino sugars from the neutral sugars was carried out according to the ion exchange method of Boas (1953) i n which the amino sugars were adsorbed to a cation exchange column and subsequently eluted with 2N HC1. The only modification of t h i s procedure was the substitution of AG 50W-X2 (Bio-Rad Laboratories) for Dowex 50-X4 r e s i n . The Elson-Morgan reaction and the hydrolysis procedure are outlined i n d e t a i l i n Appendix I I . 3. Ultracentrifugation a) Sedimentation v e l o c i t y . A sample of approximately 1% 12S protein i n 0.IM borate buffer pH 9.2 was run at 55,000 rpm i n a Beckman L2-65B ultracentrifuge equipped with a 2-place An-D a n a l y t i c a l rotor. The mobility of the protein was observed through a schlieren o p t i c a l system and sedimentation patterns recorded photo-graphically. Similar runs were performed i n 0.IM phosphate buffers pH 7.0 adjusted to 5M and 6M urea and 0.1% 2-mercap-toethanol. b) Sedimentation equilibrium. Nitrogen determinations were performed upon a stock solution of 12S protein i n 0.1M borate buffer, pH 9.2. S e r i a l d i l u t i o n s of the stock solution were prepared i n the same buffer and the absorbance readings at 280 nm were plotted against protein concentrations (%N x 6.25). Equilibrium runs were performed at 7,800 rpm and 20°C i n the L2-65B u l t r a -centrifuge equipped with a 4-place An-F a n a l y t i c a l rotor and a Beckman Prep UV Scanner. The observed molecular weights for s i x concentrations were calculated and an M0 (molecular weight at i n f i n i t e dilution) determined from a least squares f i t of the M vs.%N data. 4. Disc gel electrophoresis a) Davis method. In order to assess the degree of electrophoretic homogeneity of the i s o l a t e , a solution of the freeze-dried material was subjected to polyacrylamide disc gel el e c t r o -phoresis by the method of Davis (1962). This system stacked at pH 8.9 and ran at pH 9.5 on 7% acrylamide gels. Bromphenol blue was used as a tracking dye i n the upper electrode buffer. 18 Details of the gel preparation are given i n Appendix I I I . A l l gels were run in a Pharmacia gel electrophoresis apparatus Model GE-4 at 5 mA per tube. Gels were stained with 0.25% Coomassie blue dissolved in a mixture of 5% methanol and 7% acetic acid for a period of 8 - 12 h. Gels were destained in a Pharmacia gel destainer, Model GD-4 with a mixture of 5% methanol in 7% acetic acid. Gels were scanned in a Transidyne TG 2980 automatic scanning densitometer at 550 nm. b) Electophoresis i n urea. The electrophoretic buffer system of Weber and Osborn (19 69) was modified b y the incorporation of 5M deionized urea and o . l % 2-mercaptoethanol into gels and electrode buffer to gain some information concerning the o v e r a l l charge of the urea-denatured fragments of the 12S agglomerate. Both anionic and cati o n i c runs were performed. An electrophoretic system of Reisfeld and Small (19 66) which ran at pH 9.4 i n the presence of 10M urea was also used to observe the elec-trophoretic behavior of the denatured 12S protein. c) Disc Immunoelectrophoresis and immunodiffusion. Immunoelectrophoresis and immunodiffusion have been used to study the taxonomy of several of the species of the genus Brassica (Vaughan et a l . , 1966; Vaughan and Waite, 1967; and Vaughan and Denford, 1968). A technique termed "disc immunoelectrophoresis" by Catsimpoolas and Meyer (19 68), and Catsimpoolas et a^ L. (1968) was used to establish the homogenei of the 11S soybean protein and to follow changes i n reserve proteins i n germinating soybeans. Disc immunoelectrophoresis was undertaken i n the present study to establish the immunological i d e n t i t y of the 12S rapeseed protein. i) Preparation of antisera. Two species were selected for the production of p r e c i p i t a t i n g antibody. In a preliminary experiment, 18 white mice were injected with a crude extract of whole rapeseed protein • (WRE) . WRE was prepared by the over night extraction of commercial rapeseed meal with 0.IM borate buffer pH 9.2. The f i l t e r e d extract was s t i r r e d at 4°C and the proteins precipitated with 9 8 g granular ammonium sulfate added over a period of 30 min. The slurr y was centrifuged at 5,000 x g for 30 min, and the p e l l e t was resuspended i n a small amount of water and placed i n a d i a l y s i s bag. The sample was dialyzed against cold running water for 72 h and then 0.01M sodium phosphate buffer for 48 h at 4°C. Protein concentration was determined by micro -Kjeldahl analysis (%N x 6.25). A 1.30% protein suspension was mixed 1:1 with Freund's complete adjuvant and 0.1 ml of the mixture administered i n t r a p e r i t o n e a l l y . Two subsequent injections of the same size were given at one week i n t e r v a l s . After a rest period of 4 weeks, the animals were given a booster i n j e c t i o n of 0.1 ml and bled by cardiac puncture 2 weeks l a t e r . The blood was pooled and allowed to c l o t at room temperature for approximately 1 h and then refri g e r a t e d 20 overnight. The clo t s were c a r e f u l l y cut and separated from the serum by centrifugation. In a l a t e r experiment, 2 Single-Comb White Leghorn roosters were injected with a 1:1 mixture of Freund's complete adjuvant and WRE (0.90% protein) prepared as before. In addition, an aqueous sample of 12S protein (0.54% protein) was prepared i n the usual manner and dialyzed against 0.01M sodium phosphate buffer, pH 7.6, and administered 1:1 i n Freund's complete adjuvant to 2 Leghorn roosters. The four roosters were given 1 ml antigen on the f i r s t week, 2 ml on the second week and 3 ml on the t h i r d , by intraperitoneal i n j e c t i o n . A b i r d that had received the 12S antigen died during a t r i a l bleeding. The three remaining roosters were boosted with 5 ml intraperitoneal injections after a 30 day rest period and, again, 1 week l a t e r . After 2 weeks, the birds were bled by cardiac puncture and the serum collected, i i ) Electrophoresis and d i f f u s i o n . Samples of the 12S f i s o l a t e were electrophoresed by the method of Davis (1962) on 4% acrylamide gels (Appendix I I I ) . Gels were loaded with 130 to 400 mg protein and after running were ejected from t h e i r tubes and placed i n disposable P e t r i dishes. Each dish was f i l l e d with enough agar to cover each gel. The agar solution was composed of 1% lonagar No. 2 (Oxoid) i n pH 8.6 sodium diethylbarbiturate-sodium acetate buffer, io n i c strength 0.05. The agar contained 0.02% sodium azide as a preservative. After s o l i d i f i c a t i o n , trenches were cut a few mm deep, 21 running p a r a l l e l to the gel columns but separated at a distance of approximately 1 cm and f i l l e d c a r e f u l l y with the mouse anti-WRE antiserum. The reactants were allowed to d i f f u s e at room temperature i n a glass desiccator containing a small amount of water. Gels containing the same antigen loads were electrophoresed along with the above gels and stained with Amido black 10B i n order to locate the protein band. Antigen (12S protein) was also diffused against anti-WRE by the Ouchterlony (1958) method u t i l i z i n g the above agar s o l u t i o n . Similar procedures were c a r r i e d out with the rooster anti-WRE and the anti-12S antisera. WRE was f i r s t d i a l i z e d against electrode buffer and immunoelectrophoresed against rooster anti-WRE serum. Lyophilized 12S protein was dissolved d i r e c t l y i n electrode buffer and immunoelectrophoresed against rooster anti-WRE serum. Protein loads ranged from 50 to 200 yg per gel for the 12S i s o l a t e and 20 to 80 yg for the WRE. Control gels were stained with Coomassie blue, which was found to be more sensitive and yielded superior resolution as compated to Amido black. Double d i f f u s i o n of the anti-12S antiserum was car r i e d out against a wide range of concentrations of the s i x component sugars of the 12S glycoprotein (Section C.2.) i n order to test the p o s s i b i l i t y that any of the sugars would be immunodeterminants. Aqueous solutions containing 1%, 0.1% and 0.01% of each sugar were diffused against p r e c i p i t a t i n g a n t i -body on agar plates. 22 d) Electrophoresis i n the presence of sodium dodecyl s u l f a t e . Sodium dodecyl sulfate (SDS) gel electrophoresis was ca r r i e d out i n a buffer system described by N e v i l l e (1971) which produced improved resolution compared with the method of Weber and Osborn (19 69). The stock solutions were prepared as in Appendix IV. Reagent grade sodium l a u r y l sulfate (Fisher S c i e n t i f i c Co.) was r e c r y s t a l l i z e d three times i n absolute ethanol. Urea solutions were deionized to a conductivity of 5 umhos or l e s s , however, no differences in electrophoretic patterns were observed between samples dissociated with non-deionized urea and those treated with deionized urea. Acrylamide gels were tested for resolving power at 7%, 10% and 15% acrylamide concentrations, the best results observed with the 10% gels. Samples of 12S protein and standard molecular weight markers were prepared as described i n Appendix IV and heated i n a b o i l i n g water bath for 2 to 5 min to ensure complete denaturation and to prevent proteolysis due to impurities or any inherent p r o t e o l y t i c a c t i v i t y of the sample i t s e l f (Weber, et a l . , 1972). The discontinuous buffer system of N e v i l l e (1972) stacked at pH 8.64 and ran at pH 9.50. Gels were run at 2 mA/tube. Temperature was held constant at about 15°C by c i r c u l a t i n g tap water through the countercurrent heat exchanger i n the base of the unit. At the end of each run, the position of the bromphenol blue tracking dye i n the gel was marked with a hypodermic needle containing dr a f t i n g ink. Since i t was 23 suspected that one of the fragments of the 12S oligomer migrated with the tracking dye, a run was performed i n which the bromphenol blue was omitted from the upper electrode buffer and some of the sample gels. Gels were stained and destained as described i n Section C.4.a. In order to i d e n t i f y the carbohydrate-containing components i n SDS gels, a modification of a procedure described by Glossmann and Ne v i l l e (1971) was employed. These authors reported that for several glycoproteins (such as ovomucoid and a^-glycoprotein) the Coomassie-stained bands represent minor contaminants, whereas PAS-reactive bands represented the true molecular subunits. Many carbohydrate-rich glyco-proteins stained only f a i n t l y with Coomassie blue. To avoid any p o s s i b i l i t y of a r t i f a c t u a l staining with Coomassie blue, the glycoprotein molecular weight markers (Table I) were located by both Coomassie blue and S c h i f f reagent. The PAS staining procedure was performed as follows. Gels were washed i n the GD-4 destainer with 40% methanol i n 7% acetic acid overnight (2 changes) i n order to remove bound and unbound SDS. A l l gels, except controls, were oxidized for 1 h i n 1% periodic acid i n 7% acetic acid i n the dark and transferred to a 0.5% sodium arsenite i n 5% acetic acid (Fairbanks et a l . , 1971) for 1 h. Three 20 min changes of 0.1% sodium arsenite in_5% acetic acid were followed by a 20 min wash i n 5% acetic acid. A l l gels, including controls, were stained with S c h i f f reagent (Appendix IV) overnight at 4°C in the dark. The unreacted S c h i f f reagent was removed with several rinses of 24 1% sodium metabisulfite i n 0.IN HC1. A l l gels were subsequently stored i n the dark at 4°C i n the metabisulfite solution. Gels were scanned i n a Transidyne TG 29 80 automatic scanning densitometer at 550 nm for Coomassie blue gels and 560 nm for PAS gels. Relative m o b i l i t i e s compared to the bromphenol blue tracking dye were calculated from the densitometer traces. Regression l i n e s were f i t t e d for the r e l a t i o n : log MW = a + bR^ where MW, a, b, and R f represent subunit molecular weight, intercept, slope and r e l a t i v e mobility, respectively. Apparent molecular weights of the subunits of the 12S glycoprotein were estimated from the regression equation derived from the mobility of molecular weight markers (Table I) . TABLE I: MOLECULAR WEIGHT MARKERS FOR SDS ELECTROPHORESIS Marker Source % Carbohydrate"1" Subunit MW1 Bovine thyro-globulin Sigma 8.5 — 335,000 Conalbumin NBC 2.5 86,180 Ovalbumin ICN 3.2 43,500 RNase (Bovine pancreatic) Sigma 11. 3 12,64.0 from Sober (1970) 25 e) SDS gel immunoelectrophoresis. The electrophoretic separation of the 12S glyco-protein fragments remained unchanged i f SDS was eliminated from running and stacking gels thus presenting the p o s s i b i l i t y of running gels with reduced levels of SDS. Although immuno-electrophoresis i s not normally carried out i n the presence of SDS due to the induction of conformational changes i n both antigen and antibody, i t has been found that glycoproteins ( p a r t i c u l a r l y those containing s i a l i c acid) bind substantially less SDS than proteins containing no carbohydrate (Segrest and Jackson, 1972). Immune SDS gels were run by the method of Glossmann and Neville\pL971) as previously described. Gels were loaded with samples ranging from 10 to 400 yg of the dissociated 12S i s o l a t e (Appendix IV). After electrophoresing for approximately 1.5 h at 2 mA per gel, the positions of the bromphenol blue tracking dye were marked and the gels embedded in Ionagar No. 2. Trenches were cut and f i l l e d with rooster anti-12S antiserum and the reactants allowed to d i f f u s e as previously described. After the p r e c i p i t i n bands had formed, the P e t r i dishes were washed for several days i n saline and stained with Amido black (Grabar and Burtin, 1964). Results were, recorded photographically. 26 5. Gel f i l t r a t i o n on Sepharose 6B and examination  of a high molecular weight component High speed ul t r a c e n t r i f u g a t i o n and disc gel electro-phoresis (Sections IV.C.3. and IV.C.4.)- indicated the presence of a high molecular weight component which sedimented at 17.4S i n 0.1M borate buffer pH 9.2. Although the component could be detected by disc gel electrophoresis, i t did not appear as a separate p r e c i p i t i n arc i n disc Immunoelectrophoresis (Section IV.C.4.) or immunodiffusion experiments. Such observations suggested the possible presence of a 12S dimer rather than a protein impurity. In order to investigate t h i s p o s s i b i l i t y , a 12S peak was c o l l e c t e d from the Sephadex G-100 column.(Section III.B.2.) and concentrated in an u l t r a f i l t r a t i o n apparatus equipped with a PM-30 f i l t e r (molecular weight exclusion>30,000 daltons). An aliquot of the 12S material was applied to a 26 x 790 mm column packed with Sepharose 6B (Pharmacia) i n 0.1M borate pH 9.2. The column was run at 2 ml min 1 and two well-defined peaks coll e c t e d . The UV-absorbing peaks were water dialyzed and l y o p h i l i z e d . The l y o p h i l i z e d material was subjected to SDS gel electrophoresis as described i n Section III.C.4.d. 27 D. Microstructural and U l t r a s t r u c t u r a l Examination of the 12S Glycoprotein. 1. Light microscopy Aqueous pastes of the 12S f r a c t i o n were fixed with 2.5% glutaraldehyde i n phosphate buffer at pH 7.0 for 1 h. Dehydration, embedding, sectioning, and S c h i f f staining were carried out as described for the seeds and meal. Slides were examined and the images recorded with a 35 mm camera system. 2. Transmission electron microscopy Samples of the 12S i s o l a t e were prepared for u l t r a -s t r u c t u r a l examination by the above procedure except that glutaraldehyde f i x a t i o n was followed by a 60 min f i x a t i o n with 1% Os0 4 i n pH 7.0 phosphate buffer. Samples of an i s o e l e c t r i c a l l y precipitated rapeseed i s o l a t e prepared by Keshavarz (1974) were included i n the study so that comparisons between the two iso l a t e s could be made. A l l sections were mounted on 300 mesh copper grids and stained with uranyl acetate (Watson, 1958J and lead c i t r a t e (Reynolds, 1963). Grids were examined i n an AEI Corinth 275 transmission electron microscope operating at 60 kV. 28 An attempt was made to examine the u l t r a s t r u c t u r a l d e t a i l of the i s o l a t e treated with an electron-dense s t a i n which would react s p e c i f i c a l l y with the carbohydrate moieties of the protein. U l t r a t h i n sections of glutaraldehyde-fixed i s o l a t e were mounted on 300 mesh gold grids, oxidized with periodic acid and stained with s i l v e r methanamine as described by Rambourg (1967). This reagent i s believed to react with periodate-oxidized sections i n a manner analogous to S c h i f f reagent; however, due to i t s reaction with copper, gold or platinum grids are necessary. A negative staining technique, similar to that described by Haschemeyer and Myers (1972) was employed for the examination of the structure of the 12S agglomerate. Copper grids (400 mesh) were washed thoroughly with acetone, dried, and dropped onto a f l o a t i n g layer of collodion. F i l t e r paper was used to pick up the collodion f i l m which supported the grids. After a i r drying, the collodion-coated grids were placed i n a Balzers Micro BA3 high vacuum coating unit and coated with carbon. Immediately before use, the collodion was removed from the grids with,a 1.5 min dip i n acetone. A drop of protein solution (approximately 0.001% w/v i n 0.lM borate buffer pH 9.2) was placed on each g r i d and the excess removed by touching the edge of the gr i d with a torn piece of f i l t e r paper after 4.5 min. A drop of 0.5% aqueous uranyl acetate (freshly dissolved) was placed on each g r i d for 2.5 min and the excess l i q u i d removed as before. After a i r drying, 29 the negatively-stained grids were examined on a Zeiss 10 transmission electron microscope with an accelerating voltage of 60 kV and images recorded at magnifications from 40,000 to 60,000 diameters. 3. Scanning electron microscopy Gels were prepared as described i n Section I I I . E . l . with 4.5% protein dispersions adjusted to various pH l e v e l s . Pieces of gel were immersed i n l i q u i d nitrogen and cryo-fractured. The frozen fragments were immediately transferred into a 2.5% glutaraldehyde solution buffered to pH 7.0 and fixed for 2 h. The gels were subsequently dehydrated i n ethanol solutions of increasing strength (30, 50, 70, 95 and 100%) and transferred into ascending concentrations of propy-lene oxide i n ethanol (1:1, 2:1, 3:0). The specimens were then dried i n a c r i t i c a l - p o i n t dryer, glued to aluminum specimen stubs and coated with a gold-palladium a l l o y i n a sputter coating device (Technics Inc.). The gels were examined i n an ETEC Autoscan scanning electron microscope at 20 kV and images recorded on Polaroid Positive/Negative 4 x 5 in f i l m . 30 E. Rheological Properties of Dispersions and Thermally-Induced Gels of the 12S Glycoprotein. 1. Sample preparation In an i n i t i a l study, samples of the 12S protein were collected from a Sephadex G-100 column and dialyzed against running water for at least 72 h. The contents of the d i a l y s i s bags were concentrated by pervaporation and subsequently dialyzed against 0.01M borate buffer, pH 9.2. Protein levels of the concentrates were determined by micro Kjeldahl analysis (%N x 6.25). This procedure was chosen instead of rehydration of the dried i s o l a t e because the l y o p h i l i z e d product appeared to lose some of i t s s o l u b i l i t y . The steady shear flow behavior of concentrates ranging from 1 to 5.4% protein was examined over a wide range of shear rates. The ef f e c t of heating upon the viscometric properties of the dispersions was also investigated. Heated samples were prepared by placing 2 ml of each sample into a 12 x 125 mm test tube which was then loosely stoppered. The tubes were weighed and placed i n a L i t t o n microwave oven (frequency, 2450 MHz) for approximately 1 sec to heat u n t i l b o i l i n g . Samples were removed immediately and cooled i n an ice water bath for about 5 min. In order to determine the possible contribution of s u l f h y d r y l - d i s u l f i d e interchange on thermally-induced gelation, a sample of 1% protein was reacted with 0.08 mM p-mercuribenzoate (PCMB) before heating. 31 Additional experiments were designed to characterize the gelation phenomenon exhibited by dispersions of the 12S glycoprotein. Samples were concentrated to approximately 5% protein (w/v) by pervaporation and subsequently dialyzed against B r i t t o n and Robinson-type universal buffers (Dawson et a l . , 1969) of pH 2, 4, 6, 8 and 10. The f i n a l concentration of each dispersion was adjusted to 4.5% (%N x 6.25) with buffer. A sample of protein dispersed in 0.01M borate buffer pH 9.2 was also prepared as previously described. Aliquots of t h i s dispersion were adjusted to 0, 0.5, and 1.OM sodium chloride, IM urea and 0.15M d i t h i o t h r e i t o l i n order.^tQ observe the effects of varying ionic strength, the presence of a diss o c i a t i n g agent, and a reducing agent, respectively. A formaldehyde treatment described by Means and Feeney (1968, 1971) was used for the selective reductive a l k y l a t i o n of the e-amino groups of ly s i n e . A protein suspension (10 mg/ml) was prepared i n 0.2M borate buffer, pH 9.0, and 0.5 mg/ml NaBH^ added at 0°C. Aqueous formaldehyde (37%) was added in fi v e increments t o t a l l i n g 0.5 ul/ml suspension over a period of 30 min. The suspension was dialyzed against water to remove the excess formaldehyde and pervaporated to approximately 5% protein (w/v), then dialyzed against 0.01M borate pH 9.2. After d i a l y s i s , the ' f i n a l concentration was adjusted to 4.5% protein (%N x 6.25) with borate buffer i n preparation for rheological characteri-zation. Samples of water-dialyzed, derivatized protein as w e l l as u n d e r i v a t i z e d 12S p r o t e i n were l y o p h i l i z e d and the e-amino groups determined by the t r i n i t r o b e n z e n e s u l f o n i c a c i d (TNBS) method of Eklund (1976). A l l samples i n the second experiment were g e l l e d i n stoppered 12 x 125 mm t e s t tubes by h e a t i n g i n b o i l i n g water f o r 5 min and c o o l i n g i n i c e water f o r 2 min. 2. R h e o l o g i c a l c h a r a c t e r i z a t i o n Steady shear flow p r o p e r t i e s of the d i s p e r s i o n s i n the i n i t i a l experiment were evaluated over more than three decades of shear r a t e with a Model R.18 Weissenberg Rheo-goniometer (Sangamo C o n t r o l s L t d . , 1971) u t i l i z i n g 10 cm diameter 0.25° cone/plate f i x t u r e s . In experiments designed to t e s t the e f f e c t o f pH and v a r i o u s a d d i t i v e s on g e l a t i o n , the g e l s were t e s t e d under steady shear to measure flow behavior and dynamic c o n d i t i o n s o f sma l l amplitude o s c i l l a t o r y shear i n order to determine v i s c o e l a s t i c p r o p e r t i e s . Both 10 cm diameter 0.25° and 5 cm diameter 2° cone/plate f i x t u r e s were employed. The shear s t r a i n i n p u t s i g n a l s , the r e s u l t a n t sheaf s t r e s s output s i g n a l s and the phase d i f f e r e n c e between the two s i n u s o i d a l l y v a r y i n g f u n c t i o n s were monitored with a Tronotec Model 703A d i g i t a l a n a l y z e r (Tronotec Inc., F r a n k l i n , N.J.) i n the dynamic shear experiments. Because these t e s t s r e q u i r e d r e l a t i v e l y l a r g e q u a n t i t i e s of the p r o t e i n , o n l y one run was performed f o r most treatments. IV RESULTS AND DISCUSSION A. Microstructure and Ultrastructure of the Rapeseed Kernel (Brassica campestris) and Rapeseed Meal. The study of the microstructure and ultrastructure of the rapeseed kernel was i n i t i a t e d to elucidate the c e l l u l a r organization and l o c a l i z a t i o n of the proteinaceous materials. The intact seed i s about 1 to 2 mm i n diameter, spherical and black or reddish brown depending upon the variety. A section through a rapeseed kernel as observed with the l i g h t micro-scope i s i l l u s t r a t e d i n Figure I I . The section demonstrates the fact that rapeseed, unlike many other seeds, contains almost no endosperm. Instead, the major protein and l i p i d -containing tissues are found i n the cotyledons (C) which are the embryonic leaves and the meristematic tissue (M), from which the.radicle (root), hypocotyl (stem) and e p i c o t y l (bud) w i l l develop. The endosperm, which usually serves a storage function to provide energy during germination and early stages of development i s r e s t r i c t e d to a single layer of c e l l s (E) located beneath the epidermis. The thick c e l l walls and seed coat contribute to the high crude f i b e r content (up to 11.5% on dry ba s i s ) . The tissue section i l l u s t r a t e d i n Figure II was stained by the PAS method and i t i s apparent that no areas of concentration of S c h i f f - r e a c t i v e material ex i s t . 33 34a FIGURE II: Light micrograph of a section of rapeseed stained by PAS method. Note cotyledons (C), meristematic tissue (M) and endosperm (E). FIGURE II I : Light micrograph of rapeseed tissue stained with toluidene blue and showing the protein-r i c h aleurone grains (A). Note the l i g h t e r staining and fragmentation of the aleurones in an area of rapid c e l l d i v i s i o n (arrows). 34b 35 Figure III i s a photomicrograph of a section of a rapeseed kernel which was stained with toluidene blue. The major proteins of rapeseed (and most other oilseeds) are located i n subcellular packages c a l l e d aleurone grains (A) that are considered to have a storage function supplying the seedling with organic nitrogen rather than s t r u c t u r a l or c a t a l y t i c functions (Appelqvist and Ohlson, 1972; Dieckert and Dieckert, 1976). The term "aleurone" was f i r s t used by Hartig (1855) who is o l a t e d protein bodies from various o i l -seeds by a nonaqueous technique. Since the defatted p a r t i c l e s resembled f l o u r , he named them "aleuron" a f t e r the Greek word for f l o u r . In several sections i t was observed that rapidly dividing c e l l s contained many small and poorly-defined aleurones (arrows) as opposed to the t y p i c a l globular inclusions i n non-dividing tissue. C e l l s about to divide or undergoing cytokinesis contain aleurones which stain weakly for protein, presumably due to the u t i l i z a t i o n of the aleurone protein for the formation of new tissue. Available experimental evidence indicates that the "aleurins" or aleurone proteins are synthesized on the rough endoplasmic reticulum and then transported by way of the lumina of the endoplasmic reticulum to the dictyosomes (Dieckert and Dieckert, 1976), where the protein i s concen-trated into droplets. The membrane-bounded protein droplets migrate through the cytoplasm to the aleurone vacuoles and pass through the vacuolar membrane by a process of membrane 36 fusion. F i n a l l y , the mature aleurone grains form by dehydration of the aleurone vacuoles. Since the 12S glycoprotein i s the major rapeseed protein and i s highly reactive to Schiff reagent, i t would be reasonable to expect that those aleurones containing large quantities of thi s protein would themselves be PAS-positive. Figure IV i l l u s t r a t e s that there are indeed aleurones which contain high levels of S c h i f f - r e a c t i v e material suggesting that for some reason, the 12S glycoprotein i s synthesized by some c e l l s but not others. The PAS-positive aleurones were dis t r i b u t e d randomly throughout the tissue. A micrograph of rapeseed meal stained with S c h i f f reagent (Figure V) i l l u s t r a t e s that both fragmented (FA) and intac t aleurones (A) are present. Fragmented c e l l walls (CW) appear to comprise a sizeable portion of the meal. Figure VI i s an electron micrograph of rapeseed showing the inter n a l structure of the c e l l . The mature rape-seed, l i k e many other oilseeds, i s remarkably free of cyto-plasmic organelles, the major c e l l u l a r components being vacuolated l i p i d (L) and aleurone grains (A). Note also the thick c e l l wall (C), nucleus (N) as well as the presence of globoid bodies (G) within the aleurone grains. Dieckert.et a l . (1962) found peanut globoids contained phytic acid, potassium, magnesium and copper. Similar r e s u l t s were found for cotton. (Lui and Atschul, 1967) and cucurbita (Lott, 1975). 37a FIGURE IV: Light micrograph of a rapeseed kernel tissue c e l l r i c h i n PAS-positive aleurone grains (A). The PAS-rich c e l l s appeared to be evenly distributed throughout the entire seed. FIGURE V: Light micrograph of commercial rapeseed meal stained by the PAS procedure. Note that both fragmented (FA) and intact (A) PAS-positive aleurones are present. 37b FIGURE VI: Electron micrograph of rapeseed showing in t e r n a l c e l l u l a r structure. Note the thick c e l l wall (C), aleurone grains (A), nucleus (N) and l i p i d vacuoles or spherosomes (L). FIGURE VII: Electron micrograph demonstrating the aleurone grain (A) surrounded by a single membrane (M) and l i p i d bodies (L). 39 Similar to other oilseeds, the aleurone grains of rapeseed are surrounded by a unit membrane (M i n Figure VII). L i p i d bodies (L) constitute a major portion of the cytoplasm. The importance of ef f e c t i v e m i l l i n g should be emphasized i f meal i s to be used for the purpose df protein recovery. Optimum protein extraction would not l i k e l y be achieved as long as the aleurone membrane was in t a c t . Figure V has i l l u s t r a t e d that i n some cases, commercial m i l l i n g i s inadequate to fragment a l l aleurones. B. Recovery of the 12S Protein from Rapeseed Meal. The solvent-extracted meal is. a by-product of the vegetable o i l industry. I t has t r a d i t i o n a l l y been used as f e r t i l i z e r or as an animal feed supplement. The current study was carried out on t h i s highly denatured material because i t i s l i k e l y that rapeseed w i l l always be cul t i v a t e d primarily as a source of o i l and that secondary protein production w i l l u t i l i z e the inexpensive meal. Although the terms "rapeseed protein" and "12S rapeseed protein" are referred to i n the text, i t i s not intended that the reader confuse t h i s material obtained from a highly denatured source with that obtained from the .intact rapeseed kernel. The p u r i f i c a t i o n procedure was a modification of that described by Goding et a l . (1970). In t h i s procedure, 40 a high-speed centrifugation step was included i n order to concentrate the high molecular weight components. This step was eliminated i n the present study for the following reason. Ultracentrifugation indicated that the major contaminant of the 12S glycoprotein was determined to be of a higher molecular weight (17S), thus high speed centrifugation would tend to enrich the 17S contaminant. Since gel f i l t r a t i o n rather than protein extraction was a r a t e - l i m i t i n g step i n recovery, NaCl was eliminated from the extraction buffer described by Goding et al.. The nitrogen recoveries for extraction and p u r i f i c a t i o n of the 12S i s o l a t e are shown in Figure VIII. The commercial defatted meal contained 5.5% nitrogen and yielded a crude alkaline extract containing 0.22% nitrogen of which approximately 50% appeared i n the f i r s t peak when subjected to gel f i l t r a t i o n (Figure IX). The column void volume was approximately 500 ml so that the 12S peak was at least p a r t i a l l y excluded by the column. Further p u r i f i c a t i o n to remove the 17S or other contaminants was not attempted for p r a c t i c a l purposes. The freeze-dried i s o l a t e produced by t h i s procedure was f l u f f y and white, contained 12.5% nitrogen and although quite bland, possessed a s l i g h t l y nutty aft e r t a s t e . The term " i s o l a t e " may perhaps be confusing when applied to the rapeseed 12S glycoprotein since i t has been suggested by Burrows et a l . (1972) that t h i s term be r e s t r i c t e d to a preparation containing a minimum of 90% 41a FIGURE VIII: Flow diagram for the preparation of 12S rapeseed i s o l a t e and concentrated dispersions. FIGURE IX: Elution p r o f i l e of the gel f i l t r a t i o n of crude rapeseed extracts on Sephadex G-100 in 0.1M borate buffer pH 9.2. Fractions were col l e c t e d in 10 ml f r a c t i o n s . 41b PREPARATION OF 12 S ISOLATE 5 . 5 % N DEFATTED MEAL 6.3% N SIFTING (60 flESH) EXTRACTION CENTRIFUGATION 0.22% N SUPERNATE GEL FILTRATION 12 S RECOVERY 50% OF TOTAL N APPLIED TO COLUMN D I A L Y S I S vs HoO \ PERVAPORAT X ON OR LYOPHILIZATION - ISOLATE D I A L Y S I S OF CONCENTRATE (O.OLN BORATE, PH 9,2) E c Tube No. 42 protein (N x 6.25) on a moisture-free basis when applied to manufactured soybean products. However, the Kjeldahl factors for glycoproteins may be higher than 6.25, p a r t i c u l a r l y when a s i g n i f i c a n t l e v e l of neutral sugar i s present. Thus, the nitrogen content of a protein does not necessarily r e f l e c t the degree of purity i n a preparation. C. Chemical Characterization of the 12S Rapeseed Globulin. j • 1. Amino acid composition The r e s u l t s of the amino acid analysis are tabulated i n Table II. Five samples representing f i v e hydrolysis times (24 to 36 h) were analyzed and the nitrogen recoveries calculated. The data i n Table II represents a 27 h hydrolysis in which the nitrogen recovery was 94%. Moore and Stein (1960) have suggested that data for serine, threonine, tryptophan and half cystine be extrapolated to zero hydrolysis time, however, these values were found to maximize at d i f f e r e n t times. The t o t a l nitrogen recoveries were 68, 78, 82 and 87% for the 24, 36, 25 and 28 h hydrolysis times, respectively. Results from the runs i n which lower recoveries were observed are l i s t e d i n Appendix V. The 12S protein would appear to be a t y p i c a l o i l -seed aleurin (Dieckert and Dieckert, 1976) in that r e l a t i v e l y TABLE I I : AMINO ACID COMPOSITION OF THE 12S GLYCOPROTEIN FROM RAPESEED MEAL Amino Acid , g R e s i d u e P e r ^ per 16g N log N recovered recovered Aspartic acid 9.56 83.0 Threonine 3.82 37. 7 Serine 4.91 56.4 Glutamic acid 20.9 162 Proline 4. 20 4 3.3 Glycine 4.65 81.4 Alanine 3.87 54.4 Valine 3.86 38.9 Methionine 1.88 14.3 Isoleucine 3.60 31. 8 Leucine 7.42 65.6 Tyrosine 3.13 19.2 Phenylalanine 4. 93 33. 5 Ammonia 2.68 158 Lysine 2.96 23.1 Histidine 2.87 20.9 Tryptophan 0 0 Arginine 6. 03 38.6 Pyridylethy1-L-cystine Trace Trace Total recovery (%N) 94 44 high lev e l s of glutamic acid, aspartic acid and arginine are present. The amino acid composition of the aleurins i s usually biased toward the more re a d i l y u t i l i z a b l e acids-arginine, glutamic acid (glutamine) or aspartic acid (asparagine). Other notable features of the 12S amino acid p r o f i l e are the absence of tryptophan and the s c a r c i t y of 1/2 cystine and methionine. These trends were observed by Finlayson et a l . (1969) and Goding et a l . (1970) for a 12S globulin extracted from Brassica campestris and by MacKenzie and Blakely (1972) for a s i m i l a r protein extracted from B. nigra, B. juncea and B. h i r t a . Goding et aJL. (1970) reported 0.145 and 0.05 mM of 1/2 cystine and tryptophan, respectively, per gram 12S protein is o l a t e d from B. campes-t r i s compared to means of 0.002 and 0 mM of these amino acids found per gram i s o l a t e i n the present study. Although precautions were taken to protect these l a b i l e amino acids, degradation during acid hydrolysis may account for the differences i n composition. 2. Carbohydrate composition Goding et a l . (1970) reported the presence of 0.15 to 0.20% galactosamine and 1.0 to 1.5% glucose and arabinose, with the major neutral sugar being glucose. These re s u l t s were reported for the 12S globulin i s o l a t e d from B. campestris L. var. Echo and B. napus L. var. Target. The 12S protein was separated into four chromatographically d i f f e r e n t 45 components at pH 2.8 i n the presence of 2M urea by the same authors. A fragment containing 20% of the t o t a l nitrogen (2.7S) also contained 0.5% of i t s nitrogen as galactosamine and 4 to 5% reducing sugar. The 2.7S fragment did not reduce to smaller subunits i n the presence of 0.01M mercaptoethanol. The present investigation was i n i t i a t e d in order to obtain similar data on the 12S glycoprotein i s o l a t e d from commercial rapeseed meal (B. campestris L. var. Span). Results of the phenol-sulphuric acid determination of t o t a l sugar yielded an estimated 8% on the basis of a standard curve for glucose. I t should be emphasized that because various sugars y i e l d d i f f e r e n t r e s u l t s , t h i s method cannot be considered quantitative. Results of the s i a l i c acid determination (N-acetyl neuraminic acid) indicated the absence of t h i s material i n the i s o l a t e although i t s presence was suspected because of electrophoretic behavior. The gas chromatographic analysis of both neutral and amino sugars indicated the presence of 6 components. A t y p i c a l gas chromatogram of the monosaccharide derivatives ( a l d i t o l acetates) i s i l l u s t r a t e d i n Figure X. The peaks i d e n t i f i e d by retention times as compared to those of authentic sugars were (a) arabinose, (c) mannose, (d) galac-tose, (e) glucose, and (f) i n o s i t o l . A peak (b), emerging shortly a f t e r arabinose, was found to have a retention time 46a FIGURE X: A t y p i c a l gas chromatogram of the a l d i t o l acetate derivatives of the neutral and amino sugars in the 12S glycoprotein. a) arabinose b) unidentified c) mannose d) galactose e) glucose f) i n o s i t o l Minutes 47 equal to that of both glucosamine and xylose. No such other coincidences i n retention times were observed on the ECNSS-M column for a wide range of standard sugars tested. Since glucosamine and xylose were not resolvable, glucosamine content was quantitated c o l o r i m e t r i c a l l y by the Elson-Morgan reaction (1933). Results of the sugar analyses are tabulated for the freeze-dried 12S i s o l a t e i n Table III along with amino acid content (from Table I I ) , assuming 100% recovery. A c a l c u l a t i o n of t h e o r e t i c a l p a r t i a l s p e c i f i c volume (Vp) (Schachman, 1957)utilizing monosaccharide p a r t i a l s p e c i f i c volumes c i t e d by Gibbons (1966) i s included i n Table I I I . The t o t a l sugar content of the 12S glycoprotein was found to be 12.9% while the remaining 87.1% was presumed to be amino acids. This figure would appear reasonable since the l y o p h i l i z e d i s o l a t e contained 12.5% nitrogen. An oven-dried sample of the i s o l a t e contained s l i g h t l y more nitrogen (13.4%) indicating that a small amount of moisture could not be removed by conventional freeze drying. I t i s evident from these data that there are large v a r i e t a l differences or differences between seed and meal. Although amino acid p r o f i l e s are similar, the carbohydrate and nitrogen contents d i f f e r considerably from those reported by Goding et a l . (1970) for the 12S protein is o l a t e d from the seeds of B. campestris L. var. Echo and B. napus L. var. Target. Instead, the 12S protein i s o l a t e d from commercial meal more cl o s e l y resembles "f r a c t i o n A" i s o l a t e d from B. nigra, B. juncea and B. h i r t a by MacKenzie and Blakely (1972) and from B. juncea TABLE I I I : MONOSACCHARIDE* AND AMINO ACID . COMPOSITION OF THE 12S GLYCO-PROTEIN FROM RAPESEED MEAL (B. CAMPESTRIS L. VAR. SPAN) Component % (w/w) P a r t i a l s p e c i f i c 7 } • - volume (Vi) v ' Arabinose 7.24 +0.16 0.613 4.44 Glucosamine 0.29 +0.042 0.666 0.193 Mannose 0.340+0.030 0. 613 0. 208 Galactose 3.32 +0.12 0. 613 2.04 Glucose 1 0.870+0.019 0.613 0.533 In o s i t o l 0.840+0.077 0.613 0.515 S i a l i c acid 0 Aspartic acid 9.40 0.59 5.55 Threonine 3.75 0. 70 2.63 Serine 4.83 0. 63 3.04 Glutamic acid 20.6 0.66 13.6 Proline 4.13 0.76 3.14 Glycine 4.57 0.64 2.92 Cont'd. TABLE I I I : Continued Component % (w/w) P a r t i a l s p e c i f i c volume (Vi) % (w/w) x Vi Alanine 3.80 0.74 2.81 Valine 3.79 0.86 3.26 Methionine 1.85 0.75 1. 39 Isoleucine 3.54 0.90 3.19 Leucine 7. 29 0.90 6. 56 Tyrosine 3.08 0.71 2.19 Phenylalanine 4. 85 0.77 3.73 Lysine 2.91 0.82 2. 39 Hist i d i n e 2.82 0.67 1.89 Tryptophan 0 Arginine 5.93 0.70 4.15 Pyridylethy1-L-cystine Trace . .. Cont'd. TABLE I I I : Continued Theoretical p a r t i a l s p e c i f i c volume (see Schachman, 1957) V P = I \ ("/"j * V i = 0.704 Z % (w/w) * A l l monosaccharide data except glucosamine reported as a mean of 4 determinations ± standard error of the mean. Glucosamine reported as a mean of 6 determinations - standard error of the mean. + Amino acid values calculated from those of Table II assuming 100% recovery. o 51 byMacKenzie (1975). The n i t r o g e n content of t h i s f r a c t i o n ( c o n t a i n i n g mainly 12S p r o t e i n ) was 12.7, 13.7 and 14.9% f o r B. n i g r a , B. juncea and B. h i r t a , r e s p e c t i v e l y ; Despite the s i m i l a r i t y i n amino a c i d and n i t r o g e n content between the 12S p r o t e i n i s o l a t e d from rapeseed meal and t h a t i s o l a t e d by the above authors, l a r g e d i s c r e p a n c i e s remain i n the carbo-hydrate c o n t e n t s . A major f a c t o r i n t h i s d i s c r e p a n c y c o u l d be the accuracy of the p h e n o l - s u l f u r i c a c i d method used by MacKenzie and B l a k e l y (1972) f o r t h i s m a t e r i a l . Only s l i g h t l y more than 50% of the t r u e sugar content was d e t e c t e d with t h i s technique i n the p r e s e n t study. The d i f f e r e n c e s i n carbohydrate content of the 12S aggregates c o u l d a l s o be e x p l a i n e d by the e f f e c t s o f commercial f a t e x t r a c t i o n . I t has been r e p o r t e d t h a t the content of r e d u c i n g sugars and a v a i l a b l e l y s i n e i n commercial meal drops a p p r e c i a b l y due to the h i g h temperatures employed ( J o s e f s s o n , 1972). T h i s phenomenon was e x p l a i n e d as a r e s u l t of the M a i l l a r d r e a c t i o n ( M a i l l a r d , 1912) i n which re d u c i n g sugars and b a s i c . amino a c i d s form condensation products v i a S c h i f f base formation. However, i f the 12S aggregate i n the p r e s e n t study were being e x t e n s i v e l y m o d i f i e d through non-enzymatic browning of the meal, such e f f e c t s would be expected to be r e f l e c t e d i n the sedimentation c h a r a c t e r i s t i c s and i n the homogeneity of the p r e p a r a t i o n . The i m p l i c a t i o n s of J o s e f s s o n ' s theory are d i s c u s s e d f u r t h e r i n S e c t i o n IV.C.4. 52 The two most notable features of the carbohydrate composition are the r e l a t i v e l y large amounts of arabinose and the presence of i n o s i t o l . Although there i s a lack of detailed information on carbohydrate components of rapeseed, Appelqvist (1972) has c i t e d the presence of sugars i n aqueous extracts of white mustard. The sugars of white mustard were predominantly arabinose, galactose and glucuronic acid and were discovered i n both hot and cold water-soluble f r a c t i o n s . The presence of i n o s i t o l i n the 12S protein may possibly be explained as follows. Phytic acid, a phosphate ester of myoinositol, has been isol a t e d from the globoid bodies of the aleurone grains of various oilseeds (Dieckert et a l . , 1962; Lui and A l t s c h u l , 1967; Lott, 1975). Rapeseed aleurones would be expected to have a s i m i l a r composition, thus there would be a p o s s i b i l i t y for i n o s i t o l incorporation into the major aleurin proteins. 3. Ultracentrifugation The 12S glycoprotein i s o l a t e d from meal was examined by sedimentation v e l o c i t y u l t r a c e n t r i f u g a t i o n . The schlieren pattern for the protein dissolved i n extraction buffer i s shown i n Figure XI.A. The major and minor components had observed sedimentation c o e f f i c i e n t s (s„_ _ £ J- ) of 12.45 v 20, Buffer and 17.25S. I t i s also apparent from t h i s photograph that a small quantity of lower molecular weight material exists in the 12S f r a c t i o n . Figure XI.B. demonstrates the e f f e c t 53a FIGURE XI: Schlieren patterns of undissociated and dissociated 12S protein extracted from commercial rapeseed meal. (A) Pattern after 17.25 min at 55,000 rpm (0.1M borate buffer pH 9.2). (B) Pattern after 82.5 min at 55,250 rpm (0.1M phosphate buffer pH 7.0 + 5M urea and 0.1% 2-mercaptoethanol). (C) Pattern after 321 min at 55,100 rpm (0.1M phosphate buffer pH 7.0 + 6M urea and 0.1% 2-mercaptoethanol). 53b 54 of 5M urea and 2-mercaptoethanol on the 12S aggregate. The photograph i l l u s t r a t e s the presence of at least 3 d i s t i n c t fragments: a high molecular weight fragment = 11.55), presumably undissociated material, and two smaller fragments whose uncorrected sedimentation c o e f f i c i e n t s were 6.3S and 0.8S. The degree of d i s s o c i a t i o n would appear dependent upon urea concentration. Figure XI.C. shows that only one low molecular weight peak i s present i n 6M urea and 0.1% 2-mercap-toethanol. The uncorrected sedimentation c o e f f i c i e n t for thi s peak was 0.64S. Results of sedimentation v e l o c i t y runs are similar to those published by Bhatty et a l . (1968) and Goding et a l . (1970) for rapeseed globulins as well as those published by MacKenzie and Blakely (1972) for f r a c t i o n A extracted from B. juncea. According to previous studies and the present data, the 12S fractions extracted from both seeds and meal are heterogeneous. I t i s d i f f i c u l t at the present time to ascertain whether the higher and lower molecular weight components of the 12S protein i s o l a t e s are minor contaminants or products of a system which r e a d i l y dissociates and self-associates, depending upon pH and temperature. The major contaminant i n a l l previous studies of rapeseed 12S globulins has been a 17S component. This component was not observed i n the urea-dissociated protein at pH 7.0. MacKenzie and Blakely (1972) also reported the presence of higher molecular weight components i n the 12S i s o l a t e s produced from B. juncea, 55 B. h i r t a and B. nigra and separated the 12S component of B. juncea from the higher molecular weight contaminants. The nitrogen content of the protein increased from 13.7% to 16 -17% during the p u r i f i c a t i o n procedure on Sephadex G-200 superfine. In a subsequent publication, however, MacKenzie (1975) stated that the amino acid composition of the high . molecular weight contaminant was indistinguishable from that of the p u r i f i e d 12S f r a c t i o n which suggests that the 15S protein i n B. juncea i s merely a dimer of the 12S component. These two points would appear contradictory, however, were not discussed by the author(s). The value of the 13.7% nitrogen for the "unpurified" 12S i s o l a t e of MacKenzie corresponds much more clos e l y to the value obtained for the 12S i s o l a t e extracted from rapeseed meal i n the present study. U l t r a c e n t r i f ugation i n urea-' as, well as re s u l t s to be discussed i n Sections IV.C..4. and IV.C. 5. suggest that the 17S and lower molecular weight "contaminants" are the products of s e l f - a s s o c i a t i o n and fragmentation of the 12S glycoprotein of rapeseed meal. Dieckert and Dieckert (1976.) have suggested that the evolutionary significance of the aleurins, often being multi-subunit s e l f - a s s o c i a t i n g proteins, i s that such assemblies reduce the i n t e r n a l osmotic pressure of the c e l l . Molecular weight determinations of such systems are tedious because of the d i f f i c u l t i e s encountered i n assessing the purity of a preparation. The 12S protein aggregate i n the present study was obviously heterogeneous by sedimentation v e l o c i t y u l t r a c e n t r i f u g a t i o n (schlieren optics) and yielded c u r v i l i n e a r p l o t s of l o g c vs. r ^ (c = c o n c e n t r a t i o n a t any p o i n t i n the c e l l , r = d i s t a n c e from cent e r of r o t a t i o n ) i n e q u i l i b r i u m runs. C o n v e n t i o n a l sedimentation e q u i l i b r i u m u l t r a c e n t r i f u g a t i o n y i e l d e d a weight average molecular weight of 129,000 d a l t o n s when s i x molecular weights were e x t r a p o l a t e d to zero c o n c e n t r a t i o n . 4. D i s c g e l e l e c t r o p h o r e s i s The p r o t e i n s of the 12S aggregate of rapeseed are o l i g o m e r i c . D i s s o c i a t i o n of the complex may be brought about by the use of urea or s o l u t i o n s a t pH values below 3.5 (Goding e t a_l. , 1970) . Assessment of e l e c t r o p h o r e t i c homo-gen e i t y of the i n t a c t complex i s , t h e r e f o r e , necessary i n an a n i o n i c system and a t a l k a l i n e pH's. However, complete assurance of e l e c t r o p h o r e t i c homogeneity cannot be o b t a i n e d a t o n l y one pH l e v e l . In a d d i t i o n to the problems i n h e r e n t i n the e v a l u a t i o n of homogeneity of o l i g o m e r i c p r o t e i n s which tend to d i s s o c i a t e and s e l f - a s s o c i a t e , other problems i n v o l v e d with the e l e c t r o p h o r e s i s of g l y c o p r o t e i n s have been w e l l documented. "Mi c r o h e t e r o g e n e i t y r e p r e s e n t s t h a t v a r i a t i o n i n the carbohydrate groups of g l y c o p r o t e i n s produced by p a r t i a l s u b s t i t u t i o n of sugar r e s i d u e s on a b a s i c a l l y s i m i l a r core s t r u c t u r e " (Montgomery, 1972). For example, the a ^ - g l y c o p r o t e i n was deemed monodisperse by a l l c o n v e n t i o n a l methods i n c l u d i n g immunological c h a r a c t e r i z a t i o n (Schmid e t a l . , 1962), however, separated i n t o 7 v a r i a n t s d u r i n g s t a r c h g e l 57 electrophoresis. Similar phenomenon have been observed i n human plasma 3-^-glycoprotein (Labat et a].. , 1969) and fet u i n (Oshiro and Eylar, 1968). Montgomery (1972) stated that the microheterogeneities found i n so many glycoproteins may represent oligosaccharides in varying stages of completion, or r e s u l t i n g from transglycosylations that do not have an absolute s p e c i f i c i t y . One would expect that the microhetero-geneities could be possible i n a system which has been heat denatured i n the presence of reducing sugars. Heterogeneities of t h i s type could occur by means of non-enzymatic browning reactions i n the defatted meal. The degree of such differences in carbohydrate composition r e s u l t i n g from Mail l a r d condensation could be expected to depend upon the types of reducing sugars present i n the meal. The l y o p h i l i z e d 12S i s o l a t e was subjected to the disc electrophoretic method of Davis (1962). Figure XII i l l u s t r a t e s a densitometric scan of a t y p i c a l gel stained with Coomassie blue. At pH 9.5, the protein migrates only slowly and i s c l e a r l y separated into a major component, and a slower moving minor component, presumably i n the 17S protein described i n the previous section. Preliminary studies u t i l i z i n g Amido black did not demonstrate a separation between these two components. Electrophoresis at pH 7.0 i n the presence of 5M urea and 0.1% 2-mercaptoethanol resulted in. a broad band which migrated slowly toward the anode as well as a second fragment which was either neutral or basic i n 58 nature. When run c a t i o n i c a l l y under the same conditions, only one band was evident and t h i s component remained close to the o r i g i n . In 10M urea and at alk a l i n e pH, the 12S protein did not appear to enter the running gel when electrophoresed a n i o n i c a l l y . MacKenzie (.1975) reported that the major dissociated fragments of the 12S protein i s o l a t e d from B. juncea were basic, with the largest proportion of the protein possessing an i s o e l e c t r i c point of 9.15. If the protein i n the present study was si m i l a r , the electrophoretic behavior could possibly suggest that the surface of the 12S aggregate occludes a highly basic amino acid backbone. An explanation for the apparent charge occlusion could be that the carbohydrate portion i s located on the surface of the structure. The amino acid p r o f i l e reported i n Section IV.C.l. suggests that the 12S rapeseed protein would be a c i d i c because of the presence of large quantities of both glutamic and aspartic acids. From the amino acid p r o f i l e s of the various i s o e l e c t r i c a l l y - s e p a r a t e d components of B. juncea, MacKenzie (1975) concluded that aspartic and glutamic acids must be present largely as the amides asparagine and glutamine. It may be reasonable to expect s i m i l a r r e s u l t s from the fragments of the 12S globulin i s o l a t e d from rapeseed meal since the amino acid p r o f i l e s are s i m i l a r , although investigation by means of i s o e l e c t r i c focussing would be necessary to confirm such speculation. Since the 12S protein undergoes d i s s o c i a t i o n , and because of the possible confusion of subunit structure with 59 the presence of contaminants, immunochemical methods of characterization were undertaken. Immunodiffusion and disc Immunoelectrophoresis were carr i e d out with p r e c i p i t a t i n g antibody produced from two d i f f e r e n t species. Figure XIII shows that the 12S protein prepared from commercial rapeseed meal was immunologically homogeneous. This photograph demon-strates the re s u l t s of an experiment i n which a wide range of concentrations of the 12S i s o l a t e were electrophoresed and the disc gels subsequently diffused against rooster anti-12S antiserum. Although two components were c l e a r l y present i n the disc gels, close examination of the p r e c i p i t i n arcs (even i n gels containing up to 400 yg protein) f a i l e d to reveal the presence of a contaminant. Similar r e s u l t s were obtained with mouse anti-12S antiserum. Diffusion of disc gels on which whole rapeseed extract (WRE) had been e l e c t r o -phoresed against rooster anti-12S antiserum resulted i n the formation of only one pair of p r e c i p i t i n arcs. Double d i f f u s i o n experiments revealed i d e n t i c a l r e s u l t s . If a foreign 17S contaminant were present i n the i s o l a t e , i t would l i k e l y have been detected by Immunoelectrophoresis since 12S and 17S components were f i r s t separated on the basis of electrophoretic mobility. Such evidence strongly suggests that the immuno-chemically homogeneous preparation of 12S glycoprotein i n the present study self-associates probably to form dimers which are immunologically i d e n t i c a l to the monomer aggregate. Since glycoproteins i n aqueous solution are often oriented such that the carbohydrate moieties^are located on 60a FIGURE XII: Densitometric scan of a disc gel of the 12S glycoprotein isolated from commercial rape-seed meal. Electrophoresis c a r r i e d out at pH 9.5, indicated that two components were separated near the o r i g i n , presumably the 12S and 17S proteins. (t) marks the position of the bromphenol blue tracking dye. FIGURE XIII: Photograph of the disc immunoelectrophoresis pattern of the 12S rapeseed glycoprotein which was diffused against rooster anti-12S antiserum. (t) marks the po s i t i o n of the bromphenol blue tracking dye. 60b :::: TT . . . . 4 :: "~ ~' ~ '"j '[•••[ !'T" ^ t | 4 t f f [ : b i b - t ; Y • tr-:::z±4:t i 1 -i 1 1 . ' "IT i 11 • [ L 1 1. . : : : [ : : : / -61 the surface of the molecule, antibodies are sometimes formed against t h i s portion rather than the protein portion located in the i n t e r i o r of the molecule. A set of double d i f f u s i o n plates were prepared i n which the p r e c i p i t a t i n g anti-12S antibody was diffused against various concentrations of the component sugars of the 12S protein. No p r e c i p i t i n arcs formed afte r one week and the plates were discarded. These results suggest that antibody production was either directed toward di-or polysaccharides or that the synthesis of a n t i -body was not directed against the carbohydrate moieties. The accuracy of the molecular weight determination of proteins by electrophoresis i n sodium dodecyl sulfate depends upon two factors: proteins must bind a constant amount of SDS r e s u l t i n g i n a constant charge to mass r a t i o , and the proteins which are reacted with SDS must assume a rod-l i k e conformation such that the lengths of the structures are proportional to t h e i r polypeptide chain lengths '(Segrest and Jackson, 1972). The anomalous behavior of glycoproteins on SDS gels has been well documented (Segrest and Jackson, 1972; Anderson et a_l, 1974; and Voyles and Moskowitz, 1974). This e f f e c t i s believed to be due to the decreased binding of SDS to the carbohydrate portion of the molecule and often results i n slower electrophoretic migration and thus over-estimates of molecular weight. The degree of molecular weight anomaly i s generally a l i n e a r function of i t s carbohydrate content, however, non-sialoproteins bind less SDS than those containing s i a l i c acid (Segrest and Jackson, 1972). In 62 oligomeric glycoproteins i n which the carbohydrate i s present in some but not a l l polypeptide fragments, the non-glyco-protein subunits would be expected to behave normally. The discontinuous buffer system of Glossmann and Nev i l l e (1971) was used for the polyacrylamide gel e l e c t r o -phoresis of the 12S glycoprotein. Figure XIV i s a densito-metric scan of an SDS gel on which the reduced, dissociated 12S aggregate was electrophoresed. Apparent molecular weights of the separable 12S fragments were 37,300±400, 30,100±1,100, 17,400±400 and 12,200±1,000 where the l i m i t s ,represent the standard errors of the estimates for four determinations. The smallest subunit t r a v e l l e d with the bromphenol blue tracking dye although i t s presence was confirmed from experiments i n which no tracking dye was used. In one of the runs, the component with the highest molecular weight was c l e a r l y separated into two components with apparent molecular weights of 42,000 and 37,600 daltons. Figure XV (upper) i l l u s t r a t e s a densitometric scan of the dissociated 12S complex which was not reduced with 2-mercaptoethanol. The major component (a) of the unreduced protein has an apparent molecular weight of 60 to 70 thousand daltons, although minor peaks which correspond to the reduced fragments (b, c and d) were observed. A high molecular weight component (e) was observed which may correspond to a small amount of unfragmented 12S protein. The lower portion of Figure XV demonstrates the e f f e c t of a 63a FIGURE XIV: Densitometric scan of an SDS disc gel (12S glycoprotein). The apparent molecular weight scale was determined with the standard proteins bovine thyroglobulin, conalbumin, ovalbumin and RNase. FIGURE XV: Densitometric scans of SDS gels which were run with unreduced dissociated 12S glyco-protein (upper) and 12S protein which was reduced with 0.15M 2-mercaptoethanol (lower). 63b 64 reducing agent on the fragmentation of the 12S aggregate. Although only trace amounts of cystine were recovered i n the amino acid analyses, i t i s evident that intermolecular d i s u l f i d e bonds are present. Similar r e s u l t s were observed when 0.15M 2-mercaptoethanol was substituted with d i t h i o t h r e i t o l . Goding et a l . (1970) reported that 0.02M 2-mercaptoethanol had no e f f e c t on extraction chromatography or electrophoresis of the 12S globulin prepared from rapeseed, however, MacKenzie (1975) reported that 0.IM mercaptoethanol did contribute to the d i s s o c i a t i o n of the B. juncea 12S aggregate, although 0.05M mercaptoethanol had no e f f e c t . Thus, i t i s possible that the eff e c t s reported i n the e a r l i e r study resulted from an i n s u f f i c i e n t l e v e l of disulfide-reducing reagent. A method similar to that of Glossmann and N e v i l l e (1971) was used to detect the presence of carbohydrate-containing fragments i n SDS gels. The technique involves the t o t a l removal of both bound and unbound SDS from the protein and gel before staining with S c h i f f ' s reagent. Figure XVI i l l u s t r a t e s a t y p i c a l densitometric scan. The only PAS-positive fragment (g) r e s u l t i n g from the di s s o c i a t i o n of the 12S aggregate migrated with the tracking dye (t) which was marked with dr a f t i n g ink before periodate oxidation and Schiff staining. A control gel which was not oxidized with .periodic acid did not react. Band (g) was broader than the lowest molecular weight fragment (12,200 daltons) observed with Coomassie blue staining technique and, i n some cases 65 ( p a r t i c u l a r l y gels with large sample loads), appeared to precede the tracking dye s l i g h t l y . One explanation for t h i s phenomenon could be that the fragment containing the carbo-hydrate contains l i t t l e protein. Glossmann and N e v i l l e (1971) reported that many true glycoproteins react only f a i n t l y with Coomassie blue, and molecular weight determinations by t h i s method were sometimes inaccurate since the Coomassie blue bands occasionally represented non-glycoprotein impurities in a preparation. Since peak f(.g) contains a l l the PAS-reactive material, i t may be possible that t h i s fragment possesses a molecular weight lower than 12,000 daltons. On the other hand, the S c h i f f - r e a c t i v e fragment may indeed be a part of the low molecular weight peak detected with Coomassie blue. Since t h i s fragment must contain a major proportion of i t s weight as carbohydrate, Coomassie blue may not react with the entire fragment but only a s p e c i f i c end of the glycoprotein complex. Iso l a t i o n of the glycopeptide (s) perhaps by i s o e l e c t r i c focussing, may resolve t h i s question. An experiment designed to test the hypothesis that the carbohydrate-containing fragment i s located on the surface of the 12S aggregate was performed by the use of SDS gel immunoelectrophoresis. SDS gels on which the dissociated 12S protein had been run were subsequently diffused against rooster anti-12S p r e c i p i t a t i n g antibody. A technique was described i n Section III.4.e. i n which the l e v e l of chemical denaturant i n the gel was reduced. Conformational changes induced by SDS i n either antigen"; or IgG could c e r t a i n l y lead to a loss i n the a b i l i t y of the antigen-antibody complex to form. After 4 or 5 days at room temperature, dense, white prec i p i t a t e s formed around the PAS-, posit i v e band i n a l l of the plates. Sample loads on the gels ranged from 10 to 400 ug 12S protein which had received extensive denaturation i n hot SDS. Upon extensive washing in saline, a technique commonly used to remove unreacted antibody (Grabar and Burtin, 19 6 4), the arcs disappeared from some of the plates. The plates which had received the highest antigen loads retained t h e i r o r i g i n a l pattern. Figure XVII i s a photograph of such a plate which was stained with Amido black to increase the contrast of the bands. The bands i n the SDS immune gels precipitated nearer to the agar-aeryl.amide Interface than i n ordinary immune gels and are aty p i c a l since they appeared somewhat feathery, possibly due to the p a r t i a l d i s s o l u t i o n of the p r e c i p i t i n arcs by the SDS. The white p r e c i p i t a t e formed i n the same place i n a l l gels. This i s not to say, however, that other fragments were not immunoresponsive since the antigen reaction with p r e c i p i t a t i n g antibody would be expected to be reduced i n the presence of the d i s s o c i a t i n g agents, urea and SDS. It i s understandable, however, that i f any fragment were expected to complex with antibody, i t would be a fragment which bound a minimum of SDS, namely, the carbohydrate-containing moiety. This experiment, although not conclusive, suggests that the 6 7 a FIGURE XVI: Upper: a photograph of a Sch i f f - s t a i n e d SDS gel showing the PAS-positive fragment which electrophoresed s l i g h t l y ahead of the tracking dye, compared with a control gel (c) which received no periodic acid treatment. Lower: a densitometric scan of a PAS-treated gel showing the position of the carbohydrate-containing band (g) and the tracking dye ( t ) . FIGURE XVII: The p r e c i p i t i n arcs formed by the PAS-positive fragment of the 12S protein i n Ionagar No. 2. A densitometric scan of a Coomassie blue-stained SDS gel i s superimposed on the plate so that the locations of the fragments could be marked. Note the "feathery" appearance of the arcs i n the presence of SDS (compare with Figure XIII). 67b T 68 / carbohydrate-containing fragment i s situated on the surface of the 12S aggregate. Perhaps confirmatory evidence could have been obtained i f the experiment was ca r r i e d out i n the absence of SDS. Such a separation may have been achieved on polyacrylamide gradient gels, however, such an experiment was beyond the scope of the present study. 5. Gel f i l t r a t i o n on Sepharose 6B and examination  of a high molecular weight component Since elution of rapeseed protein on Sephadex G-100 would not successfully separate the 12S protein from the 17S protein (Section IV.C.3.), a 12S peak from such a run was applied to a Sepharose 6B column. The elu t i o n p r o f i l e of the sample i n 0.1M borate buffer pH 9.2 i s shown in Figure XVIII. Two peaks were observed and col l e c t e d . The f i r s t peak (a) was l y o p h i l i z e d and analyzed by both sedimentation v e l o c i t y u l t r a c e n t r i f u g a t i o n and SDS gel electrophoresis. It possessed a sedimentation c o e f f i c i e n t (s„. „ ) of 33.9S i n 0.1M . 20 Buffer borate buffer and was evidently an aggregate of the o r i g i n a l 12S f r a c t i o n since no such material had previously been observed i n the ultracentrifuge. No 17S material was detected from the elution p r o f i l e . The presence of the newly formed aggregated material may possibly be explained by the u l t r a -f i l t r a t i o n step following chromatography on Sephadex G-100 and i n which the 12S sample was constantly s t i r r e d under pressure for 2 days. SDS electrophoresis was used to examine 69a FIGURE XVIII: The elution p r o f i l e of a concentrate of 12S rapeseed glycoprotein. The sample was chromato-graphed o n a 2 6 x 7 0 m m column packed with Sepharose 6B. Elution buffer was 0.IM borate, pH 9.2 run at 2 ml per min. Peaks (a) and (b) were coll e c t e d i n 3 ml fractions and l y o p h i l i z e d for further characterization. \ FIGURE XIX: Densitometric scans of SDS gels loaded with 12S (upper) and 33.9S (lower) rapeseed protein fractions prepared by chromatography on Sepharose 6B. The location of the tracking dye (t) was marked with drafting ink. 70 both peaks (a) and (b) i n order to confirm that the 33.9S peak was an aggregate of 12S material or a mixed aggregate of 12S and 17S fra c t i o n s . Figure XIX i l l u s t r a t e s two densito-meter tracings: 12S (upper) and 33.9S (lower). Both tracings appear to represent the same major fragments although the electrophoretic pattern of the 33.9S protein i s not as we l l -defined as that of the 12S protein. This may be due to the incomplete d i s s o c i a t i o n of the larger agglomerate. Although thi s experiment does not add to the evidence that the 17S protein i s a dimer of the 12S aggregate, i t does indicate that the 12S protein self-associates. MacKenzie (1975) has shown that the 12S protein of B. juncea self-associates when stored i n the cold. It has also been observed i n the present study that solutions: of the 12S rapeseed i s o l a t e become turbid at 0°C. D. Microstructural and U l t r a s t r u c t u r a l Examination of the 12S Glycoprotein. 1. Light microscopy Light micrographs of sections stained by the PAS technique (Figures XX, XXI, and XXII) indicate that the is o l a t e contains an appreciable amount of PAS-reactive carbo-hydrate. The protein lacked w e t t a b i l i t y at pH 7.0 and large pockets, believed to contain water before dehydration, are indicated i n the micrographs as "w". In many cases, globules 71a FIGURE XX: L i g h t micrograph of hydrated 12S rapeseed p r o t e i n i s o l a t e o x i d i z e d w i t h p e r i o d i c a c i d and s t a i n e d w i t h S c h i f f ' s reagent. S l i g h t l y more r e a c t i v i t y appears near the water d r o p l e t (w)-protein i n t e r f a c e (arrow). FIGURE XXI: P A S - p o s i t i v e rapeseed p r o t e i n present as g l o b u l a r masses w i t h i n the water d r o p l e t (w). Increased PAS r e a c t i v i t y occurs at the water-p r o t e i n i n t e r f a c e (arrow). 71b 72a FIGURE XXII: Light micrograph showing the water-protein interface (arrow). 7 2 b of protein were located within these pockets as shown i n Figure XXI. For some reason, the PAS reaction appeared more intense at the protein-water interfaces (arrows). One explanation for t h i s could be that the carbohydrate moiety i s more hydrophilic and thus i s attracted to the protein-water interface. 2. Transmission electron microscopy Figure XXIII i s an electron micrograph of the hydrated i s o l a t e which had been p o s i t i v e l y stained with uranyl acetate and lead c i t r a t e . It demonstrates areas of protein agglomeration (A). The 12S aggregate exhibited minimum s o l u b i l i t y between pH 3.5 and 7.2. It i s l i k e l y that the i s o e l e c t r i c point of the undissociated complex i s located within t h i s range, ^although i t may be that the i s o e l e c t r i c points of the major fragments of the dissociated complex are somewhat higher (Section IV.C.4.). Since the protein represented i n Figure XXIII was fixed at pH 7.0, i t i s possible that the tendency to clump was due to the absence of o v e r a l l e l e c t r o s t a t i c repulsion. The photomicrograph may be compared with Figure XXIV which demonstrates the u l t r a -structure of an i s o e l e c t r i c a l l y p recipitated protein produced by Keshavarz (1974). Fibrous structures composed of spherical p a r t i c l e s 4 - 5 nm i n diameter present i n i s o e l e c t r i c a l l y p recipitated a l k a l i n e extract of commercial meal were not present i n the 12S aggregate. 74a FIGURE XXIII: Electron micrograph of a po s i t i v e l y - s t a i n e d (lead c i t r a t e , uranyl acetate) section of hydrated i s o l a t e fixed at pH 7 i n 2.5% glutaraldehyde followed by 1% OsC>4. The protein appears highly agglomerated (A), perhaps as a r e s u l t of the pH of f i x a t i o n . FIGURE XXIV: Electron micrograph of a p o s i t i v e l y - s t a i n e d section of i s o e l e c t r i c p r e c i p i t a t e of rapeseed protein alkaline extract. Note fibrous structures which were not found i n the 12S is o l a t e (Figure XXIII). 75 Transmission electron microscopy of the unfixed i s o l a t e applied to carbon films i s demonstrated i n Figures XXV and XXVI. The specimens were negatively stained with aqueous uranyl acetate. Figure XXV i l l u s t r a t e s a f i e l d of view i n which several hundred of the 12S p a r t i c l e s are di s t r i b u t e d . The 12S aggregate v i s u a l i z e d by this procedure would appear to be a much more complicated structure that the 11S protein of soybean which has been examined with a si m i l a r technique by Badley et a l . (1975). The soybean globulin i s an oligomer composed of 12 subunits packed into two i d e n t i c a l hexagons placed one upon the other with a maximum p a r t i c l e diameter of 110$;; The rapeseed glycoprotein would appear to be a morula-like structure composed of more than 12 subunits and much more i r r e g u l a r l y shaped than the soybean protein. The 12S rapeseed protein has a maximum diameter of 120A and i s more or less spherical. However, caution should be excercised i n making conclusions concerning molecular size and shape from such micrographs. It i s possible that conforma-t i o n a l changes take place when a macromolecule i s transferred from solution to i t s dehydrated state. The recent work of MacKenzie (1975) on the subunit structure of the 12S globulin isolated from B. juncea showed that at least 11 d i f f e r e n t fractions could be obtained from i s o e l e c t r i c focusing experiments whereas only 6 d i s t i n c t subunits have been isol a t e d from g l y c i n i n (11S soybean protein) by a similar procedure (Catsimpoolas et a l . , 1971). An i s o e l e c t r i c focusing study was not included i n the examination of rapeseed, however, i n Electron micrograph of 12S rapeseed glyco-protein. The specimen was negatively stained and supported on a carbon f i l m . A higher magnification of the 12S glyco-protein aggregate. Mean p a r t i c l e diameter ^120A. 76b 77 view of the u l t r a s t r u c t u r a l complexity and evidence reported by MacKenzie (1975) on a related species, i t would not be surprising to f i n d the 12S glycoprotein to be more chemically heterogeneous than the IIS soybean globulin. The sta i n of Rambourg (1967) used to detect carbo-hydrate at the u l t r a s t r u c t u r a l l e v e l was found to be non-s p e c i f i c i n i t s r e a c t i v i t y as compared to Schiff reagent used i n the l i g h t microscopy of rapeseed protein. 3. Scanning electron microscopy The 12S rapeseed glycoprotein readily self-associates upon heating i n pH's of 4 and above. Thermally induced gelation was observed i n dispersions at 4.5% protein concen-t r a t i o n and measurable thickening occurred at the 1% protein l e v e l . This phenomenon w i l l be more thoroughly discussed in Section IV.E. In order to examine the effects of varying pH on the u l t r a s t r u c t u r a l properties of the thermally induced gels, cryofractured samples of the gels were examined by means of scanning electron microscopy (SEM). Figures XXVII and XXVIII represent gels which were prepared at pH's of 4 - 10 in B r i t t o n and Robinson type universal buffers (Dawson et a_l. , 1969). Gels A, B, C, and D were prepared at pH 4, 6, 8, and 10, respectively. At pH 4, the 12S protein was extremely insoluble and a thermally-induced gel matrix was unstable. 78a FIGURE X X V I I : Scanning electron micrographs of thermally-induced, cryofractured gels formed from 4.5% dispersions of 12S rapeseed glycoprotein. A - pH 4.0 B - pH 6.0 C - pH 8.0 D - pH 10.0 78b 79a FIGURE XXVIII: Higher magnifications of gel structure formed from heated 4.5% rapeseed protein dispersions showing the e f f e c t of pH on the 3-dimensional ordering. A - pH 4.0 B - pH 6.0 C - pH 8.0 D - pH 10.0 79b 80 It remained as a gel only for a few minutes and subsequently reverted back to a sol form. The pH 4.0 gel (Figures XXVII.A. and XXVIII.A.) appears as an amorphous mass. Although highly aggregated at t h i s pH (presumably near i t s i s o e l e c t r i c point), very l i t t l e space i s provided between the p a r t i c l e s of protein for the entrapment of water. At pH 6.0, the beginning of a 3-dimensional structure i s evident (Figures XXVII.B. and XXVIII.B.). Many more c a v i t i e s of smaller size are observed and thus the gel more e f f e c t i v e l y immobilizes water. Figures XXVII.C. and XXVIII.C. as well as XXVII.D. and XXVIII.D. i l l u s t r a t e a progression i n 3-dimensional ordering and a decrease i n pore size as the pH of the protein i s raised from 8 to 10. An increase i n intermolecular cross-l i n k i n g i s evident at high pH values although large differences i n e l a s t i c properties were not observed i n gels at pH 6 or higher (Section IV.E.). Part of the reason for this phenomenon could be that i n s o l u b i l i t y of the protein at pH 6 resulted i n anomalously high gel strengths. E. Rheological Properties of Dispersions and Gels of the 12S Glycoprotein. Gelation phenomena of globular proteins are poorly understood, although numerous studies concerning fibrous proteins appear i n l i t e r a t u r e . Ferry (1948) proposed that thermally induced gelation of globular proteins occurs by way of a two-stage process. The native (corpuscular) protein 81 becomes denatured and assumes a more or less extended and i r r e g u l a r form. The denatured protein often exhibits reduced s o l u b i l i t y and i s more hydrophobic than the native precursor. Under a s p e c i f i c range of pH and i o n i c strength, a gel network i s formed by the association of polypeptide chains which interact along the e n t i r e molecular length. These bonds may be covalent, e l e c t r o s t a t i c , hydrophobic or hydrogen. In a recent a r t i c l e , Tombs (1974) suggested that globular proteins form gels as a r e s u l t of aggregation of the denatured protein to form strands followed by the in t e r a c t i o n of the strands to form a gel mesh. Although complete characterization of the forces involved i n the 12S globulin gels was beyond the scope of the present study, the experiments described gave valuable information concerning the p o s s i b i l i t i e s for thermally-induced i n t e r a c t i o n . Rheological measurement has been p a r t i c u l a r l y useful i n the examination of protein in t e r a c t i o n during gel formation. The effects of heating on soybean gel formation have been assessed by C i r c l e et al_. (1964) , Wolf (1970) , Catsimpoolas and Meyer (1970), and Catsimpoolas and Meyer (1971a and b). The globulin f r a c t i o n of soybean whose major component i s an 11S oligomeric protein w i l l gel at 8% concentration or above (Catsimpoolas and Meyer, 1970). Hermansson (1975) c r i t i c i z e d the e a r l i e r methods of rheo-l o g i c a l evaluation since most of the data consisted of single point measurements and gave no i n d i c a t i o n of flow behavior 82 at various rates of shear. In addition, the term "apparent v i s c o s i t y " has been misused i n several studies since t h i s term applies to a value obtained when a non-Newtonian f l u i d i s subjected to a constant rate of shear.. The instrument used i n such studies (Brookfield Syncro-Lectric Viscometer) was equipped with d i s c - l i k e spindles which rotate i n a solution and i n which the rate of shear varies continuously across the surface of the d i s c . Such an instrument would be useful for the measurement of Newtonian l i q u i d s i n which v i s c o s i t y i s independent of shear rate but cannot provide useful information in terms of flow behavior of more complex rheological systems. The results of a preliminary experiment involving the steady shear measurements of dispersions and gels at d i f f e r e n t concentrations are demonstrated i n Figures XXIX and XXX. A l l dispersions displayed shear-thinning flow behavior c h a r a c t e r i s t i c of pseudoplastic power-law f l u i d s , that i s , apparent v i s c o s i t y (n, poise) decreased with increasing - 1 shear rate (y, sec ) according to the r e l a t i o n : • n-1 n = m y where m i s the consistency index (dyne sec" cm ^) and n i s the flow behavior index (dimensionless). The power law parameters for the steady shear flow behavior of the various rapeseed protein dispersions at 25°C are summarized i n Table IV. S i g n i f i c a n t increases i n v i s c o s i t y were observed when samples containing as l i t t l e as 1% protein were heated. Gelation due to microwave heating was obtained r e a d i l y at 83a FIGURE XXIX: Rheograms of 12S protein i s o l a t e dispersions in 0.01M borate buffer pH 9.2. Heated samples were prepared i n a microwave oven. • 1.0% unheated A 2.7% heated o 5.4% unheated • 5.4% heated 8 3b FIGURE XXX: Rheograms indicating the eff e c t s of heat and PCMB on v i s c o s i t y of 1% dispersions of 12S protein i s o l a t e at pH 9.2. o unheated + PCMB A heated • heated + PCMB 84b SHEAR RATE ( S E C - 1 ) TABLE IV: POWER-LAW PARAMETERS FOR STEADY SHEAR FLOW BEHAVIOR OF AQUEOUS 12S GLOBULIN DISPERSIONS AT 25°C AND pH 9.2. (EXPERIMENT I) Dispersion Consistency coef., * n -2 dyne sec cm Flow behavior index C o e f f i c i e n t of determination 1.0% Unheated 0.670 0.21 0. 96 2.7% Heated 6. 82 0.11 0.99 5.4% Unheated 4. 37 0.23 0.99 5.4% Heated 69. 6 0.17 0.99 1.0% Heated 1.09 0.20 0.98 1.0% Heated + PCMB 1.20 0.21 0.98 1.0% Unheated + PCMB 0. 315 0.073 0. 99 86 the 5.4% protein l e v e l . Water loss due to t h i s rapid method of heating as determined by weighing was not detectable. Treatment with the sulfhydryl blocking reagent, PCMB, f a i l e d to r e s u l t i n a decrease i n v i s c o s i t y after heating (Figure XXX). The effects of varying heating time were not investigated due to the d i f f i c u l t y with which samples of the 12S i s o l a t e were obtained. In a second experiment, however, samples were heated much more slowly. A l l gels i n the second study were prepared i n sealed test tubes which were placed i n b o i l i n g water for 5 min. Figure XXXI demonstrates the e f f e c t of various additives on the steady shear flow behavior of pH 9.2 gels. Perhaps the most notable feature of the rheogram i s that samples of 4.5% protein gelled on d i f f e r e n t days yielded different:flow properties. The gel which was formed from a dispersion stored for 4 days at 4°C, had a n 2 consistency c o e f f i c i e n t of 104 dyne sec cm , whereas the consistency c o e f f i c i e n t of a gel produced 4 days e a r l i e r from n — 2 the same stock dispersion was only 16.3 dyne sec cm . A possible explanation for t h i s phenomenon could be that the protein self-associates i n the cold to form higher molecular weight complexes which i n * turn produce firmer gels. The temperature dependent s e l f - a s s o c i a t i o n of the 12S protein of B. juncea has been reported by MacKenzie (1975). Because of t h i s phenomenon, i t i s d i f f i c u l t to make conclusions concerning the effects of additives on thermally-induced intermolecular bonding. 87a FIGURE XXXI: Rheograms of pH 9.2 12S protein gels (4.5%.) measured under steady shear at 23°C. Samples were prepared by placing i n 100°C water bath for 5 min. • no additives • + IM urea • + .15M d i t h i o t h r e i t o l T + .5M NaCl O + IM NaCl A no additives but aged 4 days at 4°C 87b 88 The e f f e c t s of IM urea and 0.15M d i t h i o t h r e i t o l on gel formation were not as dramatic as those of varying ion i c strength and pH. The intera c t i n g e f f e c t of aging the dispersions in the cold makes the assessment of contributions of hydrogen and d i s u l f i d e bonding d i f f i c u l t , however, neither of the reagents were capable of preventing gelation when added at the stated l e v e l s . Gels containing urea or d i t h i o t h r e i t o l possessed higher consistency c o e f f i c i e n t s than the pH 9.2 gel containing no additives but exhibited lower apparent v i s c o s i t i e s (at the shear rates tested) than the gel prepared from an "aged" dispersion. Unlike g e l a t i n gels which are believed to be primarily cross-linked with hydrogen bonds (Bello and Vinograd, 1958), the rapeseed protein gels remained r e l a t i v e l y uneffected. Refrigerated g e l a t i n was found to gel at the 4.5% protein l e v e l i n the presence of IM urea, however, reverted to a sol when warmed to 22°C. Similar reversion was not observed for ge l a t i n i n the absence of urea. The pH 9.2 rapeseed protein gels were observed to be thermally i r r e v e r s i b l e , at least up to 100°C although measurements of gel strength were not taken at elevated temperatures. Attempts to dissolve the rapeseed protein gels i n 8M urea were unsuccessful although slow disruption of the gel took place when the temperature of the d i s s o c i a t i n g agent approached 100°C. Such observations suggest that although hydrogen bonding may be present, i t i s not l i k e l y to be a major factor involved i n the formation of intermolecular cross-links during the gelation of the 12S rapeseed protein. 89 The e f f e c t of the d i s u l f i d e reducing agent, d i t h i o t h r e i t o l , upon gelation was not dramatic. Although tempting, the v a l i d i t y of concluding the absence of s u l f h y d r y l -d i s u l f i d e interchange may be questionable. Catsimpoolas and Meyer (1970) found that while low concentrations of mercap-toethanol (0.1%) inh i b i t e d the gelation of soy protein, high concentrations (10%) actually enhanced i t . The same authors reported that the addition of 0.1% N-ethylmaleimide (a sulfhydryl blocking reagent) to soybean dispersions had no e f f e c t on gelation. Although the existence of intermolecular d i s u l f i d e bonding has been demonstrated i n the 12S glyco-protein complex (Section IV.C.4.), i t i s d i f f i c u l t to imagine that the small amount of cystine reported i n the amino acid p r o f i l e could r e f l e c t such a highly cross-linked network within the gel. Figure XXXI also i l l u s t r a t e s the e f f e c t of increased i o n i c strength on the apparent v i s c o s i t y of the gels. Gels which were adjusted to 0.5M and 1.0M NaCl demonstrated dramatically higher consistency c o e f f i c i e n t s (39 6 and 472 dyne n ~ 2 sec cm , respectively) and s l i g h t l y higher flow behavior indices than gels with no additives. These data are l i s t e d in Table V and contrast the r e s u l t s published for soybean iso l a t e s (Catsimpoolas and Meyer, 1970, and Hermansson, 1972). It was postulated i n these studies that lowering the i o n i c strength of a soybean protein dispersion resulted in strong intermolecular forces of replusion, r e s u l t i n g i n d e s t a b i l i -zation of the quaternary structures of the major globulins. 90 If i o n i c bonds were of major importance i n rapeseed protein gels, increased io n i c strength would be expected to r e s u l t i n reduced gel strength due to competition of the ions for the interacting functional groups of the protein. However, i t may be that the 12S aggregate becomes much more soluble at high io n i c strength, allowing more e f f e c t i v e overlapping of functional groups. No experiments were performed to further investigate the e f f e c t of ions on gelation. In the previous section, the e f f e c t s of varying pH on the ultrastructure of rapeseed gels were discussed. Unfortunately, rheological data on a l l of the gels were not obtained, however, some of the r e s u l t s are presented i n Figure XXXII and Table V. The highest apparent v i s c o s i t i e s were obtained from gels prepared at pH 10. Although not shown on the graph, a gel formed at pH 4 was unstable and quickly reverted to a sol form. No gel formation was observed at pH 2 although the 12S glycoprotein was much more soluble at pH 2 than at any of the other pH l e v e l s . The gel strengths as r e f l e c t e d by the rheograms indicate l i t t l e difference between pH 6, 8 and 9.2 gels. The data from Table suggests that the pH 6.0 gel was firmer than both pH 9.2 and pH 8.0 gels. This r e s u l t does not appear l o g i c a l and i s not consistent with the u l t r a s t r u c t u r a l evidence presented i n Section IV.D.3. I t i s possible that the high apparent v i s c o s i t i e s observed for the pH 6.0 gel resulted from protein i n s o l u b i l i t y rather than an increase i n s t r u c t u r a l i n t e g r i t y . Catsimpoolas and Meyer (1970) reported that the gelation of 91a FIGURE XXXII; Rheograms of 4.5% rapeseed protein gels measured under steady shear at 23°C. pH adjustments were made by d i a l y s i s against Britton-Robinson universal buffers. • pH 10 O pH 8 A pH 6 • pH 2 • modified with NaBH^ and formaldehyde A "aged" 12S protein, pH 9.2 91 b TABLE V: POWER-LAW PARAMETERS FOR STEADY SHEAR FLOW BEHAVIOR OF 4.5% 12S GLOBULIN GELS AT 2 3°C. (EXPERIMENT II) Consistency coef., Flow behavior Co e f f i c i e n t of dyne sec cm index determination pH 9.2 16. 3 0.30 0.99 pH 9.2 "aged"* 104 0. 25 0.99 pH 9.2 + IM urea 54.1 0.15 0.99 pH 9.2 + 0.15M DTT 71.1 0.26 0.99 PH 9.2 + 0.5M NaCl 396 0 . 36 0.99 pH 9.2 + IM NaCl 472 0. 38 0.99 pH 10 300 0.13 0.99 pH 8.0 70.8 0. 20 0.99 PH 6.0 133 0.28 0.99 pH 2.0 0.109 0.89 0.99 pH 9.2 "modified"** 8.35 0.43 0.99 Cont'd. TABLE V: Continued * pH 9.2 "aged" gel refers to a sample prepared from a 4.5% rapeseed protein dispersion which had been stored for 4 days at 4°C. ** pH 9.2 "modified" gel refers to a sample prepared from a 4.5% rapeseed protein dispersion which had been reduced and alkylated by the technique of Means and Feeney (1971). 94 soybean globulins was pH-dependent, gel strengths increasing with corresponding increases in pH below pH 10. The e f f e c t of increasing pH was explained p a r t i a l l y by an increased s o l u b i l i t y and molecular expansion due to an increase i n net negative charge (Hermansson, 1972). In order to determine the possible involvement of lysine i n some form of cr o s s - l i n k i n g reaction, a sample of the 12S protein was reduced with low l e v e l s of sodium boro-hydride and subsequently treated with formaldehyde i n order to methylate the free e-amino groups (Means and Feeney, 1971). The 12S protein (4.5% protein) modified by t h i s procedure did not gel to form a s o l i d self-supporting matrix (Figure XXXII). When the content of free e-amino groups of lysine was determined by the method of Eklund (1976), no differences were detected between modified and unmodified 12S protein, indicating that modification of some other functional group ess e n t i a l for gelation had taken place i n preference to e-amino. No further experimentation was carried out i n order to determine the i d e n t i t y of t h i s functional group. I t may be that the crosslinking reactions involve carbohydrate as well as amino acid functional groups, however, more research i s required to e s t a b l i s h such a re l a t i o n s h i p . The steady shear rheological data are commonly used for the characterization of gelatinous semi-solids. Gels, however, behave neither i n a purely viscous nor purely 95 e l a s t i c manner. A v i s c o e l a s t i c material i s one which simultaneously exhibits both e l a s t i c and viscous responses to applied stress. This complex rheological behavior i s displayed by a l l polymeric materials (Wohl, 1968). The dynamic response which i s a manifestation of the v i s c o e l a s t i c material properties was measured for rapeseed gels in the Weissenberg Rheogoniometer. This instrument measures the shear stress r e s u l t i n g i n a material which i s subjected to a sinuso i d a l l y varying shear s t r a i n . The sample i s located in a gap between a c i r c u l a r f l a t plate and a cone of equal diameter, providing a small angle between the cone and plate. The applied s t r a i n measured i n the d r i v i n g mechanism of the lower platen and the r e s u l t i n g stress transferred by the sample to the upper platen are transduced as e l e c t r i c a l signals which can be monitored,. A t y p i c a l input s t r a i n wave and output stress wave i s shown i n Figure XXXIII. In t h i s diagram, the shear stress and s t r a i n signals are separated by a phase difference, (f», the tangent of which i s a d i r e c t measurement of the r a t i o of energy l o s t as heat due to viscous flow and the energy stored due to e l a s t i c deformation. The e l a s t i c and viscous components may be separated and expressed as the dynamic shear moduli, G' and G", which describe the v i s c o e l a s t i c behavior of a material. G1 i s the storage modulus which i s a measure of the energy stored and recovered i n a cycle of deformation, and G" i s the loss modulus which i s associated with d i s s i p a t i v e e f f e c t s . Both moduli may be expected to depend on the o s c i l l a t o r y frequency FIGURE XXXIII: Time p r o f i l e of a dynamic shear experiment on a v i s c o e l a s t i c material as measured with the Weissenberg Rheogoniometer. The shear s t r e s s (a) and shear s t r a i n (y) waves are shown separated by the phase angle (f>. of the dynamic t e s t s . Figures XXXIV and XXXV i l l u s t r a t e the e f f e c t s of frequency of o s c i l l a t i o n on the storage and loss moduli, respectively, for the pH 9.2 gels containing various a d d i t i v e s . The high degree of c r o s s - l i n k i n g i n a l l gels, p a r t i c u l a r l y those prepared i n high s a l t concentrations, i s indicated by the r e l a t i v e l y constant values of the storage modulus over the range of o s c i l l a t o r y frequency. Even at low frequencies 97a FIGURE XXXIV: Dynamic shear storage moduli as a function of o s c i l l a t o r y frequency for 4.5% rapeseed protein gels at pH 9.2 and 23°C. • no additives • + IM urea • + 0.15M d i t h i o t h r e i t o l A + 0.5M NaCl O + IM NaCl A modified by reductive a l k y l a t i o n 97b 98 (long relaxation times), the gels prepared with NaCl retain a constant l e v e l of stored energy. The gels with d i t h i o t h r e i t o l and urea appear to store less energy i n o s c i l l a t o r y shear than the pH 9.2 gel with no additives. The shapes of these three curves are similar, with a small i n f l e c t i o n evident at low frequency. This phenomenon i s sometimes due to entanglement coupling (Ferry, 1970) i n which extended l i n e a r fragments interact i n a s p e c i f i c frequency range such that an increase in e l a s t i c behavior i s observed. This e f f e c t i s less pronounced in the 0.5M NaCl sample and not detectable in the IM NaCl gel. The modified sample which did not form a s o l i d self-supporting matrix demonstrates a much greater dependence on frequency of o s c i l l a t i o n . At high frequency, the l i q u i d demonstrates a plateau or region of more t y p i c a l e l a s t i c behavior. At lower frequencies, the uncross-linked l i q u i d also shows the ch a r a c t e r i s t i c maxima perhaps due to entanglement coupling. Figure XXXV i l l u s t r a t e s the ef f e c t of frequency on the energy dissipated or l o s t as heat per cycle of sinusoidal deformation. Gels containing NaCl, urea, d i t h i o t h r e i t o l and no additives exhibited only small increases in dissipated energy with increasing frequency of o s c i l l a t i o n . The only major e f f e c t on the v i s c o e l a s t i c properties of the 12S gel at pH 9.2 was the reductive a l k y l a t i o n procedure. Although the addition of NaCl increased the absolute strength of the pH 9.2 gel system, i t s e f f e c t s on the rubberlike e l a s t i c i t y were minimal. 99a FIGURE XXXV: Dynamic shear l o s s moduli as a f u n c t i o n o f o s c i l l a t o r y frequency f o r 4.5% rapeseed p r o t e i n g e l s a t pH 9.2 and 23°C. © no a d d i t i v e s 0 + 1M urea • + 0.15M d i t h i o t h r e i t o l A + 0.5M NaCl • O + 1M NaCl A m o d i f i e d by r e d u c t i v e a l k y l a t i o n 9 9 b FREQUENCY ( S E C - 1 ) 100 Figures XXXVI and XXXVII demonstrate the e f f e c t of o s c i l l a t o r y frequency on storage and loss moduli of samples at d i f f e r e n t pH's. Gels prepared at pH 6, 9.2 and 10 indicated only small changes in both storage and loss moduli over a r e l a t i v e l y wide range of frequency. The r e l a t i v e values of storage and loss moduli for the pH 6 gel compared with pH 9.2 and 10 are not i n agreement with the steady shear data reported in Figure XXXII since under steady shear, the pH 6 gel exhibited lower apparent v i s c o s i t i e s than the pH 10 gel . I t may be that a s i g n i f i c a n t amount of structure was l o s t during steady shear and would account for the lower observed apparent v i s c o s i t i e s in the steady shear experiments. It may also be that since the pH 6 gel had a much "lumpier" texture than the gels at higher pH, highly e l a s t i c l o c a l i z e d aggregates could have been formed due to e l e c t r o s t a t i c forces in the pH 6 g e l . Such aggregates could have been responsible for the highly e l a s t i c recoveries i l l u s t r a t e d i n Figure XXXVI. Upon steady shearing, however, the aggregates may have been able to move with respect to one another since each aggregate would not necessarily be cross-linked with others. Thus, under conditions of high coulombic a t t r a c t i o n and minimum s o l u b i l i t y , the moduli observed need not necessarily r e f l e c t the true gel strength. Scanning electron micrographs presented i n Section IV.D.3. would tend to support t h i s explanation and suggest that an increase in three dimensional gel structure occurs with increases i n pH rather than a maximum observable structure at pH 6. 101a FIGURE X X X V I : D y n a m i c s h e a r s t o r a g e m o d u l i a s a f u n c t i o n o f o s c i l l a t o r y f r e q u e n c y f o r 4.5% r a p e s e e d p r o t e i n g e l s p r e p a r e d a t v a r i o u s pH i e v e l s a n d m e a s u r e d a t 23°C. • p l l 2 A pH 9.2 O pH 10 A pH 6 101b 102a FIGURE XXXVII: Dynamic shear loss moduli as a function of o s c i l l a t o r y frequency for 4.5% rapeseed protein gels prepared at various pH l e v e l s and measured at 23°C. • pH 2 A pH 9.2 ® pH 10 A pll 6 102 b 103 Other problems e x i s t w i t h the method used f o r r h e o l o g i c a l c h a r a c t e r i z a t i o n of g e l s t r u c t u r e . Since i t was necessary to cut the gels s e v e r a l times and then force them i n t o a narrow gap by compression, i t i s l i k e l y t h a t much of the three dimensional s t r u c t u r e was destroyed before r h e o l o g i c a l measurements could be taken. This problem c o u l d perhaps be e l i m i n a t e d i f the g e l were formed i n a narrow gap between the platens of the rheometer. Such an experiment was not undertaken i n the present study but should be considered f o r f u t u r e s t u d i e s of g e l a t i o n phenomena. V SUMMARY AND CONCLUSIONS The 12S glycoprotein extracted from commerical rapeseed meal (B. campestris L. var. Span) was recovered by gel f i l t r a t i o n and characterized by chemical, microstructural and rheological methods. The aggregate was not a sialoprotein; however, i t contained arabinose, galactose, glucose, i n o s i t o l glucosamine and mannose and strongly reacted when oxidized and exposed to Schiff reagent. Microstructural evidence suggested that the 12S aleur i n was located within some but not a l l of the c e l l s . The PAS-positive aleurone grains were dis t r i b u t e d randomly throughout the meristematic tissues and cotyledons which comprise a large proportion of the seed. The aleurone grains of rapeseed contain globoid bodies which suggest the presence of phytic acid. This observation may be related to the presence of i n o s i t o l i n the protein aggregate. I t may be that t h i s sugar i s added to the protein backbone some time after protein synthesis i s completed or perhaps could be complexed with the protein by way of a Mai l l a r d condensation reaction i n the meal during l i p i d removal. I t i s also possible that the differences observed between th i s and previous reports of carbohydrate composition of 12S glycoprotein recovered from seed could have resulted from non-enzymatic browning reactions. The amino acid p r o f i l e of the 12S globulin was dominated by the a c i d i c amino acids glutamic and aspartic. However, i t may be that the a c i d i c components are largely 104 105 present i n th e i r amide forms. There was a s c a r c i t y of the sulfur-containing amino acids 1/2 cystine and methionine although the 12S agglomerate would appear at le a s t p a r t i a l l y held together with intermolecular d i s u l f i d e bonds. Trypto-phan was not detected from a p-toluenesulfonic acid hydrolyzate of the protein. The protein aggregate i s morula-like and has a o maximum p a r t i c l e diameter of 120A as determined from electron-micrographs of negatively-stained specimens. The complex yielded weight average molecular weight of 129,200 daltons as determined by conventional sedimentation equilibrium u l t r a c e n t r i f u g a t i o n . The 12S i s o l a t e appeared heterogeneous according to polyacrylamide gel electrophoresis and ultracent-r i f ugal methods, however, was immunologically homogeneous as determined by disc immunoelectrophoresis and immunodiffusion experiments. Since the 12S protein has been observed to self-associate over time at low temperature, the available evidence suggests that the high molecular protein fra c t i o n s observed i n preparations of t h i s material are s e l f - a s s o c i a t i o n products. The 12S protein dissociated into f i v e components of lower molecular weight i n the presence of urea, SDS and mercaptoethanol. The fragments had apparent molecular weights of approximately 42,000, 37,600, 30,100, 17,400 and 12,200. SDS electrophoresis revealed that only one fragment contained a l l of the the S c h i f f - r e a c t i v e material and migrated with or 106 s l i g h t l y a h e a d o f t h e s m a l l e s t s u b u n i t d e t e c t a b l e w i t h C o o m a s s i e b l u e . The d i s c I m m u n o e l e c t r o p h o r e s i s o f S D S - t r e a t e d p r o t e i n a g a i n s t a n t i - 1 2 S a n t i s e r a s u g g e s t s t h a t t h e g l y c o p e p t i d e p o r t i o n o f t h e c o m p l e x i s l o c a t e d o n t h e s u r f a c e a n d i s p e r h a p s t h e m a j o r i m m u n o d e t e r m i n a n t g r o u p . D i s p e r s i o n s o f t h e 12S g l y c o p r o t e i n f o r m g e l s u p o n h e a t i n g . G e l a t i o n was o b s e r v e d a t t h e 4.5% p r o t e i n l e v e l a n d i n c r e a s e s i n a p p a r e n t v i s c o s i t y w e r e d e t e c t e d a t t h e 1% p r o t e i n l e v e l . T h e g e l s t r e n g t h s w e r e e f f e c t e d b y c h a n g e s i n pH a n d s o d i u m c h l o r i d e c o n c e n t r a t i o n s , t h e s t r o n g e s t g e l s b e i n g f o r m e d a t h i g h pH a n d i o n i c s t r e n g t h . U r e a , m e r c a p t o e -t h a n o l a n d p - m e r c u r i b e n z o a t e h a d l i t t l e e f f e c t o n t h e r m a l l y -i n d u c e d p o l y m e r i z a t i o n . G e l f o r m a t i o n i n t h i s s y s t e m i s o b v i o u s l y a c o m p l e x phenomenon w h i c h may i n v o l v e c o v a l e n t , i o n i c , h y d r o p h o b i c a n d h y d r o g e n b o n d i n g . T h e p r e s e n c e o f s u c h a h i g h l e v e l o f c a r b o h y d r a t e (12.9%) may a l s o s u g g e s t t h e p o s s i b i l i t y o f p r o t e i n - c a r b o h y d r a t e i n t e r a c t i o n d u r i n g g e l f o r m a t i o n . 107 REFERENCES Agriculture Canada, 1974. 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Schmid, K., B i n e t t e , J.P., Kamiyama, S., P f i s t e r , V., and Takahashi, S., 1962. S t u d i e s on the s t r u c t u r e of o ^ - a c i d g l y c o p r o t e i n . I I I . Polymorphism of a ^ - a c i d g l y c o p r o t e i n and the p a r t i a l r e s o l u t i o n and c h a r a c t e r i z a t i o n of i t s v a r i a n t s . Biochem. 1:959. Segrest, J.P., and Jackson, R.L., 1972. M o l e c u l a r weight de t e r m i n a t i o n of g l y c o p r o t e i n s by p o l y a c r y l a m i d e g e l e l e c t r o -p h o r e s i s i n sodium dodecyl s u l f a t e , In V. Ginsburg ( E d i t o r ) , Methods i n Enzymology, V o l . XXVIII, Academic Press, New York, p. 60. 112 Sheehan, D.C., and Hrapchak, B.B., 1973. The Theory and  Practice of Histotechnology. C.V. Mosby Co., St. Louis, Mo. Spurr, A.R., 1969. A low v i s c o s i t y epoxy res i n embedding medium for electron microscopy. J. U l t r a s t . Res. 26:31. Sober, H.A., (Editor), 1970. Handbook of Biochemistry, 2nd Edition, Chemical Rubber Company, Cleveland Ohio. Sorensen, 1909. Cited In,H.A. Sober (Editor), 1968. Handbook  of Biochemistry, 2nd Edition, Chemical Rubber Company, Cleveland, Ohio, p. J-234. Tombs, M.P., 1974. Gelation of globular proteins. Faraday Discuss. Chem. Soc. 57:158. Vaughan, J.G., and Denford, K.E., 1968. An acrylamide gel electrophoretic study of the seed protein of Brassica and Sinapis species, with special reference to taxonomic value. J. Exp. Bot. 19:724. Vaughan, J.G., and Waite, A., 1967. Comparative electrophoretic studies of the seed proteins of certain species of Brassica and Sinapis. J. Exp. Bot. 18:100. Vaughan, J.G., Waite, A., Boulter, D., and Waiters, S., 1966. Comparative studies of the seed proteins of Brassica campestris, Brassica oleracea, and Brassica nigra, J. Exp. Bot. 17:332. Voyles, B.A., and Moskowitz, M., 1974. Polyacrylamide gel electrophoresis of glycoproteins on single concentration and gradient gels. Biochem. Biophys. Acta, 351:178. Warren L., 1959. The t h i o b a r b i t u r i c acid assay of s i a l i c acids. J. B i o l . Chem. 234:1971. Watson, M.L., 1958. Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem Cytol. 4:475. Weber, K., and Osborn, M., 1969. The r e l i a b i l i t y of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. B i o l . Chem. 244:4406. Weber, K., Pringle, J.R., and Osborn, M., 1972. Measurement of molecular weights by electrophoresis on SDS-acrylamide gel, In C.H.W. Hirs and S.N. Timasheff (Editors), Methods in  Enzymology, Vol. XXVI, Academic Press, New York. pp. 8-9. Winzler, R.J., 1969. In R.T. Smith and R.A. Good (Editors), C e l l u l a r recognition, Appleton- Century -Crofts, New York, pp. 11-17. 113 Wohl, M.H., 1968. Rheology of non-Newtonian materials. Chem. Eng. Feb. 12:130. Wolf, W.J., 1970. Soybean proteins: t h e i r functional, chemical and physical properties. J. Agr. Food Chem. 18:969. Woyewoda, A.D., and Nakai, S., 1974. Detoxification of rape-seed protein i s o l a t e s by an activated carbon treatment, Paper G-2, presented at the 17th Annual Conference, C.I.F.S.T., Montreal, P.Q. APPENDIX I 114 115 Sample P r e p a r a t i o n f o r  Amino A c i d A n a l y s i s C y s t e i n e a l k y l a t i o n . One hundred mg of i s o l a t e were d i s s o l v e d i n 10 ml b u f f e r : 0.4 g T r i s 19 mg KC1 0.25 ml 0.01% EDTA s o l u t i o n 12.1 g urea - d i l u t e to about 2 0 ml and a d j u s t pH to 7.5 w i t h n i t r i c a c i d and f u r t h e r d i l u t e to 25 ml. The sample was reduced w i t h 0.1 ml 2-mercaptoethanol and s t i r r e d f o r 16 h under a n i t r o g e n atmosphere. D e r i v i t i z a t i o n was accomplished with the a d d i t i o n of 0.15 ml 4 - v i n y l p y r i d i n e ( f r e s h l y d i s t i l l e d ) and s t i r r e d f o r 2 h under n i t r o g e n . The pH was a d j u s t e d to 3 and the sample d i a l y z e d a g a i n s t 2 changes of 0.01N a c e t i c a c i d f o l l o w e d by exhaustive d i a l y s i s a g a i n s t s e v e r a l changes of d i s t i l l e d water. The sample was subsequently f r o z e n and l y o p h i l i z e d . H y d r o l y s i s i n p - t o l u e n e s u l f o n i c a c i d . The h y d r o l y s i s mixture was prepared by d i s s o l v i n g 2.85 g p - t o l u e n e s u l f o n i c a c i d and 10 mg 3-(2-aminoethyl) i n d o l e i n 2.5 ml d i s t i l l e d water. T h i s mixture was p l a c e d i n a b o i l i n g water bath and d i l u t e d to 5 ml. Each 2.5 mg 12S i s o l a t e was d i s s o l v e d i n 1 ml of the h y d r o l y s i s mixture, p l a c e d i n an ampoule, and f r o z e n i n an acetone-dry i c e bath. Each ampoule was then evacuated to 20 to 30 ym Hg and heat s e a l e d . A P P E N D I X I I 116 117 Hexosamine Determination - Elson-Morgan Reaction Samples weighing between 1.5 and 5.5 mg were placed in hydrolysis v i a l s and hydrolyzed as described i n the experimental section III.C.2.C. The contents of each v i a l (sample and 200-400 mesh AG 50W-X2 resin) were transferred to a 17 x 155 mm test tube with a ground glass j o i n t and each v i a l rinsed several times with d i s t i l l e d water, the washings transferred each time to the test tube. The contents of each tube were evaporated to dryness at 50°C in a rotary evaporator in order to remove the HCl used i n hydrolysis. Ion exchange columns prepared from 10 ml pipets were plugged at the t i p with glass wool and f i l l e d with 7.5 ml of an aqueous suspension (l:l;w/v) of AG 50W-X2 (200-400-mesh, H + form). The dried hydrolyzate was transferred i n each case to a column with water and the neutral sugars eluted from the column with 15 ml of d i s t i l l e d water, and the effl u e n t discarded. The hexosamines were then eluted with 10 ml 2N HCl, the eluate c o l l e c t e d i n a 50 ml round bottom flask and the contents of the flask were evaporated to dryness as before. Two ml of water were added to each evaporation flask and the dried material dissolved. One-ml aliquots of the dissolved samples were then placed i n 16 x 125 mm screw-capped culture tubes using caps with Teflon l i n e r s . One ml of 2% (v/v) 2,4-pentanedione (freshly d i s t i l l e d ) i n IN aqueous sodium carbonate was added to each tube. The tubes were sealed and placed i n a 90°C water bath for 45 min and cooled. Four ml of 95% 118 ethanol were added and the contents were thoroughly mixed. One ml of a p-dimethylaminobenzaldehyde (PDMAB) solution (677.5 mg PDMAB dissolved i n 25 ml of 1:1 ethanol-concentrated HC1) was mixed into each sample and afte r 1 h the absorbance readings of samples and standards were taken at 54 0 nm. A standard curve of A,-^ VS glucosamine concentration was prepared and a b e s t - f i t t i n g straight l i n e determined by the least squares method. The concentration of glucosamine in each sample was determined from the regression equation: uM glucosamine = 0.000135 + (0.449) {Au.An) APPENDIX I I I 1.19 120 S t o c k S o l u t i o n s ( p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s ) Upper r e s e r v o i r b u f f e r : pH 8 . 5 3 . 0 g T r i s 1 4 . 4 g g l y c i n e - d i l u t e t o 1:1 w i t h d i s t i l l e d w a t e r and u s e 1 :10 d i l u t i o n o f s t o c k s o l u t i o n f o r b u f f e r . Lower r e s e r v o i r b u f f e r : same as above A R u n n i n g g e l b u f f e r : pH 8 . 9 , r u n n i n g pH 9 . 5 2 4 ml IN H C l 1 8 . 1 g T r i s 0 . 1 2 ml N j N ^ N ' / N ^ t e t r a m e t h y l e t h y l e h e d i a m i n e (Temed) - d i l u t e t o 100 ml w i t h d i s t i l l e d w a t e r B S t a c k i n g g e l b u f f e r : pH 6 . 7 , r u n n i n g pH 8 . 9 5 . 9 8 g T r i s 0 . 4 6 ml Temed - a d j u s t pH t o 6 . 7 w i t h IN H C l and d i l u t e t o 100 ml w i t h d i s t i l l e d w a t e r C R u n n i n g a c r y l a m i d e (7% g e l s ) : 2 8 . 0 g a c r y l a m i d e 0 .74 g N , N 1 - m e t h y l e n e b i s a c r y l a m i d e ( B i s ) - d i l u t e t o 100 ml w i t h d i s t i l l e d w a t e r C R u n n i n g a c r y l a m i d e (4% g e l s ) : 1 6 . 0 g a c r y l a m i d e 0 .74 g B i s - d i l u t e t o 100 ml w i t h d i s t i l l e d w a t e r 121 D Stacking acrylamide: 20.0 g acrylamide 5.0 g Bis - d i l u t e to 100 ml with d i s t i l l e d water E Riboflavin: 4. 0 mg - d i l u t e to 100 ml with d i s t i l l e d water F Sucrose: 40.0 g - d i l u t e to 100 ml with d i s t i l l e d water G Ammonium persulfate c a t a l y s t : 0.14 g - d i l u t e to 100 ml with d i s t i l l e d water H 0.005% Bromphenol blue tracking dye Running gel preparation: 1 part stock solution A + 1 part stock solution B + 2 parts c a t a l y s t G, overlay with water and allow 30 min to gel. Stacking and sample gel preparation: 1 part stock solution B 1 part stock solution D 1 part stock solution E 4 parts stock solution F 1 part d i s t i l l e d water 122 - remove water from top of running gel and add stacking gel followed by overlay - photopolymerize with fluorescent l i g h t APPENDIX IV 123 Stock Solutions (SDS gel electrophoresis) Upper reservoir buffer: pH 8.64 0. 0.4M boric acid 0.041M T r i s 0.1% SDS Lower reservoir buffer: pH 9.18, running pH 9.50 0.0.31M HC1 0.42M T r i s A Running gel buffer: pH 9.18, running pH.9.50 0.12M HC1 1.7M T r i s 0.12% N,N,N 1,N'-tetramethyethylenediamine (Temed) B Stacking gel buffer: pH 6.1, running pH 8.64 0.21M H 2S0 4 0.43M T r i s C Running acrylamide (10% gels): 3.60 g N,N'-methylenebisacrylamide (Bis) 36.4 g acrylamide - d i l u t e to 100 ml with d i s t i l l e d water D Stacking acrylamide (3.2% gels) 1.60 g Bis 23.4 g acrylamide - d i l u t e to 100 ml with d i s t i l l e d water 125 E Riboflavin: 4 mg - d i l u t e to 100 ml with d i s t i l l e d water F Sucrose: 40 g - d i l u t e to 100 ml with d i s t i l l e d water G Ammonium persulfate c a t a l y s t : 0.14 g - d i l u t e to 100 ml with d i s t i l l e d water H Sch i f f reagent: Dissolve 16 g potassium metabisulfite i n 2 1 H^ O and add 21 ml concentrated HCl. Add 8 g basic fuchsin and s t i r slowly for 2 h. Let solution stand for 2 h and decolorize with a small amount of Darco G60 charcoal f i l t e r . 1 part stock solution A 1 part stock solution B 2 parts catalyst G This mixture may be made 0.1% i n SDS or the SDS may be omitted without any change in the ele c t r o -phoretic pattern. Running gel preparation: 126 Stacking and sample gel preparation: 1 part stock solution B 1 part stock solution D 1 part stock solution E 4 parts stock solution F 1 part d i s t i l l e d water - add 0.1% SDS and expose to fluorescent l i g h t to polymerize Sample preparation: 10 mg of each protein was dissolved i n 0.2 ml buffer B and 1.8 ml 8M urea, the f i n a l mixture containing 1% SDS and 0.14M 2-mercaptoethanol or dithio t h r e i t o l . . No difference in the electophoretic pattern could be detected when the sample solution was adjusted to 10% SDS or when d i t h i o t h r e i t o l was substituted for 2-mercaptoethanol. Best r e s u l t s were obtained when 10 to 50 yg of protein were applied to each gel. A P P E N D I X V 127 128 Amino Acid Analysis Results* Using Various Hydrolysis Times . for the 12S Glycoproti ein Hydrolysis Time at 110°C (h) Amino Acid 24 25 28 36 Aspartic acid 10.2 10.9 9.90 9. 57 Threonine 4.15 4.27 4.06 4. 36 Serine 4.54 4.50 4.35 3.92 Glutamic acid 19.3 19.9 19.5 18.7 Proline 4.89 4.71 5.51 3.82 Glycine 5. 02 5. 03 4.93 4.76 Alanine 4.21 4. 39 3.92 3.86 Valine 4 . 2 9 4.83 4.30 4. 62 Methionine 2.09 1.78 1.82 1.78 Isoleucine 3.95 4.12 3.87 3.92 Leucine 8.10 8.54 7.74 7. 68 Tyrosine 3.17 3.08 3.17 3.32 Ammonia 2.35 1.04 2.15 2.77 Phenylalanine 5.01 5.41 4.91 . 4.80 Lysine 3.00 3.33 2.94 2.92 Histidine 2.97 3.70 3.49 3.35 Tryptophan 0 0 0 o Arginine 5.93 6.86 6.10 5.93 Pyridylethyl-L-•cystine 0 0.212 0.0232 0 Total recovery (%N) 68 82 87 78 * A l l values reported in g amino acid residue per 16 g N recovered. 

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