UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

A sedimentation equilibrium study of ovomucin Miller, Steven Michael 1981

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1981_A6_7 M55.pdf [ 3.89MB ]
Metadata
JSON: 831-1.0095244.json
JSON-LD: 831-1.0095244-ld.json
RDF/XML (Pretty): 831-1.0095244-rdf.xml
RDF/JSON: 831-1.0095244-rdf.json
Turtle: 831-1.0095244-turtle.txt
N-Triples: 831-1.0095244-rdf-ntriples.txt
Original Record: 831-1.0095244-source.json
Full Text
831-1.0095244-fulltext.txt
Citation
831-1.0095244.ris

Full Text

A SEDIMENTATION EQUILIBRIUM STUDY OF OVOMUCIN by STEVEN MICHAEL MILLER B . S c , U n i v e r s i t y of Washington, 1977 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Food Science) We accept t h i s t h e s i s as conforming to the r equ i r ed standard THE UNIVERSITY OF BRITISH COLUMBIA February, 1981 (c) Steven M i chae l M i l l e r , 1981 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t 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 t h a t t h e 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 t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a 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 n o t 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 . Department o f FOOD SCIENCE  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 2075 Wesbrook P l a c e V ancouver, Canada V6T 1W5 n 3 f o MARCH 2 1 , 1 9 8 1 . DE -6 (2/79) ABSTRACT Sedimentat ion 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 was used to de tec t heterogeneous p r o t e i n - p r o t e i n a s soca t ions i n i n t e r a c t i n g mixtures of lysozyme and ovomucin and to determine the change i n mo lecu la r weight of ovomucin du r i ng egg wh i te t h i n n i n g . Mo lecu la r weight d i s t r i b u t i o n (MWD) pa t te rn s c a l c u l a t e d from sed imentat ion e q u i l i b r i u m data u s i ng m u l t i p l e r e g r e s s i o n a n a l y s i s revea led the presence of a t empe ra tu r e - s en s i t i v e i n t e r a c t i o n at pH 6.9 between lysozyme and ovomucin a t i o n i c s t rengths of 0.13 and below. The extent of i n t e r -a c t i o n d i f f e r e d depending on the method of p r epa r a t i on of ovomucin. The i n t e r a c t i o n of lysozyme was s t ronger w i t h 3-ovomucin than w i t h a-ovomucin and removal of s i a l i c a c i d re s i dues from 3-ovomucin d i d not decrease t h i s i n t e r a c t i o n but a c e t y l a t i o n of lysozyme d i d . S e l f -a s s o c i a t i o n of a-ovomucin a t low i o n i c s t reng th was a l s o observed. The apparent molecu la r weight of n a t i v e ovomucin i s o l a t e d from blended f r e s h egg wh i te by g e l f i l t r a t i o n on Sepharose 4B was 5.64 x 10 at pH 6.95 and i o n i c s t r eng th 0.13. D e t a i l e d u l t r a c e n t r i f u g a l . . a n a l y s i s i n d i c a t e d a remarkable dependence of molecu la r weight on p r o t e i n c o n c e n t r a t i o n . The apparent molecu la r we ight , amino a c i d and carbohydrate compos i t ions of n a t i v e ovomucin were s i m i l a r to those of ovomucin i s o l a t e d from egg wh i te that had been s to red f o r 166 h at 30 C. The mo lecu la r weights of ovomucin, i s o l a t e d by g e l f i l t r a t i o n on Sepharose 4B of f r e s h egg wh i te reduced w i t h 0.02% 2-mercaptoethanol , were 309,500 and 726,200- I t i s thus cons idered that d i s u l f i d e c leavage of ovomucin does not occur dur ing n a t u r a l t h i n n i n g . The r e l a t i o n of the r e s u l t s obta ined i n the present study of ovomucin to the mechanism of egg wh i te t h i n n i n g was a l s o d i s cu s sed . i i TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v LIST OF FIGURES v i LIST OF SYMBOLS v i i i ACKNOWLEDGEMENTS i x INTRODUCTION 1 LITERATURE REVIEW 4 EXPERIMENTAL 12 M a t e r i a l s 12 Methods 12 A. P r e p a r a t i o n of Ovomucin Complex 12 B. Reduct ion of P u r i f i e d Ovomucin Complex 13 C. P r e p a r a t i o n of Na t i ve Ovomucin 13 D. P r e p a r a t i o n of Ovomucin from Stored Egg White 14 E. P r e p a r a t i o n and F r a c t i o n a t i o n of Reduced F resh Egg White 15 F. M o d i f i c a t i o n of Lysozyme 15 G. M o d i f i c a t i o n of Ovomucins 16 H. Dete rminat ion of N a t i v e , Stored and Reduced Ovomucin Concent ra t ions 16 I. Amino A c i d A n a l y s i s of Ovomucin 17 J . Carbohydrate Composit ion of Ovomucins 17 1. Hexose 17 2. Amino Sugars 18 3. S i a l i c A c i d 18 K. Sedimentat ion 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 18 L. Ovomucin-Lysozyme I n t e r a c t i o n s 20 M. Mo lecu l a r Weight D i s t r i b u t i o n s 21 N. Mo lecu l a r Weight D e t e r m i n a t i o n s . . . . 22 RESULTS 24 A. I n t e r a c t i o n Between Egg White Lysozyme and Ovomucin 24 1. E f f e c t of I on i c S t rength 24 (a) RA Ovomucin Complex-Lysozyme 24 (b) Na t i ve Ovomucin-Lysozyme 26 2. E f f e c t of Chemical M o d i f i c a t i o n 27 (a) M o d i f i c a t i o n of Lysozyme 27 (b) M o d i f i c a t i o n of Ovomucin 28 i i i 3. E f f e c t of Temperature 30 B. Mo lecu l a r Weight of Ovomucin Dur ing Egg White T h i n n i n g . . . 30 1. P r e p a r a t i o n of N a t i v e , Stored and Reduced Ovomucins.. 30 2. Amino A c i d Composit ion of Ovomucin 31 3. Carbohydrate Composit ion of Ovomucins 31 4. Mo lecu la r Weight of Ovomucins 32 DISCUSSION 34 A. I n t e r a c t i o n Between Lysozyme and Ovomucin 34 1. E f f e c t of I on i c S t rength 34 (a) RA Ovomucin Complex-Lysozyme 34 (b) Na t i ve Ovomucin-Lysozyme 36 2. E f f e c t of Chemical M o d i f i c a t i o n 38 (a) Lysozyme 38 (b) Ovomucin 39 3. E f f e c t of Temperature 41 B. Mo lecu l a r Weight of Ovomucin Dur ing Egg White T h i n n i n g . . . 42 GENERAL DISCUSSION. 46 1. Lysozyme-Ovomucin Complex 46 2. Chemical or P h y s i c a l Change i n Ovomucin.„During Th i nn i n g . . 48 3. D i s s o c i a t i o n of an Ovomucin Complex S t a b i l i z e d by Lysozyme. .' 51 CONCLUSION 55 REFERENCES 56 TABLES 64 FIGURES 70 i v LIST OF TABLES page Table 1 Amino a c i d compos i t ion of whole ovomucin 64 Table 2 Amino a c i d compos i t ion of ovomucins from n a t i v e and s to red egg wh i te • 65 Table 3 Carbohydrate compos i t ion of ovomucins from f r e s h and s to red egg wh i te 66 Table 4 M of n a t i v e ovomucin as a f u n c t i o n of p r o t e i n w app concen t r a t i on 67 Table 5 M of s to red ovomucin as a f u n c t i o n of p r o t e i n w app concen t r a t i on 68 Table 6 Mo lecu la r weights of s o l ub l e ovomucins, 69 v LIST OF FIGURES page F i g u r e 1 Mo lecu la r weight d i s t r i b u t i o n of an RA ovomucin complex-lysozyme mix tu re a t i o n i c s t rengths 0.13 and 0.07 . , 7 1 F i gu re 2 Mo lecu la r weight d i s t r i b u t i o n of an RA ovomucin complex-lysozyme mix tu re a t i o n i c s t r eng th 0 . 1 3 . . . . . 7 ^ F i gu re 3 Mo lecu l a r weight d i s t r i b u t i o n of an RA ovomucin complex-lysozyme mix tu re a t i o n i c s t r eng th 0 . 0 7 . . . . . . . . . . 75 F i g u r e 4 Mo lecu l a r weight d i s t r i b u t i o n of a n a t i v e ovomucin-lysozyme mix tu re at i o n i c s t rengths 0.13 and 0.07 77 F i gu re 5 Mo lecu la r weight d i s t r i b u t i o n s of ovomucin-a c e t y l a t e d lysozyme mix tures at i o n i c s t reng th 0.07 79 F i gu re 6 Mo lecu la r weight d i s t r i b u t i o n of an a s i a l o , RA ovomucin complex-lysozyme mix tu re at i o n i c s t r eng th 0.07 81 F i gu re 7 Mo lecu la r weight d i s t r i b u t i o n of an a s i a l o , n a t i v e ovomucin-lysozyme mix ture a t i o n i c s t r eng th 0-07 83 F i gu re 8 Mo lecu la r weight d i s t r i b u t i o n s of RA ovomucin complex-lysozyme mixtures (1:4) and lysozyme at 20 and 3°C 8 5 F i gu re 9 Ge l f i l t r a t i o n of egg wh i te on Sepharose 4B. 87 F i gu re 10 Sedimentat ion e q u i l i b r i u m pa t te rn s of ovomucins, In A vs r 89 v i P a g e F i gu re 11 Apparent mo lecu la r weight (M ) of ovomucins w app as a f u n c t i o n of p r o t e i n concen t r a t i on i n 0.07 M sodium phosphate c on t a i n i n g 0.02% sodium a z i d e , pH 6.95, 20°C 91 F i gu re 12 Sedimentat ion 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 of f r a c t i o n s 24 and 37 from g e l f i l t r a t i o n of egg wh i te reduced w i t h 0.02% 2 - m e r c a p t o e t h a n o l . . . . . . . , 93 v i i LIST OF SYMBOLS A absorbance A^ area under peak i n molecu la r weight d i s t r i b u t i o n C c o n c e n t r a t i o n , g/1 E molar e x t i n c t i o n c o e f f i c i e n t M^ a p p apparent weight average molecu la r weight MOL WT molecu la r weight MWD molecu la r weight d i s t r i b u t i o n R u n i v e r s a l gas cons tant , 8.315 x 10^ ergs/degree mole RA reduced and a l k y l a t e d T abso lu te temperature, °K UV u l t r a v i o l e t T/2 i o n i c s t r eng th In n a t u r a l l o ga r i t hm r r a d i a l d i s t a n c e from cent re of r o t a t i o n , cm. rpm r e v o l u t i o n s per minute p den s i t y of s o l u t i o n , g/ml v p a r t i a l s p e c i f i c volume of s o l u t e , ml/g 03 angular v e l o c i t y , rad ians per second v i i i ACKNOWLEDGEMENTS I would l i k e to express my s i n ce re a p p r e c i a t i o n and g r a t i t u d e to Dr. S. Naka i and Dr. A. Kato f o r i n i t i a t i n g t h i s p r o j e c t and f o r t h e i r constant adv i ce , he lp and encouragement dur ing the course of t h i s s tudy, and i n the p r e p a r a t i o n of the t h e s i s . The adv i ce and he lp of P ro fe s so r s W.D. Powrie and B . J . Skura are s i n c e r e l y app rec i a t ed . The author wishes to express h i s a p p r e c i a t i o n to Mary Ban f o r the e x c e l l e n t job i n the t yp i ng of t h i s manuscr ipt . i x 1 INTRODUCTION Egg wh i te t h i n n i n g , a w e l l known phenomenon, i s a decrease i n the v i s c o s i t y of t h i c k wh i t e . As f r e s h eggs are s to red at room temperature the g e l s t r u c t u r e of the t h i c k wh i t e , composed main ly of ovomucin, i s g r a d u a l l y destroyed w i t h the i nc rea se i n pH of egg wh i te and changes i n t o t h i n wh i te . Many workers have been concerned w i t h e l u c i d a t i n g the mechanism of t h i s n o n - m i c r o b i a l d e t e r i o r a t i o n and i t i s g e n e r a l l y assumed tha t the po l yd i s pe r s e g l y c o s u l f o p r o t e i n ovomucin i s d i r e c t l y i n vo l v ed . Thus c h a r a c t e r i z a t i o n of the p h y s i c a l and chemica l p r o p e r t i e s of ovomucin i s e s s e n t i a l . Many s t ud i e s have been c a r r i e d out on ovomucin which has been chem ica l l y mod i f i ed w i t h 2-mercaptoethanol because of the poor s o l u b i l i t y of ovomucin i n non-denatu r ing s o l v e n t s . However, i n f o rma t i on on the p r o p e r t i e s of n a t i v e ovomucin, t ha t i s ovomucin s o l ub l e i n non-denatur ing so l ven t s w i thout chemica l m o d i f i c a t i o n , i s necessary to understand the j in v i v o process of egg wh i te t h i n n i n g . The p r epa r a t i on of a s o l ub l e ovomucin by g e l f i l t r a t i o n of blended egg wh i te has been r epo r t ed , a l l o w i n g s o l u b l e ovomucin to be prepared w i thout chemica l m o d i f i c a t i o n and cleavage of cova lent bonds. Of s e v e r a l hypotheses proposed to e x p l a i n the r o l e of ovomucin i n egg wh i te t h i n n i n g , one that has r e ce i ved much support s t a t e s tha t the i n t e r a c t i o n of ovomucin w i t h lysozyme i s r e s pon s i b l e f o r the ge l a t i nou s s t r u c t u r e of t h i c k wh i te and the decrease i n t h i s i n t e r -a c t i o n du r i ng s torage r e s u l t s i n t h i n n i n g . Evidence f o r t h i s suggest ion has been obta ined u s ing t u r b i d i m e t r i c measurements at 450 nm and 550 nm of mix tu res of lysozyme and reduced or a l k y l a t e d ovomucin. S ince not a l l i n t e r a c t i o n products cou ld cause t u r b i d i t y , these data may not be r e l i a b l e . Mo lecu la r weight d i s t r i b u t i o n s of i n t e r a c t i n g 2 p r o t e i n s c a l c u l a t e d by m u l t i p l e r e g r e s s i o n a n a l y s i s of sed imentat ion e q u i l i b r i u m data can be used to p rov ide d i r e c t evidence of a lysozyme-ovomucin i n t e r a c t i o n . An advantage of t h i s technique over t u r b i d i m e t r i c measurements i s tha t s o l u b l e p r o t e i n - p r o t e i n i n t e r a c t i o n products can' be de tec ted . Moreover use of a UV scanning system prov ides a d i r e c t measure of p r o t e i n c o n c e n t r a t i o n , s i nce p r o t e i n s absorb s t r ong l y at 280 nm. To t h i s end i t would be i n t e r e s t i n g to compare r e s u l t s obta ined u s i ng sed imentat ion 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 to those obta ined u s ing t u r b i d i m e t r i c methods, p a r t i c u l a r l y under those c o n d i t i o n s (pH 7.0, i o n i c s t r eng th 0.13) which approximate the i n v i v o c o n d i t i o n , where no lysozyme-ovomucin i n t e r a c t i o n can be detected by t u r b i d i m e t r i c measurement. I t would a l s o be i n t e r e s t i n g to see i f there i s any d i f f e r e n c e i n the extent of i n t e r a c t i o n of lysozyme w i t h chem ica l l y mod i f i ed versus unmodif ied ovomucin as a l l p rev ious measurements of the lysozyme-ovomucin have been conducted u s i ng c hem i ca l l y mod i f i ed ovomucin. I t i s known that ovomucin i s a complex of at l e a s t two d i s t i n c t g l y c o p r o t e i n s , a - and 8-ovomucin, and the i n t e r a c t i o n of lysozyme w i t h B-ovomucin has been repor ted to be s t ronger than::that w i t h a-ovomucin. Moreover, the b i nd i n g s i t e s i n the ovomucin-lysozyme i n t e r a c t i o n have been s t ud i ed . S ince these determinat ions were done us ing t u r b i d i m e t r i c methods i t seems necessary to repeat the experiments u s ing sed imentat ion 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 as a d i r e c t measure of p r o t e i n - p r o t e i n i n t e r a c t i o n s . I t has been proposed tha t du r i ng egg wh i te t h i n n i n g , ovomucin i s depolymer ized by the r e d u c t i o n or a l k a l i n e h y d r o l y s i s of d i s u l f i d e bonds. Thus to e l u c i d a t e more c l e a r l y the mechanism of egg wh i te t h i n n i n g i t i s important to determineethe molecu la r weight of n a t i v e 3 ovomucin and the change i n mo lecu la r weight of ovomucin dur ing t h i n n i n g . Sedimentat ion 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 i s a c l a s s i c technique f o r de te rm ina t i on of molecu lar weight of macro-molecules i n s o l u t i o n and has an advantage over other techniques i n t ha t the dete rminat ions can be done under c o n d i t i o n s of pH and i o n i c s t r eng th approx imat ing the p h y s i o l o g i c a l c o n d i t i o n . I f c leavage of d i s u l f i d e bonds i n ovomucin by r educ t i on or a l k a l i n e h y d r o l y s i s does occur du r i ng n a t u r a l t h i n n i n g i t should be p o s s i b l e to detect a r e l a t i v e l y low molecu la r weight ovomucin i n s to red and th inned egg wh i t e . The s p e c i f i c o b j e c t i v e s , t h e r e f o r e , of the present i n v e s t i g a t i o n were: (.1) To study the i n t e r a c t i o n of lysozyme w i t h mod i f i ed and un -mod i f i ed ovomucin under c o n d i t i o n s of pH and i o n i c s t reng th c l o s e to the jLn v i v o c o n d i t i o n and to determine the b i nd i ng groups i n the i n t e r a c t i o n . (2) To study the change i n chemica l and p h y s i c a l p r o p e r t i e s of n a t i v e ovomucin du r i ng n a t u r a l t h i n n i n g . 4 LITERATURE REVIEW Ovomucin, a h i gh molecu la r weight g l y c o p r o t e i n present main ly i n the t h i c k wh i te f r a c t i o n of a v i an eggs (McNal ly, 1933) i s the e s s e n t i a l component i n ma in ta i n i ng the f i r m g e l - l i k e cons i s tency of t h i c k egg wh i te ( H i l l et a l . , 1949). S ince Lann i and Beard (1948) and Go t t s cha l k and L i nd (1949 a, b) showed that the i n h i b i t o r y a c t i v i t y of egg wh i te aga ins t hemagg lut inat ion by i n f l u e n z a v i r u s e s was conta ined i n the ovomucin f r a c t i o n , much e f f o r t has been made to c h a r a c t e r i z e ovomucin. Lann i et_ a l . (1949) s tud ied the p r o p e r t i e s of the s e m i p u r i f i e d i n h i b i t o r and found i t to have a molecu lar weight of 7.6 x 10^ u s ing sed imentat ion 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 . D e t a i l e d e l e c t r o n m ic ro scop i c examinat ion of i n h i b i t o r p repa ra t i on s revea led the presence of s t r u c t u r e s of h i g h l y asymmetric shape i n the form of f i l a m e n t s or f i b r e s (Sharp et^ a l . , 1950), and the asymmetry of p a r t i c l e shape was i n accord w i t h p r e v i o u s l y demonstrated h i gh v i s c o s i t y of i n h i b i t o r p r epa ra t i on s . Sharp et a l . (1951) repor ted tha t these p repa ra t i on s con s i s t ed e s s e n t i a l l y of ovomucin and tha t ovomucin i s a m ix tu re of exceed ing ly l a r g e mucoproteins, not a l l of which possess i n h i b i t o r y a c t i v i t y . Chemical a n a l y s i s of ovomucin by Young (1937) showed the presence of hexose and hexosamines, a h i gh content of c y s t e i n e , and that ovomucin was s i m i l a r i n compos i t ion to the cha lazae. Feeney et: a l . (1960) repor ted tha t ovomucin conta ined 40 per cent of the t o t a l s i a l i c a c i d of egg wh i t e . Brooks and Hale (1961) a l s o repor ted on the presence and amount of hexosamine i n ovomucin p repa ra t i on s . A p r e l i m i n a r y repor t on the chemica l compos i t ion of ovomucin was g iven by Robinson and Monsey (1966) who repor ted that ovomucin was a 5 g l y c o p r o t e i n c r o s s - l i n k e d by d i s u l f i d e bonds and conta ined 33 per cent (w/w) carbohydrate composed of g a l a c t o se , mannose, g lucosamine, ga lactosamine, and N-acety lneuramin ic a c i d . Kato et^ a l . (1970 a , b) repor ted on the d i f f e r e n c e i n hexose, hexosamine, and s i a l i c a c i d between ovomucin i s o l a t e d from t h i c k versus t h i n egg wh i te and on the change i n chemica l compos i t ion of ovomucin du r i ng s torage. The l a t t e r study a l s o repor ted on the amino a c i d compos i t ion of ovomucin i s o l a t e d from f r e s h t h i c k wh i t e . A d e t a i l e d chemica l and p h y s i c a l c h a r a c t e r i z a t i o n of an ovomucin p r ep a r a t i o n f r e e of other recogn ized egg wh i t e p r o t e i n s was g i ven by Donovan e t a l . (1970) who repo r ted the presence of e s t e r s u l f a t e groups that accounted f o r a l l of the s u l f u r i n ovomucin. Robinson and Monsey (1971) and Robinson (1972) repor ted on the d e t a i l e d chemica l compos i t ion of ovomucin, i n c l u d i n g amino a c i d s , N -acety lg lucosamine, N -acety l ga lac tosamine, g a l a c t o se , mannose and s i a l i c a c i d . They a l s o repor ted the presence of e s t e r s u l f a t e and phosphate. Fu r the r s t ud i e s on the changese in fchemica l compos i t ion of ovomucin du r i ng l i q u e f a c t i o n of t h i c k egg wh i te have been repor ted (Robinson and Monsey, 1972 a , b ) . Young and Gardner (1972) repo r ted on the chemica l compos i t ion of ovomucin prepared by g e l f i l t r a t i o n of egg wh i te and found genera l agreement w i t h p rev ious r e p o r t s . The chemica l compos i t ion of ovomucin prepared w i thout chemica l m o d i f i c a t i o n was repo r ted by Adachi et^ al. (1973) and that of an ovomucin f r a c t i o n i s o l a t e d by h i gh speed c e n t r i f u g a t i o n was repo r ted by S l e i g h ej: al. (1973). In both r e p o r t s , the y i e l d and p u r i t y of ovomucin compared f avou rab l y w i t h o the r s . A d e t a i l e d study on the carbohydrate of egg wh i te ovomucin p repa ra t i on s was repor ted by Smith e^t a_l. (1974). These authors found tha t the p ropo r t i on s of hexose, 6 hexosamine and s i a l i c a c i d were w ide l y v a r i a b l e i n d i f f e r e n t p repa -r a t i o n s and that t h i s he te rogene i t y may be due to the presence of s e v e r a l d i f f e r e n t g l y c o p r o t e i n s and to v a r i a t i o n s i n the s i z e and compos i t ion of the carbohydrate groups. Kato el: a i . (1970 a) repor ted the presence of two components -a f a s t moving j(peak F) and a slow moving (peak S) component, i n the e l e c t r o p h o r e t i c p a t t e r n of ovomucin from t h i c k egg wh i t e . Fu r the r s t ud i e s (Kato and Sato, 1971; Kato et a l . , 1971; Kato e t a l . , 1977) showed the presence of two components by f r e e boundary e l e c t r o p h o r e s i s , d e n s i t y g rad ien t column e l e c t r o p h o r e s i s and chromatography on lysozyme-Sepharose 4B, r e s p e c t i v e l y . Subsequent chemica l a n a l y s i s of the two components showed peak F to be a c a r b o h y d r a t e - r i c h and peak S a carbohydrate-poor component, c o n s i s t i n g of about 60 per cent and 15 per cent carbohydrate, r e s p e c t i v e l y . At the same t ime, Robinson and Monsey (1971, 1975) repor ted on the presence of a homogeneous carbohydrate-poor g l y c o p r o t e i n (a-ovomucin) and a heterogeneous c a r b o h y d r a t e - r i c h g l y c o p r o t e i n f r a c t i o n (3-ovomucin) obta ined from p repa ra t i on s of reduced ovomucin by sed imentat ion e q u i l i b r i u m u l t r a -c e n t r i f uga t i on i n a den s i t y g rad ient of cesium c h l o r i d e i n the presence of 4 M guanid ine h yd roch l o r i de . The o l i g o s a c c h a r i d e moiety of a-ovomucin was found to c on t a i n main ly N-acety lg lucosamine and mannose w h i l e the 3-ovomucin f r a c t i o n was d i s t i n g u i s h e d from a l l other egg wh i te p r o t e i n s by i t s h igh carbohydrate content and s i g n i f i c a n t amounts of N -acety l ga lac to samine , e s t e r s u l f a t e and l a r g e amounts of s i a l i c a c i d . Hayakawa and Sato (1976) repor ted on d i s s o c i a t i o n of s o l u b l e ovomucin by sonicat- ion and the s epa ra t i on i n t o two components, peak 1 and peak I I , by DEAE -ce l l u l o se column chromatography. The chemica l compos i t ion of these subun i t s were compared to those repo r ted by 7 Kato ert a l . (1971) and Robinson and Monsey (1971). The ca rbo -hydrate and amino a c i d compos i t ions of peak I, a-ovomucin and peak S a re almost the same as are those of peak I I , 8-ovomucin and peak F. The nature of the carbohydrate s i de cha ins and t h e i r l i n kage to the p r o t e i n of ovomucin was repor ted by Kato et^ a l . (1973). I t was shown by these authors t ha t the re are a t l e a s t three types of ca rbo -hydrate s ide cha ins i n ovomucin: a cha in composed of g a l a c t o se , ga lactosamine, s i a l i c a c i d and s u l f a t e i n a molar r a t i o of about 1:1:1:1; a cha in composed of mannose and glucosamine i n a molar r a t i o of about 1:1; and a cha in composed of ga lac to se and glucosamine i n a molar r a t i o of a b o u t L l : l . The ca rbohydrate -pept ide l i n kage i n carbohydrate-poor ovomucin (a-ovomucin) was i d e n t i f i e d as N - g l y c o s i d i c l i n k a g e between N-acety lg lucosamine and asparag ine, but the s t r u c t u r e of i t s carbohydrate sequence was riot determined. The ca rbohydra te -pept ide l i n kage i n c a r b o h y d r a t e - r i c h (8-ovomucin) ovomucin was i d e n t i f i e d as 0 - g l y c o s i d i c between th reon ine or s e r i ne and N - a c e t y l -ga lactosamine. The s t r u c t u r e of i t s carbohydrate sequence was determined (Kato e t a l . , 1978 b, c) as N -ace ty l neu ram iny l - ( 2—>3 ) -g a l a c t o s y l - ( 1—>3 ) . -N - ace t y l g a l a c t o s am in i t o l - 6 - s u l f a t e . The p o s s i b l e r o l e of ovomucin i n the d e t e r i o r a t i o n of t h i c k wh i te du r i ng s torage of s h e l l eggs has r e ce i ved much a t t e n t i o n . Brant ejt al. (1955) repo r ted tha t egg wh i te i s e s s e n t i a l l y a s o l u t i o n of p r o t e i n s c o n t a i n i n g a l i t t l e s a l t and sugar w i t h pa r t of the s o l u t i o n enmeshed i n a ge l a t i nou s s t r u c t u r e , and tha t t h i c k egg wh i te i s appa ren t l y ge l a t i nou s because i t conta in s a h i gh content of the ovomucin f r a c t i o n . Moreover, Feeney et a l . (1955) found that ovomucin s o l u t i o n s th inned at r a t e s s i m i l a r to those of blended egg wh i t e . 8 MacDonnell et^ a l . (1950, 1951) found that sma l l amounts of reduc ing agents s imu la te c e r t a i n e f f e c t s of prolonged storage of eggs, no tab l y t h i n n i n g of the t h i c k wh i te and suggested that t h i n n i n g might be due to r e d u c t i o n of the d i s u l f i d e bonds of ovomucin. Feeney et^ a l . (1951) s tud ied the d e t e r i o r a t i o n of the separated components of eggs (whites and yo l k ) to t r y and i d e n t i f y the p o s s i b l e sources of reduc ing substances which cause n a t u r a l t h i n n i n g . Feeney et a l . (1956) s t ud i ed the k i n e t i c s and mechanisms of y o l k d e t e r i o r a t i o n i n s he l l - e g g s and po s t u l a t ed that t h i n n i n g may be due to s o l u b i l i t y changes i n ovomucin caused by the h i gh pH of s to red egg wh i t e , a t which pH the a c t i o n of s u l f h y d r y l reduc ing agents i s g r e a t l y a c c e l e r a t e d . Robinson and Monsey (1964) desc r ibed a method f o r p repar ing a s o l ub l e form of ovomucin by reduc ing ovomucin g e l w i t h 2-mercaptoethanol , suggest ing t ha t the i n s o l u b i l i t y of ovomucin g e l i s due to presence of d i s u l f i d e bonds. Nakamura et a l . (1969) repo r ted that the i n t r i n s i c v i s c o s i t y of ovomucin g e l decreased markedly a f t e r treatment w i t h the reduc ing agent sodium t h i o g l y c o l a t e . Donovan ejt a l . (1972) and Tomimatsu and Donovan (1972) repor ted a t h i n n i n g or decrease i n v i s -c o s i t y of the g e l - l i k e t h i c k p o r t i o n of ch i cken egg wh i t e produced by exposure of ovomucin to a l k a l i n e pH. They suggested that n a t u r a l t h i n n i n g was caused by a l k a l i n e h y d r o l y s i s of the d i s u l f i d e bonds of ovomucin as opposed to r e d u c t i o n . Th is po i n t of v iew was supported by the work of Sato et^ a l . (1976) who s tud ied the s o l u b i l i z a t i o n of i n s o l u b l e ovomucin du r i ng t h i c k wh i te t h i n n i n g . Bever idge and Naka i (1975) s tud ied by v i s c o s i t y and u l t r a c e n t r i f u g a l measurements, the e f f e c t of -SH b l o c k i n g and o x i d a t i o n on the t h i n n i n g of egg wh i te and suggested that t h e i r r e s u l t s support the hypothes i s t ha t t h i n n i n g 9 i s due to depo l ymer i za t i on of ovomucin by r e d u c t i o n of d i s u l f i d e bonds. A number of workers have proposed tha t the i n t e r a c t i o n of ovomucin w i t h other p r o t e i n s i s important to e x p l a i n the mechanism of egg wh i te t h i n n i n g . H i l l et^ a l . (1949) repor ted that the f i rmness of egg wh i te appeared to r e s u l t d i r e c t l y from the b i nd ing of ovomucin to other p r o t e i n s . Hawthorne (1950) suggested tha t egg wh i te t h i n n i n g r e s u l t e d from the slow i n s o l u b i l i z a t i o n of ovomucin due to a s t r u c t u r a l change i n ovomucin caused by a p r o t e i n - p r o t e i n i n t e r -a c t i o n of ovomucin w i t h lysozyme. Seve ra l i n v e s t i g a t o r s have supported the e x i s t ence of an i n t e r a c t i o n between ovomucin and lysozyme tha t r e s u l t s i n the fo rmat ion of an i n s o l u b l e complex (Feeney e_t a l . , 1952; C o t t e r i l l and W in te r , 1955; Dam and Bennett , 1963; G a r i b a l d i e t a l . , 1968; Robinson and Monsey, 1969 a, b ) . C o t t e r i l l and Winter (1955) and Rhodes and Feeney (1957) found a maximum i n t e r a c t i o n between ovomucin and lysozyme at pH 7. Less ovomucin-lysozyme complex was formed as the egg wh i te became more a l k a l i n e suggest ing that d i s s o c i a t i o n of the ovomucin-lysozyme complex, r a the r than i t s f o rma t i on , might cause t h i n n i n g . Wi l cox (1955) obta ined evidence f o r an a s s o c i a t i o n between the lysozyme l e v e l and q u a l i t y of egg wh i t e , support ing t h i s v iew p o i n t . Brooks and Hale (1959, 1961) s t ud i ed the mechanica l p r o p e r t i e s of t h i c k wh i t e and suggested that the r i g i d i t y of t h i c k wh i te cou ld be best exp la ined by assuming that cha ins of an ovomucin-lysozyme complex are c r o s s - l i n k e d i n t o a network. Gradual d i s s o c i a t i o n or h y d r o l y s i s of the complex cou ld account f o r the changes i n t h i c k wh i te du r i ng t h i n n i n g . 10 To determine the e f f e c t of the i n t e r a c t i o n of lysozyme w i t h ovomucin on the r i g i d i t y of t h i c k wh i te g e l , Robinson and Monsey (1969 b) r epo r ted on the s t o i c h i o m e t r i c compos i t ion of a pure lysozyme-reduced ovomucin complex. The i r r e s u l t s suggested that the i n t e r -a c t i o n was e l e c t r o s t a t i c i n nature and that the decreased i n t e r -a c t i o n observed at h i gh pH va lues was compat ib le w i t h a decrease i n the p o s i t i v e charge of the lysozyme molecu les . Dam (1971) repor ted on the e f f e c t of pH and temperature on the lysozyme-ovomucin i n t e r -a c t i o n and concluded t ha t i f lysozyme-ovomucin complexing e x i s t s i n v i v o , i t i s not i n t i m a t e l y i n vo l ved i n the process of t h i n n i n g of the t h i c k wh i t e . A l t e r n a t e l y i t was proposed that ovomucin i t s e l f p layed a major r o l e i n the s t r u c t u r e of the t h i c k wh i t e . Kato et^ a l . (1970 a) showed that the i n t e r a c t i o n of ovomucin w i t h lysozyme i n the t h i c k wh i te was s t ronger than that i n the t h i n wh i t e . As i t was subsequently shown that ovomucin con s i s t ed of carbohydrate-poor and c a r bohyd r a t e - r i c h components (Kato and Sato, 1971; Robinson and Monsey, 1971) and that there was a g radua l d i s s o c i a t i o n of the c a r b o h y d r a t e - r i c h component i n t o the l i q u i d pa r t of t h i c k wh i te du r i ng n a t u r a l t h i n n i n g (Kato and Sato, 1972; Robinson and Monsey, 1972 a) i t was proposed than an ovomucin complex c r o s s - l i n k e d by lysozyme e x i s t e d i n t h i c k wh i te and tha t t h i n n i n g of the t h i c k wh i te accompanying storage of whole eggs was due to d i s s o c i a t i o n of the complex as the pH of the wh i te inc reased (Kato €!t a l . , 1971; Robinson, 1972). Robinson and Monsey (1972 b) suggested tha t e l e c t r o s t a t i c i n t e r a c t i o n between the negat i ve charges ( i n p a r t i c u l a r those c a r r i e d by the s i a l i c a c i d re s idues ) of the ovomucin molecules and the p o s i t i v e charges of the lysozyme molecules are important f o r the lysozyme-ovomucin i n t e r a c t i o n . Kato e t a l . (1975) s t ud i ed the b i n d i n g groups i n the lysozyme-ovomucin i n t e r a c t i o n and found that the nega t i ve charges of the t e r m i n a l s i a l i c a c i d i n ovomucin and the p o s i t i v e charges of the l y s y l e-amino groups i n lysozyme are e s s e n t i a l f o r the e l e c t r o s t a t i c i n t e r a c t i o n of lysozyme w i t h ovomucin. I t was a l s o repor ted by these authors that the extent of i n t e r a c t i o n of lysozyme w i t h g-ovomucin was g rea te r thanr tha t w i t h a-ovomucin, r e f l e c t i n g on the d i f f e r e n c e i n s i a l i c a c i d content . Kato et a l . (1978) s t ud i ed the changes i n lysozyme dur ing egg wh i te t h i n n i n g and found tha t lysozyme d i s s o c i a t e s from the g e l i n t o the l i q u i d f r a c t i o n i n t h i c k wh i te dur ing s torage. I t was suggested by these authors that t h i s d i s s o c i a t i o n of lysozyme from the g e l might be caused by d i s s o c i a t i o n of g-ovomucin from the t h i c k wh i te g e l du r i ng s torage as was shown i h : . t he i r p rev ious r epo r t (Kato and Sato, 1972). Kato et a i . (1979) repor ted on the degradat ion of the O-g l yco -s i d i c a l l y l i n k e d carbohydrate u n i t s of ovomucin dur ing egg wh i te t h i n n i n g i n an attempt to e x p l a i n why the s p e c i f i c s o l u b i l i z a t i o n of g-ovomucin occurs du r i ng egg wh i te t h i n n i n g . I t was suggested tha t the 0 - g l y c o s i d i c a l l y l i n k e d carbohydrate u n i t s of g-ovomucin were important i n ma i n t a i n i n g the swo l l en r i g i d g e l s t r u c t u r e of t h i c k egg wh i te and tha t g radua l l i b e r a t i o n of these carbohydrate u n i t s du r i n g s torage of egg wh i te caused d i s s o c i a t i o n of the g-ovomucin molecu les from the t h i c k wh i t e ; hence t h i n n i n g . 12 EXPERIMENTAL M a t e r i a l s Un less otherwise i n d i c a t e d , a l l chemicals were reagent grade or the h i ghes t q u a l i t y prov ided by the s u p p l i e r , and were used wi thout f u r t h e r p u r i f i c a t i o n . Lysozyme, prepared from hen ' s egg wh i te by the d i r e c t . . c r y s t a l l i z a t i o n method (A lde r ton and Fevo ld , 1946) was r e c r y s t a l l i z e d f i v e t imes and l y o p h i l i z e d . The f o l l o w i n g m a t e r i a l s were obta ined commerc ia l l y . Neuraminidase ( C l o s t r i d u i m  p e r f r i n g e n s , type IV ) , N -acety lneuramin ic a c i d ( E s c h e r i c h i a c o l i ) , N-acety lg lucosamine and N-acety lga lactosamine were a l l purchased from Sigma Chemicals , S t . L o u i s , MO. D-ga lactose and D-mannose were products of Eastman Organic Chemicals , Rochester , N.Y. Bovine serum albumen was from Calbiochem, I n c . , La J o l l a , CA. Sephadex G-25 and Sepharose 4B were the products of Pharmacia F i ne Chemicals , Sweden. A l l eggs were c o l l e c t e d from the U n i v e r s i t y f l o c k w i t h i n 24 hours a f t e r l a y i n g and the s h e l l s s t e r i l i z e d w i t h 70% e thano l . Methods A. P r e p a r a t i o n of Ovomucin Complex Th ick egg wh i te from 25 eggs was separated by s t r a i n i n g egg wh i te through an aluminum s i eve accord ing to the method of Kato et_ a l . (1970 a ) . The egg wh i te which remained on the s i e ve was the t h i c k wh i te and tha t which d ra ined r e a d i l y through the s i e ve was the t h i n wh i t e . The t h i c k wh i te (500 ml) was blended at the s lowest speed i n a S o r v a l l Omni-mixer and crude ovomucin complex was p r e c i p i t a t e d by a d d i t i o n of 500 ml of 4% KC l as de sc r i bed by Kato et a l . (1971). The ge l a t i nou s p r e c i p i t a t e (200 ml) was separated from the supernatant by c e n t r i f u g a t i o n at 10,000 rpm f o r 20 min a t 4°C u s ing a S o r v a l l RC-2B r e f r i g e r a t e d c e n t r i f u g e and GSA r o t o r , and was washed w i t h 2% KC1 s o l u t i o n u n t i l the supernatant was f r e e from p r o t e i n s as judged by p r e c i p i t a t i o n w i t h 2% t r i c h l o r o a c e t i c a c i d . The r e s u l t i n g g e l (30 ml) was e xhau s t i v e l y d i a l y z e d at 4°C aga in s t d i s t i l l e d water , c on t a i n i n g 0.02% sodium a z i d e , to remove KC1 and i s h e r e a f t e r r e f e r r e d to as p u r i f i e d ovomucin complex. B. Reduct ion of P u r i f i e d Ovomucin Complex P u r i f i e d ovomucin complex was reduced accord ing to the method of Robinson and Monsey (1971). 10 ml of 1.0 M T r i s - HC1 b u f f e r , pH 8.2, c o n t a i n i n g 0.6 M 2-mercaptoethanol and 12 M urea were added s l ow l y w i t h s t i r r i n g to 10 ml of p u r i f i e d ovomucin complex and the mix ture a l lowed to stand at 20°C f o r 16 h. The t h i o l groups were then a l k y l a t e d by the a d d i t i o n of an equivolume (20 ml) of 2 M T r i s -HC1 b u f f e r , pH 8.0, c on t a i n i n g 0.72 M i o d o a c e t i c a c i d and 8 M u rea . The r e s u l t i n g mix ture was a l lowed to stand i n the dark at 20°C f o r 2 h. F o l l o w i n g a l k y l a t i o n the reduced ovomucin complex (50 ml) was e xhau s t i v e l y d i a l y z e d a t 4°C aga in s t d i s t i l l e d water c on t a i n i n g 0.02% sodium az i de and i s h e r e a f t e r r e f e r r e d to as reduced, a l k y l a t e d ovomucin complex (RA ovomucin complex). C. P r e p a r a t i o n of Na t i ve Ovomucin Na t i ve ovomucin was prepared u s ing a m o d i f i c a t i o n of the method of Young and Gardner (1972). Fresh egg wh i te (30 m l ) , pH 7.8, was blended f o r 1 min u s i ng a S o r v a l l Omni-mixer at the s lowest speed. The blended wh i te was then d i l u t e d to 100 ml w i t h 0.05 M T r i s - H C l b u f f e r , pH 8.6, c o n t a i n i n g 0.85% sodium c h l o r i d e , and then blended as be fo re . 10 ml were a p p l i e d to a Sepharose 4B column (2.5 x 40 cm), w i t h a 1 cm l a y e r of Sephadex G-25 on the bottom, which had been 14 p r e v i o u s l y e q u i l i b r a t e d w i t h 2 bed volumes of 0.05 M T r i s - H C l b u f f e r , pH 8.6, c on t a i n i n g 0.85% sodium c h l o r i d e . The v o i d volume of the column was determined by measuring the e l u t iO ru volume of B lue Dextran 2,000, 2 mg/ml '(Pharmacia F ine Chemicals , Sweden). The column was e l u t e d w i t h the same b u f f e r at a f l ow r a t e of 13 ml per h and 3.5 ml f r a c t i o n s were c o l l e c t e d u s ing a G i l s o n Med i ca l E l e c t r o n i c s drum-type f r a c t i o n c o l l e c t o r . F r a c t i o n s were ana lyzed f o r p r o t e i n and s i a l i c a c i d and those f r a c t i o n s c on t a i n i n g the ovomucin ( f r a c t i o n s 24 and 25) were combined and e xhau s t i v e l y d i a l y z e d at 4°C aga ins t d i s t i l l e d water or c e n t r i f u g a t i o n b u f f e r , c on t a i n i n g 0.02% sodium a z i d e . A l i q u o t s f o r hexose, hexosamines, s i a l i c a c i d and amino a c i d ana lyses were s to red at -20°C be fo re ana ly ses . A l i q u o t s f o r sed imentat ion e q u i l i b r i u m a n a l y s i s were s to red a t 4°C f o r up to 3 days. D. P r e p a r a t i o n of Ovomucin from Stored Egg White Egg wh i te from eggs c o l l e c t e d w i t h i n 24 h a f t e r l a y i n g was s to red f o r 166 h a t 30 ± 2°C i n a s t e r i l i z e d f l a s k sea led w i t h a c o t t on p lug . A l l procedures were a s e p t i c a l l y c a r r i e d out . At the end of the g iven time (166 h) the egg wh i te (pH 9.0) was removed from the f l a s k , b lended at the s lowest speed i n a S o r v a l l Omni-mixer , d i l u t e d w i t h column b u f f e r and prepared f o r f r a c t i o n a t i o n on Sepharose 4B as be f o re . 10 ml were a p p l i e d to a Sepharose 4B column and f r a c t i o n a t e d as p r e v i o u s l y de s c r i bed . F r a c t i o n s c on ta i n i n g the ovomucin were combined and d i a l y z e d and a l i q u o t s were removed f o r chemica l a n a l y s i s and sed imentat ion e q u i l i b r i u m as desc r ibed be fo re . He rea f t e r t h i s f r a c t i o n w i l l be r e f e r r e d to as s to red ovomucin. 15 E. P r e p a r a t i o n and F r a c t i o n a t i o n of Reduced Fresh Egg White 2-Mercaptoethanol (0.02%) was added to f r e s h egg wh i te (30 ml) and then blended f o r 1 min u s ing a S o r v a l l Omni-mixer a t the s lowest speed. F o l l ow i n g d i l u t i o n to 100 ml w i t h column b u f f e r and b lend ing as be f o r e , 10 ml were app l i ed to a Sepharose 4B column (2.5 x 40 cm) and f r a c t i o n a t e d as be fo re . A l i q u o t s from each f r a c t i o n were removed f o r s i a l i c a c i d de te rm ina t i on and those c on t a i n i n g s i a l i c a c i d ( f r a c t i o n s 17 to 43) were subsequently analyzed f o r hexose. On the ba s i s of the r a t i o of s i a l i c a c i d to hexose, f r a c t i o n s 24 and 37 were i d e n t i f i e d as con ta i n i n g ovo-mucin. These f r a c t i o n s , h e r e a f t e r r e f e r r e d to as reduced ovomucin, were e xhau s t i v e l y d i a l y z e d a t 4°C aga in s t c e n t r i f u g e b u f f e r c on t a i n i n g 0.02% sodium a z i d e , as be fo re , i n p r epa r a t i o n f o r sed imentat ion e q u i l i b r i u m a n a l y s i s . F. M o d i f i c a t i o n of Lysozyme A c e t y l a t i o n of lysozyme was c a r r i e d out acco rd ing to the genera l method of Yamasaki et: a l . (1968) as mod i f i ed by Kato et^ a l . (1975). 5 ml s a tu ra ted sodium ace ta te were s l ow l y added w i t h s t i r r i n g to 5 ml of 4% lysozyme i n d i s t i l l e d water and the mix ture kept on i c e . To t h i s m i x tu re , kept on i c e , 0.2 g a c e t i c anhydr ide was g r a d u a l l y added over a 1 h pe r i od (0.01 ml a c e t i c anhydr ide every 3 min) . The pH was mainta ined above 7.0 over the 1 h pe r i od by a d d i t i o n of a sma l l volume of 1 N sodium hydrox ide f o l l o w i n g each a d d i t i o n of a c e t i c anhydr ide. A f t e r a d d i t i o n of the a c e t i c anhydr ide was complete, the a c e t y l a t e d lysozyme was e xhau s t i v e l y d i a l y z e d at 4°C, aga in s t d i s t i l l e d water c o n t a i n i n g 0.02% sodium a z i d e . The extent of m o d i f i c a t i o n of f r e e amino groups i n the a c e t y l a t e d lysozyme was determined s p e c t r o -p h o t o m e t r i c a l l y u s i ng t r i n i t r o b e n z e n e s u l f o n a t e , a reagent s p e c i f i c 16 f o r amino groups, accord ing to the method of Haynes at al_. (1967). To 1 ml of a c e t y l a t e d lysozyme s o l u t i o n (1 mg/ml) were added 1 ml o f 4% sodium b i c a rbona te , pH 8.5, and 1 ml of 0.1% t r i n i t r o -benzene s u l f ona te i n water. The mix tu re was incubated at 40°C f o r 2 h and then 1 ml of 10% sodium dodecy l s u l f a t e and 0.5 ml of 1 N h y d r o c h l o r i c a c i d were added. Absorbance was read at 344 nm aga in s t a b lank t r e a t ed as above but c on t a i n i n g 1 ml of d i s t i l l e d water i n s t ead of lysozyme s o l u t i o n and was compared to the absorbance of unmodif ied lysozyme (1 mg/ml) t r e a t ed as above. G. M o d i f i c a t i o n of Ovomucins Neuraminidase (1.85 ug), E.C. 3.2.1.18 (4.2 units/mg) was added to 2 ml of a 0.3% s o l u t i o n of RA ovomucin complex or n a t i v e ovomucin i n 0.03 M sodium phosphate b u f f e r , pH 6.9, c o n t a i n i n g 0.02% sodium a z i de and then incubated at 30°C f o r 22 h. An a l i q u o t (0.2 ml) was removed f o r s i a l i c a c i d de te rm ina t i on and the remainder e xhau s t i v e l y d i a l y z e d at 4°C aga in s t c e n t r i f u g a t i o n b u f f e r . The extent of enzymatic removal of s i a l i c a c i d was determined by comparison to the t o t a l s i a l i c a c i d re l ea sed by a c i d h y d r o l y s i s of unmodif ied ovomucin w i t h 0.1 N s u l f u r i c a c i d a t 80°C f o r 1 h. H. Determinat ion of N a t i v e , Stored and Reduced Ovomucin Concentrat ions Ovomucin concent ra t i on s were est imated q u a l i t a t i v e l y by absorbance at 280 nm. Q u a n t i t a t i v e e s t i m a t i o n of ovomucin concen t ra t i on s was made by the method of Lowry eT a l . (1951) u s i ng bov ine serum albumin as a s tandard or by s pec t r opho tomet r y measurements us ing the e x t i n c t i o n c o e f f i c i e n t , E ^2g0 nm 1 cm = 7.0 which was determined e xpe r imen ta l l y . 17 I. Amino Ac i d A n a l y s i s of Ovomucin Amino a c i d ana lyses were conducted on 2 mg samples of n a t i v e and s to red ovomucins hydro lyzed w i t h 6 N h y d r o c h l o r i c a c i d a t 105 - 110°C f o r 22 h i n s ea l ed , evacuated ampules p r e v i o u s l y f l u s hed w i t h n i t r o g e n . A f t e r h y d r o l y s i s the samples were evaporated to dryness under n i t r o g e n , d i s s o l v e d i n 0.2 M sodium c i t r a t e b u f f e r (pH 2.2) and ana lyzed by. the method of Moore and S t e i n (1954) on a s i ng le - co lumn system (Durrum Chem. Corp. , Pa lo A l t o , CA) a t tached to a Phoenix Model M 6800 Amino A c i d Ana lyze r (Phoenix P r e c i s i o n Instrument Co . ) . Va lues f o r amino ac i d s are averages of d u p l i c a t e ana lyses w i t h no c o r r e c t i o n s made f o r p o s s i b l e d e s t r u c t i o n of c e r t a i n amino a c i d s which may have occurred due to the presence of carbohydrates or f o r p o s s i b l e d e s t r u c t i o n of s e r i ne and th reon ine du r i ng h y d r o l y s i s . J . Carbohydrate Composit ion of Ovomucins 1. Hexose Hexose content was determined by the o r c i n o l - s u l f u r i c a c i d method (Winz le r , 1955) u s i ng an equimolar mixture of D-ga lactose and D-mannose as a s tandard. To 0.2 ml ovomucin s o l u t i o n (99 yg ovomucin) were added 0.5 ml of 1.6% o r c i n o l i n 30% s u l f u r i c a c i d , and 3.0 ml 60% s u l f u r i c a c i d and the mix ture was heated to 80°C f o r 20 min i n the dark. F o l l ow i n g t h i s i n c u b a t i o n , the m ix tu re was coo led i n an i c e bath i n the dark. Absorbance a t 520 nm, or 540 nm f o r h i gh absorbance samples, was determined s p e c t r o -p h o t o m e t r i c a l l y . 18 2. Amino Sugars Hexosamine was determined by the method of Neuhaus and L e t z r i n g (1957) i n which ah equimolar m ix tu re of N - a c e t y l -galactosamine and N-acety lg lucosamlne was used as a s tandard. Ovomucin samples (99 yg i n 0.2 ml) were evaporated to dryness i n g l a s s - s toppered c e n t r i f u g e tubes by hea t i ng i n a b o i l i n g water bath . One m i l l i l i t r e of 3 N h y d r o c h l o r i c a c i d was added and the tubes were stoppered and heated i n a b o i l i n g water bath f o r 4 h. The remainder of the procedure was c a r r i e d out e x a c t l y as de sc r i bed by Neuhaus and L e t z r i n g (1957) except that absorbances a t 530 nm were measured i n quartz cuvet te s i n s t ead of K l e t t c o l o r ime te r tubes. 3. S i a l i c A c i d S i a l i c a c i d was measured a f t e r a c i d h y d r o l y s i s ( t o t a l s i a l i c a c i d ) or a f t e r enzymatic r e l e a s e , by the t h i o b a r b i t u r i c a c i d method of Warren (1959) w i t h N-acety lneuramin ic a c i d as a s tandard. Na t i ve or s to red ovomucin samples (99 yg ovomucin i n 0.2 ml) were hydro lyzed w i t h 0.2 ml of 0.2 N s u l f u r i c a c i d a t 80°C f o r 1 h and f r e e s i a l i c a c i d was determined e x a c t l y as desc r ibed by Warren (1959) f o r assay of s i a l i c a c i d i n t i s s u e homogenates. K. Sedimentat ion 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 A Beckman L2-65B U l t r a c e n t r i f u g e equipped w i t h a Prep UV Scanner and i n t e g r a t e d w i t h a data a c q u i s i t i o n system was employed f o r sed imentat ion e q u i l i b r i u m s t u d i e s . An e v a l u a t i o n of t h i s system f o r sed imentat ion e q u i l i b r i u m a n a l y s i s has been pub l i shed (Van de Voort and Naka i , 1978). C e n t r i f u g a l runs were performed at 20 ± 0.5°C, un le s s otherwi se s p e c i f i e d , u s ing a b l a c k anodized four p l a ce r o t o r (An-F) ho l d i n g t h ree , 12 mm f i l l e d Epon double sec to r c en te rp i e ce s , a l l o w i n g th ree samples to be ana lyzed du r i ng the same run . Separate exhaust ive d i a l y s i s of p r o t e i n s a t 4°C aga in s t phosphate b u f f e r s (NaH^PO^, Na^HPO^), pH 6.9 c on t a i n i n g 0.02% sodium a z i d e ( c e n t r i f u g a t i o n b u f f e r ) preceded a l l a n a l y t i c a l c e n t r i f u g a l runs. The outer d i a l y s a t e s were checked s p e c t r o -p h o t o m e t r i c a l l y to ensure that no p r o t e i n had leaked from the d i a l y s i s sac du r i ng d i a l y s i s and these were used as the re fe rence s o l u t i o n s i n sed imentat ion e q u i l i b r i u m experiments i n accord w i t h the requirements s p e c i f i e d by Casassa and E i senberg (1964). In gene ra l , the so l vent channel of each cen te rp i ece was f i l l e d w i t h 0.19. ml of the app rop r i a te outer d i a l y s a t e and the sample channel w i t h 0.03 ml f l uo roca rbon o i l (to p rov ide a curved bottom), f o l l owed by 0.12 ml p r o t e i n s o l u t i o n . Sedimentat ion e q u i l i b r i u m experiments were s t a r t e d by running the r o t o r a t low speed (5,000 to 10,000 rpm) f o r about 10 min and making a scan to o b t a i n the i n i t i a l s o l u t e c oncen t r a t i o n . An overspeed run of 3 h a t 1% t imes the s e l e c t e d e q u i l i b r i u m speed was used to reduce the t r a n s i e n t t ime to o b t a i n the e q u i l i b r i u m c o n d i t i o n (except f o r molecu la r weight determinat ions of n a t i v e and s to red ovomucins) and then the r o t o r was dece l e ra ted to the s e l e c t e d e q u i l i b r i u m running speed. The t ime to reach e q u i l i b r i u m was est imated us ing the Van Ho lde-Ba ldwin equat ion (Van Holde and Ba ldwin , 1958) and the system was cons idered at e q u i l i b r i u m when hour l y scans taken a f t e r the c a l c u l a t e d e q u i l i b r i u m t ime showed no observab le change. A f t e r the e q u i l i b r i u m scan was taken the r o t o r speed was inc reased to approx imate ly 46,000 rpm f o r 3 h to dep le te the meniscus and then 20 dece l e r a ted to the e q u i l i b r i u m speed where an a d d i t i o n a l scan was immediately taken to o b t a i n the b a s e l i n e absorbance, assuming tha t the absorbance i n the dep leted reg i on of the c e l l i n d i c a t e s the b a s e l i n e f o r the e n t i r e c e l l . L. Ovomucin-Lysozyme I n t e r a c t i o n s Sed imentat ion e q u i l i b r i u m was a p p l i e d to the study of the heterogeneous a s s o c i a t i o n s of ovomucin w i t h lysozyme. D i a l y zed lysozyme and ovomucin (RA ovomucin complex and n a t i v e ovomucin) s o l u t i o n s were d i l u t e d such that the absorbances at 280 nm were approx imate ly 0.30 and mixed i n an ovomucin to lysozyme volume r a t i o of 1:4, p r i o r to a n a l y t i c a l u l t r a c e n t r i f u g a t i o n . C o r r e c t i n g f o r UV a b s o r p t i v i t y d i f f e r e n c e between lysozyme and ovomucin, i . e . E 1 % „ D _ . =26 .5 ( C a n f i e l d , 1963 a) and E 1 % 0 0 . . = 7 . 0 280 nm, 1 cm 280 nm, 1 cm f o r lysozyme and ovomucin, r e s p e c t i v e l y , g i ves a c a l c u l a t e d ovomucin-to lysozyme c o n c e n t r a t i o n r a t i o of 1:1 (w/w) f o r these m ix tu re s . Th is r a t i o was chosen because an i n s o l u b l e lysozyme-ovomucin aggregate forms i n mixtures of lysozyme and ovomucin under c ond i t i o n s where there i s an excess of lysozyme (Robinson, 1972; Kato e^ t al., 1975). Robinson (1972) has c a l c u l a t e d that 1.97 g of lysozyme are r equ i r ed to c r o s s - l i n k 2 g of ovomucin a t pH 7.4. The e f f e c t of i o n i c s t r eng th on the ovomucin-lysozyme i n t e r -a c t i o n was i n v e s t i g a t e d a t i o n i c s t rengths 0.13 (0.033 M NaH^PO^, 0.033 M Na 2 HP0 4 ) and 0.07 (0.017 M NaR^PO^, 0.017 M Na HPO^), pH 6.9. The e f f e c t of temperature on the i n t e r a c t i o n was i n v e s t i g a t e d by conduct ing the sed imentat ion e q u i l i b r i u m runs at 20 ± 0.5°C and 3 ± 0.5°C f o r i o n i c s t rengths of 0.13 and 0.07. For the e q u i l i b r i u m runs a t 3°C, the p r o t e i n s o l u t i o n s were loaded i n t o the c e n t r i f u g e 21 c e l l s and the assembled r o t o r was p re - coo led to 3 ± 0.5 C p r i o r to c e n t r i f u g a t i o n . M. Mo lecu l a r Weight D i s t r i b u t i o n s Absorbance va lues were monitored at 278 nm as a f u n c t i o n of r a d i a l d i s t ance and were co r rec ted u s ing a standard curve drawn f o r ovalbumin. Mo lecu l a r weight d i s t r i b u t i o n s (MWD) were c a l -c u l a t e d by m u l t i p l e r e g r e s s i o n a n a l y s i s of sed imentat ion e q u i l i b r i u m data acco rd ing to the method of Van de Voort et a l . (1979) and Naka i and Van de Voort (1979). Rotor speeds were determined by averag ing at l e a s t 5 odometer readings taken dur ing the course of the run. The p a r t i a l s p e c i f i c volume of lysozyme was taken as 0.726 ml/g (Deonier and W i l l i a m s , 1970) and a p a r t i a l s p e c i f i c volume of 0.662 ml/g was c a l c u l a t e d f o r the RA ovomucin complex acco rd ing to the method of Howlett and N i c h o l (1973) us ing va lues of 0.701 ml/g and 0.649 ml/g f o r the p a r t i a l s p e c i f i c volumes of a - and 8- ovomucins (Robinson, 1972) assuming that RA ovomucin complex c o n s i s t s of ( a - ovomic in )^ : (B- ovomucin)^ where n i s a p o s i t i v e whole number. The area under the peaks i n the MWD i s approx imate ly p r o p o r t i o n a l to the c oncen t r a t i on of t ha t mo lecu la r weight spec ie s and p r o t e i n -p r o t e i n i n t e r a c t i o n s between ovomucin and lysozyme were detected as changes i n the MWD such as the disappearance of peaks, the appearance of new peaks and/or thej.change i n area under a p a r t i c u l a r peak. The extent of i n t e r a c t i o n between ovomucin and lysozyme was determined by measuring the area under the lysozyme peak. 22 N. Mo lecu la r Weight Determinat ions Mo lecu la r weights of ovomucins ( n a t i v e , s to red and reduced) were c a l c u l a t e d from sed imentat ion e q u i l i b r i u m data us ing the r e l a t i o n s h i p 2RT (d l n c ) M / = -„ - — (Schachman, 1959) w app •>-- . ,2 j/ 2. v* (l-vp)o) d ( r ) where: M = the apparent weight average molecu lar weight w app of the sample R = u n i v e r s a l gas constant = 8.315 x 10^ ergs/degree/mole T = abso lu te temperature i n degrees K e l v i n v = p a r t i a l s p e c i f i c volume, ml/g a) = angular v e l o c i t y of r o t o r i n rad ians per second = 0.10472 x rpm c = p r o t e i n c o n c e n t r a t i o n , g/1 r = r a d i a l d i s t a n c e from center of r o t a t i o n , cm p = d e n s i t y of the s o l u t i o n , g/ml Absorbance va lues were monitored at 278 nm as a f u n c t i o n of r a d i a l d i s t ance as be fo re and p l o t s of In absorbance aga in s t 2 ( r a d i a l d i s t ance ) were cons t ruc ted f o r ovomucin samples i n r 0.07 M sodium phosphate b u f f e r ( - = 0.13), pH 6.95, c on t a i n i n g 0.02% sodium az ide at p r o t e i n concent ra t i on s between 0.15 mg/ml and 2 0.7-0 mg/ml. The s lopes of these p l o t s (d In A/d(r )) were 'determined and the apparent mo lecu la r weight of ovomucin c a l c u l a t e d as above. The p a r t i a l s p e c i f i c volume of n a t i v e , s t o r ed , and reduced ovomucin was assumed to be the same (0.662 ml/g) as that c a l c u l a t e d f o r RA ovomucin complex. 23 The subscript "app" in this equation denotes that for a nonideal system of charged molecules (i.e. a solution of ovomucin at pH 6.95) the molecular weight is apparent, containing unknown contributions from charge and nonideality effects (Jeffrey and Coates, 1966). RESULTS A. I n t e r a c t i o n Between Egg White Lysozyme and Ovomucin 1. E f f e c t of I o n i c S t rength (a) RA Ovomucin Complex - Lysozyme E q u i l i b r i u m experiments were c a r r i e d out a t 20 ± 0.5°C on s o l u t i o n s of lysozyme, RA ovomucin complex, and RA ovomucin r complex-lysozyme mix tures (1:4) i n 0.07 M ( - = 0.13) and 0.03 M ( - = 0.07) sodium phosphate b u f f e r , pH 6.9, c on ta i n i n g 0.02% sodium a z i d e . For each run th ree samples were analyzed and because of the l a r g e d i f f e r e n c e i n mo lecu la r weight between lysozyme and ovomucin i t was necessary to use d i f f e r e n t r o t o r speeds -a low speed of approx imate ly 10,000 rpm and a h igher speed of approx imate ly 20,000 rpm, to o b t a i n good e q u i l i b r i u m pa t te rn s f o r RA ovomucin complex and lysozyme, r e s p e c t i v e l y . The mo lecu la r weight d i s t r i b u t i o n '(MWD) of p r o t e i n s computed from sed imentat ion e q u i l i b r i u m data are presented i n F i gu re s 1, 2 and 3. The r e s u l t from a t y p i c a l experiment i s shown i n F i gu re 1. Pane l A shows the MWD from the c e l l c on t a i n i n g lysozyme on ly and panels B and C show the MWD pa t te rn s from the c e l l s c on t a i n i n g RA ovomucin complex-lysozyme mixtures (1:4) a t i o n i c s t rengths of 0.13 and 0.07, r e s p e c t i v e l y . I t can be seen that the area under the lysozyme peak i n the MWD of RA ovomucin complex-lysozyme mix tu re s decreases w i t h dec reas ing i o n i c s t r eng th of s o l u t i o n . I n other, words, an i o n i c s t r eng th - dependent i n t e r a c t i o n between RA ovomucin complex and lysozyme i s i n d i c a t e d . A l though the i n t e r a c t i o n i s much s t ronger at i o n i c s t r eng th 0.07, a s l i g h t i n t e r a c t i o n can be detected at i o n i c s t r eng th 0.13 when the area under the lysozyme 25 peak i n the MWD i n pane l A ( co r rec ted f o r sampling volume) i s compared to t ha t i n pane l B. Only a lysozyme peak i s seen i n these MWD pa t t e rn s because the much l a r g e r molecules of RA ovomucin complex and RA ovomucin complex-lysozyme i n t e r a c t i o n product sediment to the c e l l bottom at the e q u i l i b r i u m speed tha t i s needed to o b t a i n a good lysozyme p a t t e r n . The extent of i n t e r a c t i o n of lysozyme w i t h ovomucin can be c a l c u l a t e d as the per cent lysozyme that forms a complex w i t h ovomucin and sediments to the c e l l bottom. In other words, the percentage decrease i n the area of the lysozyme peak i n the MWD of ovomucin-lysozyme mixtures r e l a t i v e to the area of the lysozyme peak from the c e l l c o n t a i n i n g lysozyme on l y , can be used to express the ex tent of i n t e r a c t i o n . Thus a t i o n i c s t r eng th 0.13, the extent of i n t e r a c t i o n i s 5.6% w h i l e at i o n i c s t r eng th 0.07 i t i s 70%. To o b t a i n f u r t h e r i n f o rma t i on on the i n t e r a c t i o n , e s p e c i a l l y on changes i n ovomucin, the i o n i c s t r eng th experiments were repeated u s i ng a much s lower e q u i l i b r i u m speed. F i gu re 2 shows the i n t e r -a c t i o n of RA ovomucin complex w i t h lysozyme at i o n i c s t reng th 0.13. Pane l A shows the MWD of the c e l l c on t a i n i n g lysozyme on l y . Pane l B shows the MWD of the c e l l c on t a i n i n g RA ovomucin complex, y i e l d i n g a t r i m o d a l d i s t r i b u t i o n c o n s i s t i n g of a-ovomucin peaks (component molecu la r weights 115,700 and 493,600) and a 3-ovomucin peak (component mo lecu la r weight 1,113,300). Pane l C shows the MWD p a t t e r n of the c e l l c o n t a i n i n g an RA ovomucin complex-lysozyme mix ture (1 :4 ) . When RA ovomucin complex and lysozyme were mixed, a decrease i n the area under the lysozyme and a-pvomucin peaks and a complete disappearance of the B-ovomucin peak are observed. Thus a measurable i n t e r a c t i o n (extent 6.9%) e x i s t s a t i o n i c s t r eng th 0.13 and a p r e f e r e n t i a l i n t e r a c t i o n of lysozyme w i t h g-ovomucin i s i n d i c a t e d . The i n t e r a c t i o n of RA ovomucin complex w i t h lysozyme at i o n i c s t reng th 0.07 i s shown i n F i g u r e 3. Pane l A shows the MWD of the c e l l c o n t a i n i n g lysozyme on ly and pane l B the MWD p a t t e r n of the c e l l c on t a i n i n g RA ovomucin complex on l y . S e l f a s s o c i a t i o n of a-ovomucins i s seen i n pane l B as evidenced by a b imodal d i s t r i b u t i o n c o n s i s t i n g of a s i n g l e a-ovomucin peak (component mo lecu la r weight 221,100) and a g-ovomucin peak (component mo lecu la r weight 1,149,200). Pane l C shows the e f f e c t of mix ing RA ovomucin complex and lysozyme. Complete disappearance of the g-ovomucin component can be seen as w e l l as the appearance of a new peak (component molecu la r weight 460,-500) which may represent a complex of a s s oc i a ted a-ovomucin w i t h lysozyme. A g rea te r decrease i n the area under the lysozyme peak at i o n i c s t r eng th 0.07 (extent of i n t e r a c t i o n , 73%) as compared to i o n i c s t r eng th 0.13 supports the prev ious f i n d i n g that the RA ovomucin-lysozyme i n t e r a c t i o n i s i o n i c s t r eng th dependent, be ing s t ronger at the lower i o n i c s t r eng th , (b) Na t i ve Ovomucin-Lysozyme To i n v e s t i g a t e the e f f e c t of i o n i c s t r eng th on the i n t e r a c t i o n of n a t i v e ovomucin w i t h lysozyme, e q u i l i b r i u m experiments at 20 ± 0.5°C were c a r r i e d out on s o l u t i o n s of lysozyme and n a t i v e ovomucin-lysozyme mixtures (1:4) i n 0.07 M and 0.03 M sodium phosphate b u f f e r s , pH 6.9, c on t a i n i n g 0.02% sodium a z i d e . The MWD pa t t e rn s of these s o l u t i o n s are shown i n F i gu re 4. An e q u i l i b r i u m speed of about 31,000 rpm was chosen f o r t h i s experiment i n order to l ook a t the lysozyme component on l y as a low speed run to l ook at ovomucin on l y was not p o s s i b l e because even at r o t o r speeds as low as 4,000 rpm the„large n a t i v e ovomucin molecules and molecules of n a t i v e ovomucin-lysozyme complex immediately sedimented to the c e l l bottom. Pane l A shows the MWD of the c e l l c o n t a i n i n g lysozyme on ly w h i l e panels B and C show the MWD pa t t e rn s of the c e l l s c o n t a i n i n g n a t i v e ovomucin-lysozyme mixtures (1:4) at i o n i c s t rengths 0.13 and 0.07, r e s p e c t i v e l y , I t can be seen tha t the a rea under the lysozyme peak of n a t i v e ovomucin-lysozyme mix tures decreases w i t h decreas ing i o n i c s t rengths (extents of i n t e r a c t i o n be ing 5.7% and 27% at i o n i c s t rengths 0.13 and 0.07, r e s p e c t i v e l y ) , i n d i c a t i n g an i o n i c s t r eng th dependent i n t e r a c t i o n between n a t i v e ovomucin and lysozyme. Th i s r e s u l t i s s i m i l a r to that obta ined u s ing RA ovomucin complex-lysozyme m ix tu re s . However, the extent of i n t e r a c t i o n s of n a t i v e ovomucin w i t h lysozyme at i o n i c s t r eng th 0.07 i s on l y about 0.4 t imes as s t rong as t ha t between RA ovomucin and lysozyme at the same i o n i c s t r eng th . 2. E f f e c t of Chemical M o d i f i c a t i o n (a) M o d i f i c a t i o n of Lysozyme The extent of a c e t y l a t i o n of amino groups i n lysozyme was 100 per cent as determined s p e c t r o p h o t o m e t r i c a l l y . Thus a l l seven amino groups of lysozyme were m o d i f i e d . Tbe i n ve s t i g a te the e f f e c t of m o d i f i c a t i o n of amino groups i n lysozyme on the ovomucin-lysozyme i n t e r a c t i o n , s o l u t i o n s of a c e t y l a t e d lysozyme, a c e t y l a t e d lysozyme-RA ovomucin complex (4:1) and a c e t y l a t e d l y sozyme-nat i ve ovomucin (4:1) i n 0.03 M sodium phosphate b u f f e r , pH 6.9, con ta i n i n g 0.02% 28 sodium a z i de were prepar.ed:.and e q u i l i b r i u m experiments were c a r r i e d out a t 20 ± 0.5°C us ing r o t o r speeds of about 34,000 rpm. The MWD pa t t e rn s of these s o l u t i o n s are shown i n F i gu re 5. By t h i s m o d i f i c a t i o n of lysozyme the extent of i n t e r a c t i o n w i t h RA ovomucin complex a t i o n i c s t r eng th 0.07 i s 10.6% and that w i t h n a t i v e ovomucin under s i m i l a r c o n d i t i o n s i s 6.8%. In other words, under c o n d i t i o n s (0.03 M sodium phosphate b u f f e r , pH 6.9) where the extent of lysozyme-RA ovomucin i n t e r a c t i o n i s 70%, acety la t - i on of lysozyme decreased t h i s i n t e r a c t i o n to 10.6% and where the extent of lysozyme-n a t i v e ovomucin i n t e r a c t i o n i s 27%, a c e t y l a t i o n of lysozyme decreased t h i s to 6.8%. (b) M o d i f i c a t i o n of Ovomucin The extent of removal of s i a l i c a c i d by treatment of RA ovomucin complex and n a t i v e ovomucin w i t h neuraminidase was 100% and the MWD of neuraminidase t r e a t e d RA ovomucin complex was s i m i l a r to that of un t rea ted RA ovomucin complex i n d i c a t i n g l i t t l e , i f any p r o t e o l y s i s by contaminat ing enzymes. To i n v e s t i g a t e the e f f e c t of m o d i f i c a t i o n of ovomucin on the ovomucin-lysozyme i n t e r a c t i o n , s o l u t i o n s of lysozyme, a s i a l o RA ovomucin complex, a s i a l o RA ovomucin complex-lysozyme mix tu re (1 :4 ) , a s i a l o n a t i v e ovomucin, and a s i a l o n a t i v e ovomucin-lysozyme m ix tu re (1:4) i n 0.03 M sodium phosphate b u f f e r , pH 6.91, c o n t a i n i n g 0.02% sodium a z i de were prepared and e q u i l i b r i u m experiments were c a r r i e d out at 20 ± 0.5°C. The e f f e c t c o f m o d i f i c a t i o n of RA ovomucin complex on the ovomucin-lysozyme i n t e r a c t i o n i s shown i n F i gu re 6. Pane l B shows the MWD p a t t e r n of the c e l l c on t a i n i n g an RA ovomucin complex-lysozyme mix ture y i e l d i n g a bimodal d i s t r i b u t i o n c o n s i s t i n g of a lysozyme peak and an a s soc i a ted a-ovomucin component of mo lecu la r weight 252,500 w i t h the 8-ovomucin-lysozyme complex hav ing sedimented to 'the c e l l bottom. Here the extent of i n t e r a c t i o n of lysozyme w i t h RA ovomucin i s 72% as c a l c u l a t e d from the a rea under the lysozyme peak i n pane l A. Pane l C shows the b imodal MWD p a t t e r n of the a s i a l o , RA ovomucin complex-lysozyme mix tu re c o n s i s t i n g of a lysozyme component and an a-ovomucin component of mo lecu la r weight 262,700. Probably the a s i a l o , 8-ovomucin-lysozyme i n t e r a c t i o n products have sedimented to the c e l l bottom, thus not be ing shown i n the p a t t e r n . By complete removal of s i a l i c a c i d re s i dues from RA ovomucin complex, the extent of the ovomucin-lysozyme i n t e r a c t i o n i s 76% a t i o n i c s t r eng th 0.07 and no e f f e c t on the p r e f e r e n t i a l i n t e r a c t i o n of lysozyme w i t h 8-ovomucin can be seen. In other words, under c o n d i t i o n s (0.03 M sodium phosphate, pH 6.91) where the extent of the i n t e r a c t i o n of lysozyme w i t h RA ovomucin i s 72%, removal of s i a l i c a c i d r e s i due s has on ly a s l i g h t e f f e c t . F i gu re 7 shows the e f f e c t of m o d i f i c a t i o n of n a t i v e ovomucin on the ovomucin-lysozyme i n t e r a c t i o n . Pane l A shows the MWD of the c e l l c on t a i n i n g lysozyme on l y . Pane l B shows the MWD of the c e l l c on t a i n i n g a n a t i v e ovomucin-lysozyme mix tu re (1:4) and pane l C shows the MWD of the c e l l c on ta i n i n g an a s i a l o , n a t i v e ovomucin-lysozyme mix tu re (1 :4 ) . The extent of i n t e r a c t i o n of lysozyme w i t h n a t i v e ovomucin i s 19.5%, the same as that f o r the i n t e r a c t i o n w i t h mod i f i ed n a t i v e ovomucin. In other words, complete removal of s i a l i c a c i d re s idues from n a t i v e ovomucin has no e f f e c t on the extent of the ovomucin-lysozyme i n t e r a c t i o n a t pH 6.9, i o n i c s t reng th 0.07. 30 3. E f f e c t of Temperature The e f f e c t of temperature on the i o n i c strength-dependent ovomucin-lysozyme i n t e r a c t i o n was s t ud i ed by conduct ing e q u i l i b r i u m experiments at 20 ± 0.5 and 3 ± 0.5°C us ing s o l u t i o n s of lysozyme and RA ovomucin complex-lysozyme mixtures (1:4) i n 0.07 M and 0.03 M sodium phosphate b u f f e r s , pH 6.92, c on t a i n i n g 0. 02% sodium a z i d e . The MWD pa t te rn s a re shown i n F i gu re 8. At i o n i c s t r eng th 0.13 the extent of i n t e r a c t i o n i s v i r t u a l l y the same, 6.4% and 6.8% at temperatures of 3 and 20°C, r e s p e c t i v e l y . However, a t i o n i c s t r eng th 0.07, the extent of i n t e r a c t i o n i s much s t ronger at 3°C (100%) than at 20°C (77%). B. Mo lecu l a r Weight of Ovomucin Dur ing Egg White Th inn ing 1. P r e p a r a t i o n of N a t i v e , Stored and Reduced Ovomucins F i gu re 9 shows the e l u t i o n p r o f i l e s of n a t i v e , s to red and reduced egg wh i te on Sepharose 4B. Young and Gardner (1972) and Hayakawa and Sato (1976) repo r ted that dur ing g e l f i l t r a t i o n of b lended egg wh i t e , ovomucin emerged i n the v o i d volume from columns of Sepharose 4B i n T r i s - H G l b u f f e r , pH 8.6, c on ta i n i n g 0.85% NaC l . As shown i n panels A and B, the re i s l i t t l e d i f f e r e n c e between the e l u t i o n p r o f i l e s of n a t i v e and s to red wh i t e , except f o r a l i t t l e broader v o i d volume peak i n the p r o f i l e of s to red 1% wh i t e . The e x t i n c t i o n c o e f f i c i e n t , E ° of the ovomucin 280 nm, 1 cm f r a c t i o n was 7.0 f o r a pH 8.6 s o l u t i o n and the content of the ovomucin f r a c t i o n c a l c u l a t e d from the absorbance i n F i gu re 9 was about 3 per cent of the t o t a l n a t i v e egg wh i te s o l i d s . Robinson (1972) repo r ted that ovomucins comprise 1.5 to 3.0 per cent of t o t a l egg 31 wh i te s o l i d s , hence i t appears that most of the ovomucin i n egg wh i te can be i s o l a t e d i n a s o l u b l e s t a t e by g e l f i l t r a t i o n on Sepharose 4B a f t e r b l end ing and d i l u t i o n a t pH 8.6. F i gu re 9C shows the e l u t i o n p a t t e r n of egg wh i te reduced w i t h 0.02% 2-mer-cap toe thano l . The v o i d volume f r a c t i o n decreased w h i l e the lower mo lecu la r weight f r a c t i o n s ( f r a c t i o n s 35 to 40) i n c rea sed , i n d i c a t i n g a complete ly d i f f e r e n t p r o f i l e from that of f r a c t i o n a t i o n of s to red wh i t e . 2. Amino A c i d Composit ion of Ovomucin Table 1 shows the amino a c i d compos i t ion of the ovomucin f r a c t i o n from n a t i v e egg wh i te obta ined i n t h i s experiment and the compos i t ions of ovomucin and ovomucin complex repor ted e a r l i e r a re a l s o g i ven f o r comparison. There i s g ene r a l l y good agreement between our r e s u l t s and the compos i t ion of n a t i v e ovomucin as repo r ted by Young and Gardner (1972) except f o r the s u l f u r con -t a i n i n g amino a c i d s , methionine and c y s t e i n e . With few except ions there i s reasonably good agreement between our r e s u l t s and the other seven se t s of da ta . The v a r i a b i l i t y can be a t t r i b u t e d to the d i f f e r e n t methods of p r epa r a t i o n of ovomucin. Table 2 shows the amino a c i d compos i t ions of ovomucin from n a t i v e and s to red egg wh i t e . I t can be seen that the amino a c i d compos i t ion of the ovomucin f r a c t i o n d i d not change du r ing storage at 30°C f o r 166 h under c o n d i t i o n s where the pH of the egg wh i te i nc reased from 7.8 to 9.0 and n a t u r a l t h i n n i n g occur red . 3. Carbohydrate Composit ion of Ovomucins Tab le 3 g i ve s the carbohydrate content of the same ovomucin p repa ra t i on s l i s t e d i n Table 2. The data f o r ovomucin from f r e s h whi te i s i n gene ra l agreement w i t h e a r l i e r r epo r t s (Adachi ejt a l . , 1973; Kato et a l . , 1973), however con s i de rab le v a r i a b i l i t y i n carbohydrate content of ovomucin has been repor ted (Smith et a l . , 1974) due to the f a c t that almost a l l g l y cop ro t e i n s c o n t a i n t h e i r carbohydrate u n i t s i n v a r y i n g stages of complet ion (Robinson, 1972). Hence i t may not be meaningfu l to compare carbohydrate compos i t ions amongst d i f f e r e n t ovomucin p r epa ra t i on s . I t can be seen tha t the carbohydrate compos i t ion of the ovomucin f r a c t i o n d i d not change dur ing storage f o r 166 h at 30°C under c o n d i t i o n s i n which n a t u r a l t h i n n i n g occu r red . In c o n t r a s t , p rev ious s t ud i e s repor ted a decrease i n s i a l i c a c i d , hexose and hexosamine content of ovomucin accompanying s torage of t h i c k wh i te (Kato ejt a l . , 1972; Robinson and Monsey, 1972 a, b ) . 4. Mo lecu la r Weight of Ovomucins To get i n f o rma t i on "about the molecu la r weight of ovomucin du r i ng n a t u r a l t h i n n i n g , sed imentat ion e q u i l i b r i u m a n a l y s i s was c a r r i e d out . The running speed was set a t a low speed, 4,000 rpm, to get s t a b l e e q u i l i b r i u m p a t t e r n s . At r o t o r speeds much h igher than 4,000 rpm the ovomucin molecules sedimented to the c e l l bottom q u i t e r a p i d l y . F i gu re 10 shows r e p r e s e n t a t i v e r e s u l t s of 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 l a n a l y s i s of n a t i v e and s to red ovo-mucins, a t i n i t i a l p r o t e i n concent ra t i on s between 0.15 to 0.67 mg/ml 2 and 0.14 to 0.60 mg/ml, r e s p e c t i v e l y . The p l o t s of In A versus r f o r n a t i v e and s to red ovomucin f r a c t i o n s showed l i n e a r r e l a t i o n s h i p s w i t h h i gher s lopes observed f o r lower p r o t e i n concen t r a t i on s . The 2 s lopes of the p l o t s of In A versus r , which are p r o p o r t i o n a l to mo lecu la r we ight , were used to c a l c u l a t e the molecu lar weights as a f u n c t i o n of ' ovomucin concen t r a t i on accord ing to the r e l a t i o n s h i p 33 = 2 RT (d l n c ) w app / n N 2 •, / 2. ^r (l-vp)o) d (r ) as p r e v i o u s l y de s c r i bed . Tables 4 and 5 l i s t the c a l c u l a t e d mo lecu la r weights of n a t i v e and s to red ovomucins as a f u n c t i o n of p r o t e i n concen t r a t i on and these data are p l o t t e d i n F i gu re 11. As shown i n t h i s f i g u r e , the mo lecu la r weight of both n a t i v e and s to red ovomucins a re markedly dependent on the p r o t e i n concen t r a t i on - M of 5.6 -w app 7.5 x 10 f o r ovomucin concent ra t i on s of 0.15 to 0.67 mg/ml. By u s i ng l i n e a r r e g re s s i on a n a l y s i s , the f o l l o w i n g r e l a t i o n s h i p s between apparent molecu la r weight (M ) and p r o t e i n concen t r a t i on ° w app (C) have been c a l c u l a t e d : M x 10 ^ = -4.95 C + 5.64 f o r n a t i v e w app —6 ovomucin and M x 10- = -4.89 C + 5.25 f o r s to red ovomucin, suggest ing tha t t h e r e ^ i s l i t t l e d i f f e r e n c e between the molecu la r weight of ovomucin from f r e s h versus s to red egg wh i t e . 2 Sedimentat ion e q u i l i b r i u m pa t te rn s ( In A vs r ) of reduced ovomucin f r a c t i o n s 24 and 37 from g e l f i l t r a t i o n of egg wh i te reduced w i t h 0.02% 2-mercaptoethanol (F igure 9C) are shown i n F i gu re 12. The molecu la r weights c a l c u l a t e d from the s lopes of these p l o t s were 726,200 and 309,500, r e s p e c t i v e l y . 34 DISCUSSION A. I n t e r a c t i o n Between Lysozyme and Ovomucin 1. E f f e c t of I on i c S t rength (a) RA Ovomucin Complex-Lysozyme I t has been shown by d i r e c t measurement of p r o t e i n con -c e n t r a t i o n s that the i n t e r a c t i o n between RA ovomucin complex and lysozyme, a t pH 6.9, i s e l e c t r o s t a t i c , judg ing by the dependence of the i n t e r a c t i o n on i o n i c s t r eng th (Kauzmann, 1959), as proposed i n e a r l i e r r epo r t s ( K l o t z and Walker, 1948; Hawthorne, 1950; C o t t e r i l l and Winter , 1955; Robinson and Monsey, 1969b; Kato and Sato, 1972; Kato et a l . , 1975; Kato et a l . , 1976; G a r i b a l d i et a l . , 1968). However p rev ious measurements of t h i s i n t e r a c t i o n ( C o t t e r i l l and W in te r , 1955; Dam and Bennett , 1963; Robinson and Monsey, 1969 b; Dam, 1971; Kato et a l . , 1971; Kato and Sato, 1972; Robinson, 1972; Kato et a l . , 1975; Kato et a l . , 1976) were based on the assumption that complexes between ovomucin and lysozyme are i n s o l u b l e , thus p o s s i b l e s o l ub l e i n t e r -a c t i o n complexes were ignored i n determin ing the extent of i n t e r -a c t i o n . Moreover, none of these methods measured p r o t e i n concen t r a t i on d i r e c t l y , hence they may not be r e l i a b l e . For example, C o t t e r i l l and Winter (1955) s t ud i ed the e f f e c t of pH on the ovomucin-lysozyme i n t e r a c t i o n by measuring the volume of p r e c i p i t a t e formed upon a d d i t i o n of s o l u t i o n s of lysozyme to ovomucin. Dam and Bennett (1963) and Dam (1971) s tud ied the ovomucin-lysozyme i n t e r a c t i o n by measuring the lysozyme b i nd i n g a b i l i t y of ovomucin p repa ra t i on s f o l l o w i n g c e n t r i f u g a t i o n of t u r b i d s o l u t i o n s formed upon mix ing lysozyme and 35 ovomucin s o l u t i o n s . T u r b i d i t y dete rminat ions a t 450 nm (Robinson and Monsey, 1969 b; Robinson, 1972), 550 nm (Kato et a l . , 1975; Kato e t a l . , 1976) and 600 nm (Kato et a l . , 1971; Kato and Sato, 1972) of m ix tu res of ovomucin and lysozyme were used as a measure of the amount of ovomucin-lysozyme complex, hence the extent of i n t e r a c t i o n between lysozyme and ovomucin, assuming that a l l i n t e r a c t i o n products produce t u r b i d i t y . The u se fu lnes s of sed imentat ion 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 f o r s tudy ing heterogeneous p r o t e i n - p r o t e i n a s s o c i a t i o n s i n i n t e r -a c t i n g mix tu res of RA ovomucin complex and lysozyme, under con -d i t i o n s (0.07 M phosphate b u f f e r , pH 6.9) where an i n t e r a c t i o n cou ld not be detected us ing t u r b i d i m e t r i c measurements (Kato et a l . , 1975; Kato et a l . , 1976) .can be seen i n F i gu re 2. Here i n -fo rmat ion on both the extent of i n t e r a c t i o n and the p a r t i c i p a t i n g components i n the i n t e r a c t i o n can be ob ta ined . For example, by comparing the area under the lysozyme peak i n the MWD p a t t e r n of the c e l l c o n t a i n i n g lysozyme on ly to tha t i n the c e l l c on t a i n i n g a lysozyme-ovomucin m i x tu re , an extent of i n t e r a c t i o n of 6.9% was c a l c u l a t e d . . Furthermore, by comparison of the MWD p a t t e r n of the c e l l c o n t a i n i n g RA ovomucin complex on ly to that of the c e l l c o n t a i n i n g lysozyme-ovomucin m i x tu re , a p r e f e r e n t i a l i n t e r a c t i o n between the 8-ovomucin component and lysozyme i s seen. Kato et_ a l . (1975) have repo r ted tha t the i n t e r a c t i o n of 8-ovomucin (F-ovomucin) w i t h lysozyme was much s t ronger than that w i t h a-ovomucin (S-ovomucin) i n 0.07 M phosphate, pH 5.4. However, t h i s pH i s very f a r removed from the i n v i v o egg wh i te pH of approx imate ly 7.4 to 7.8. 36 In F i gu re 3 i t can be seen that the i n t e r a c t i o n between RA ovomucin complex and lysozyme i s s t ronger a t i o n i c s t reng th 0.07 and tha t s e l f - a s s o c i a t i o n of a-ovomucin occur s . Robinson and Monsey (1971) have repor ted s e l f - a s s o c i a t i o n of reduced a-ovomucins a t pH 7.5 i n the absence of the d i s s o c i a t i n g agent, guanid ine h y d r o c h l o r i d e . The i o n i c strength-dependent i n t e r a c t i o n between a-ovomucins a t pH 6.9 may be e l e c t r o s t a t i c as i n d i c a t e d by the present r e s u l t s , a l though Van der Waals f o r ce s and hydrogen bonds may a l s o be i n vo l ved (Waugh, 1954). The importance of the s e l f -a s s o c i a t i o n of a-ovomucins to the s t r u c t u r e of t h i c k egg wh i te and mechanism of egg wh i t e t h i n n i n g needs i n v e s t i g a t i o n , (b) Na t i v e Ovomucin-Lysozyme A l though the i n t e r a c t i o n of RA ovomucin complex w i t h lysozyme has been s t u d i e d , t he re have been no p rev ious r e p o r t s on the i n t e r a c t i o n of n a t i v e ovomucin, t ha t i s ovomucin s o l u b i l i z e d a t n e u t r a l pH w i thout chemica l m o d i f i c a t i o n , w i t h lysozyme. The importance of such a study to the understanding of the iri v i v o i n t e r a c t i o n of lysozyme w i t h ovomucin i s obv ious. I t was found i n the present study tha t the i n t e r a c t i o n between n a t i v e ovomucin and lysozyme was a l s o dependent upon i o n i c s t reng th and hence e l e c t r o -s t a t i c i n na tu re . However, w h i l e there was l i t t l e d i f f e r e n c e i n the ex tent s of i n t e r a c t i o n of lysozyme w i t h n a t i v e ovomucin compared to tha t w i t h RA ovocumin complex a t i o n i c s t r eng th 0.13, the i n t e r -a c t i o n of lysozyme w i t h n a t i v e ovomucin a t i o n i c s t r eng th 0.07 was on l y 0.4 t imes as s t rong as that w i t h RA ovomucin complex at the same i o n i c s t r eng th . I t i s known that n a t i v e ovomucin c o n s i s t s of aggregated and po lymer ized molecules of i n t e r a c t i n g a - and B-ovomucins 37 c r o s s - l i n k e d by s p e c i f i c bonds (Adachi et a l . , 1973; Hayakawa and Sato, 1976). S i m i l a r l y the s t r u c t u r e of ovomucin complex i s known to c o n s i s t of po lymer ized cha ins of a- and 8-ovomucins, the i n d i v i d u a l subun i t s be ing he l d together by d i s u l f i d e bonds (Kato et a l . , 1971; Robinson and Monsey, 1971, 1975; Tomimatsu and Donovan, 1972). Reduct ion and a l k y l a t i o n of the t h i o l groups i n t h i s complex r e s u l t s i n depo l ymer i z a t i on of the a- and 8-ovomucins to g i ve a heterogeneous m ix tu re of a- and 8 -subunits. S ince i t has been repo r ted tha t o p t i c a l r o t a t o r y d i s p e r s i o n experiments of mercaptoethanol - reduced ovomucin i n d i c a t e that the conformat ion of the po l ypep t i de cha ins i s not a l t e r e d by r e d u c t i o n (Donovan j i t a l . , 1970) i t i s assumed tha t the g rea te r i n t e r a c t i o n of lysozyme w i t h RA ovomucin complex i s due to more lysozyme b i nd i ng s i t e s be ing exposed i n the depolymer ized or p a r t l y a s s o c i a t ed a- and 8-subunits of the RA ovomucin complex than i n the much l a r g e r n a t i v e ovomucin molecu le which migrates as a s i n g l e component dur ing sed imentat ion . Th i s assumption i s supported by observat ions that pronase or t r y p s i n d i g e s t e d , reduced ovomucins i n t e r a c t more s t r o n g l y w i t h lysozyme than do unt reated reduced ovomucins (Kato je_t a l . , 1975; Kato et a l . , 1976) and t ha t the carbohydrate s i de cha ins of ovomucin which con ta i n the lysozyme b i n d i n g s i t e s seem to be concentrated a long p o r t i o n s of the p r o t e i n core (Kato jet al., 1973). In other words, i t i s proposed t ha t d i s s o c i a t i o n of subun i t s caused by r e d u c t i o n of s u l f h y d r y l bonds exposes the carbohydrate s i de cha ins of ovomucin which con ta i n the lysozyme b i nd i n g s i t e s . Zeta p o t e n t i a l or su r face charge measurements of RA ovomucin complex ver sus n a t i v e ovomucin may con f i rm t h i s p r opo sa l . 38 2. E f f e c t of Chemical M o d i f i c a t i o n (a) Lysozyme The i s o i o n i c pH of lysozyme i s 10.7 (Osuga and Feeney, 1977) w h i l e t ha t of ovomucin i s 4.5 to 5.0 (Donovan et a l . , 1970), hence at pH 6.90 lysozyme w i l l c a r r y a net p o s i t i v e charge and ovomucin a net negat i ve charge. Lysozyme has s i x l y s y l e-amino groups and an a-amino group ( C a n f i e l d , 1963 b) c a r r y i n g p o s i t i v e charges a t pH 6.9 and these groups can be chem ica l l y mod i f i ed by a c e t y l a t i o n r e s u l t i n g i n a decrease i n the net p o s i t i v e charge. I t was shown i n the present study tha t the extent of i n t e r a c t i o n of lysozyme w i t h both RA ovomucin complex and n a t i v e ovomucin decreased but was not complete ly abo l i shed f o l l o w i n g 100% m o d i f i c a t i o n of the f r e e amino groups i n lysozyme. Hence the l y s y l e-amino groups i n lysozyme are e s s e n t i a l f o r the e l e c t r o s t a t i c ovomucin-lysozyme i n -t e r a c t i o n . Kato et al. (1975, 1976) a l s o showed that the lysozyme -RA ovomucin i n t e r a c t i o n was markedly decreased by a c e t y l a t i o n of l y s y l amino groups i n lysozyme and that the i n t e r a c t i o n was complete ly abo l i s hed when 6 amino groups were m o d i f i e d . I t has been shown that no con fo rmat iona l change occurs i n the po l ypep t i de cha in as a r e s u l t of a c e t y l a t i o n of lysozyme (Yamasaki et a l . , 1968), hence i t i s p o s s i b l e tha t the r e s i d u a l i n t e r a c t i o n of a c e t y l a t e d lysozyme w i t h RA ovomucin complex and n a t i v e ovomucin represent e l e c t r o s t a t i c bonding between other p o s i t i v e l y charged groups such as h i s t i d i n e or a r g i n i n e i n lysozyme ( S t e i n e r , 1953) or hydrogen bonding (Robinson and Monsey, 1969 a ) . That t h i s r e s i d u a l i n t e r a c t i o n was not detected u s ing t u r b i d i m e t r i c measurements (Kato et a i . , 1975) i s a f u r t h e r i n d i c a t i o n of the decreased s e n s i t i v i t y of t ha t technique f o r d e t e c t i n g p r o t e i n -p r o t e i n i n t e r a c t i o n s . (b) Ovomucin I t was shown i n the present study that complete removal of s i a l i c a c i d re s i dues (which are n e g a t i v e l y charged at pH 6.9) from RA ovomucin complex and n a t i v e ovomucin by treatment w i t h neuraminidase had l i t t l e e f f e c t on the ovomucin-lysozyme i n t e r -a c t i o n a t i o n i c s t r eng th 0.07 and that a p r e f e r e n t i a l i n t e r a c t i o n of lysozyme w i t h 8-ovomucin occurred even though i t s s i a l i c a c i d r e s i due s had been removed. Dam and Bennett (1963) and Dam (1971) a l s o repor ted tha t enzymatic removal of s i a l i c a c i d re s i dues from ovomucin had l i t t l e e f f e c t on lysozyme b i n d i n g . Thus, i f the i n t e r a c t i o n between ovomucin and lysozyme i s e l e c t r o s t a t i c i n na tu re , the re must be s u f f i c i e n t n e g a t i v e l y charged po l a r groups s t i l l present to account f o r the i n t e r a c t i o n of lysozyme w i t h a s i a l o ovomucin. I t i s known tha t ovomucin a l s o conta in s e s t e r s u l f a t e which would be n e g a t i v e l y charged at pH 6.91 and that the r a t i o of s i a l i c a c i d to e s t e r s u l f a t e i s approx imate ly 1:1 (Donovan et a l . , 1970; Robinson and Monsey, 1971; Kato and Sato, 1971; Kato et a l . , 1978 c ) . S ince 8-ovomucin conta in s s i a l i c a c i d and e s te r s u l f a t e w h i l e a-ovomucin does not (Kato et a l . , 1972; Kato et a l . , 1973) and s i nce there i s a p r e f e r e n t i a l i n t e r a c t i o n of lysozyme w i t h 8-ovomucin even a f t e r complete removal of n e g a t i v e l y charged s i a l i c a c i d r e s i due s , i t seems l i k e l y that i t i s the net negat i ve charge of the ovomucin molecule made up of c o n t r i b u t i o n s from s i a l i c a c i d and e s t e r s u l f a t e , t ha t i s important f o r the e l e c t r o s t a t i c ovomucin-lysozyme i n t e r a c t i o n . In other words, there i s s t i l l s u f f i c i e n t nega t i ve charge c a r r i e d by e s t e r s u l f a t e a f t e r removal of s i a l i c a c i d re s i dues f o r a lysozyme-ovomucin i n t e r a c t i o n . Other a c i d i c 40 groups such as the c a rboxy l groups of g lu tamic and a s p a r t i c a c i d s , present i n ovomucin, may be the a c t i v e groups which r eac t w i t h lysozyme (Robinson, 1972). Th i s seems u n l i k e l y though, as a-ovomucin conta in s more of these groups than does g-ovomucin. I t i s p o s s i b l e , however, that i n a d d i t i o n to e l e c t r o s t a t i c i n t e r -a c t i o n s , a more s p e c i f i c type of i n t e r a c t i o n between the N - a c e t y l -glucosamine groups of g-ovomucin and the a c t i v e s i t e of lysozyme i s i n vo l v ed (Howlett and N i c h o l , 1973) as g-ovomucin conta in s more than 1.6 t imes as much N-acety lg lucosamine as does a-ovomucin (Smith et a l . , 1974). The r e s u l t s of the present study a re s u r p r i s i n g i n v iew of the p rev ious r epo r t s of Kato et a l . (1975) who found that the ovomucin-lysozyme i n t e r a c t i o n decreased co r re spond ing l y at a r a t e depending on the extent of m o d i f i c a t i o n of ovomucin w i t h neuraminidase and of Kato et al. (1976) who repor ted tha t s i a l i c a c i d i n ovomucin i s commonly e s s e n t i a l f o r i t s i n t e r a c t i o n w i t h ovalbumin and conalbumin and f o r i t s i n h i b i t i o n of the aggregat ion of K -case in by renn in (Kato et a l . , 1974). In f a c t a d e t a i l e d study by Kato et a l . (1976) showed tha t the b i n d i n g s i t e f o r the ovomucin-lysozyme i n t e r a c t i o n was the ca rboxy l group of the s i a l i c a c i d r e s i due . I t should be noted that these s t ud i e s were done at pH 5.4 and i o n i c s t reng th 0.13 us ing t u r b i d i m e t r i c measurements to de tec t i n s o l u b l e ovomucin-prote in i n t e r a c t i o n s . Loss of. n e g a t i v e l y charged s i a l i c a c i d groups may r e s u l t i n a change i n shape of f o l d i n g of ovomucin due to a r educ t i on i n e l e c t r o s t a t i c r e p u l s i o n (Got t s cha l k and Thomas, 1961; Sachdev e t a l . , 1979) and such a change i n conformat ion might a f f e c t the a b i l i t y of the a s i a l o ovomucin to form an i n s o l u b l e or t u r b i d i n t e r a c t i o n 41 complex w i t h lysozyme at pH 5.4 thus a f f e c t i n g i t s d e t e c t i o n by t u r b i d i m e t r i c measurements. I t seems p o s s i b l e to r e s o l v e the ques t i on of whether the e s t e r s u l f a t e i s the b i nd i n g group i n the lysozyme -3- ovomucin i n t e r a c t i o n or whether i t i s the net negat i ve charge of 3-ovomucin c on t r i bu ted by both s i a l i c a c i d re s idues and e s te r s u l f a t e tha t i s important , by enzymatic removal of e s t e r s u l f a t e . 3. E f f e c t of Temperature That the i n t e r a c t i o n between lysozyme and RA ovomucin complex was s t ronger at 3° than 20°C at i o n i c s t r eng th 0.07 i s suggest ive of an e l e c t r o s t a t i c i n t e r a c t i o n and/or hydrogen bonding between lysozyme and ovomucin (Douzou and Ba lny , 1978). That the extent of i n t e r a c t i o n a t i o n i c s t r eng th 0.13 was apparent l y una f fec ted by temperature may be due to the " s t r o n g " s a l t s o l u t i o n (h igh i o n i c s t rength ) which can reduce e l e c t r o s t a t i c p r o t e i n - p r o t e i n i n t e r a c t i o n s (Dixon and Webb, 1961) and suggests t h a t hydrogen bonding may not be important . Dam (1971) repor ted l i t t l e d i f f e r e n c e i n the e f f e c t of temperature on the amount of lysozyme bound by ch i cken ovomucin at pH 7.2 yet found tha t much more lysozyme was bound to duck ovomucin at 2° than at 40° at the same pH. As s t a ted p r e v i o u s l y , these b i nd i ng measurements were based on the assumption that a l l complexes between lysozyme and ovomucin are i n s o l u b l e , hence they may not be r e l i a b l e . The d i f f e r e n c e i n b i nd i n g observed between ch i cken and duck ovomucins may be a r e f l e c t i o n of the d i f f e r e n t p r o p e r t i e s ( e . g . , s o l u b i l i t y of ovomucins) between these p a r t i c u l a r spec ie s . 42 B. Mo lecu la r Weight of Ovomucin Dur ing Egg White Th inn ing Ovomucin, a po l yd i s pe r s e g l y c o p r o t e i n f r a c t i o n , p r e c i p i t a t e s from egg wh i te when the s a l t c oncen t r a t i on i s reduced by d i l u t i o n of the albumen w i t h water and becomes i n c r e a s i n g l y i n s o l u b l e when washed w i t h 2% KC1 du r ing f u r t h e r p u r i f i c a t i o n . S ince ovomucin prepared i n t h i s manner i s not s u f f i c i e n t l y s o l u b l e a t a c i d i c or n e u t r a l pH i n non-denatur ing s o l ven t s and i s on l y p a r t i a l l y s o l u b l e i n a l k a l i n e s o l v e n t s , c h a r a c t e r i z a t i o n has been d i f f i c u l t . Most s t ud i e s t h e r e f o r e have been done on chem i ca l l y mod i f i ed ovomucin, t ha t i s ovomucin s o l u b i l i z e d by r e d u c t i o n w i t h mercaptoethanol f o l l owed by a l k y l a t i o n of t h i o l groups w i t h i odoaceta te (Robinson and Monsey, 1964, 1971, 1975; Kato et a l . , 1971; Donovan et a l . , 1970). However, p h y s i c a l measurements on ovomucin s o l u b i l i z e d w i thout chemica l m o d i f i c a t i o n (so c a l l e d " n a t i v e " ovomucin) are necessary to determine the r e l a t i o n of ovomucin to the g e l - l i k e p r o p e r t i e s of t h i c k egg wh i t e . As c a l c u l a t e d from the absorbance va lue s i n F i gu re 9A most of the ovomucin i n egg wh i te was s o l u b i l i z e d by b lend ing egg wh i te which had been p r e v i o u s l y d i l u t e d w i t h 0.05 M T r i s - H C l b u f f e r , pH 8.6, c on t a i n i n g 0.85% Na'Cl. Ovomucin thus obta ined ( na t i ve ovomucin) has an amino a c i d compos i t ion tha t i s i n genera l agreement w i t h tha t p r e v i o u s l y repor ted (Table 1) and a carbohydrate compos i t ion (Table 2) which a l though d i f f e r e n t from prev ious r e p o r t s , r e f l e c t s on the v a r i a b i l i t y i n carbohydrate content of ovomucins ( S l e i g h et^ a l . , 1973; Smith et a l . , 1974). Na t i ve ovomucin showed an apparent molecu la r weight ( e x t r a -po l a t ed to i n f i n i t e d i l u t i o n ) of 5.64 x 10 and a remarkable 43 dependence of mo lecu la r weight on the concen t r a t i on of ovomucin which may r e f l e c t a s t rong a s s o c i a t i o n of ovomucin molecules a t d i l u t e concen t r a t i on s . A s i m i l a r dependence of sed imentat ion c o e f f i c i e n t s on the c oncen t r a t i on of ovomucin and of mucoproteins i n gene ra l has been repor ted (Adacbi e t a l . , 1973; Robinson and Monsey, 1975; Morawieck i , 1964). Table 6 shows the molecu la r weights of s o l u b l e ovomucins that have been repor ted and i t i s apparent that the molecu lar weight v a r i e s acco rd ing to the method of s o l u b i l i z a t i o n . I t should be noted tha t these pub l i shed molecu la r weight determinat ions were done at s i n g l e concent ra t i on s of ovomucin so i t i s necessary to compare them to the range of apparent mo lecu la r weights (2.43 - 5.15 x 10^) determined i n the present study. Thus n a t i v e ovomucin has a molecu la r 6 6 weight i n gene ra l agreement to the va l ue s of 7.6 x 10 and 8.3 x 10 repo r ted by Lann i et a l . (1949) and Hayakawa and Sato (1976), r e s p e c t i v e l y . The mo lecu la r weight of the ovomucin f r a c t i o n decreased on l y s l i g h t l y du r i ng the n a t u r a l t h i n n i n g of t h i c k wh i te induced by s torage of albumen at 30°C f o r one week and no change i n the amino a c i d and carbohydrate compos i t ions between the n a t i v e and s to red ovomucins was observed. Moreover, the molecu lar weight of s to red ovomucin (5.25 x 10 ) was much h igher than tha t determined f o r reduced ovomucins (mol wt. 726,000 and 309,500). S ince ovomucin g e l i n t h i c k wh i te i s s o l u b i l i z e d by low speed b l end i n g , i t seems p o s s i b l e t ha t the t h i c k wh i te g e l s t r u c t u r e c o n s i s t s of aggregated h i gh mo lecu la r weight ovomucins (mol wt. 5.6 x 10 ) l i n k e d i n t e r -mo lecu la r l y by non-cova lent f o r ce s such as hydrogen bonding or 44 hydrophobic i n t e r a c t i o n s (Robson et a l . , 1975) and that d i s agg rega t i on of these molecules might occur du r i ng n a t u r a l t h i n n i n g . S e l f -aggregat ion of mucin type g l y c o p r o t e i n s of e p i t h e l i a l s e c r e t i o n s has been observed ( H i l l jet al., 1977). Th ick §gg wh i t e i s a l s o s o l u b i l i z e d by 2-mercaptoethanol produc ing low molecu la r weight ovomucins. P rev ious s t ud i e s (Robinson and Monsey, 1971, 1975) showed t h a t r e d u c t i o n of the d i s u l f i d e bonds of ovomucin s p l i t s ovomucin i n t o a - and. 3-subunits (mol wt. 210,000 and 720,000 r e s p e c t i v e l y ) . The p r oduc t i on of s i m i l a r g l y c o -p r o t e i n subun i t s by r e d u c t i o n of t h i c k egg wh i te shows t ha t c leavage of i n t r a m o l e c u l a r d i s u l f i d e bonds i n ovomucin r e s u l t s i n the b reak -down of the i n t e r m o l e c u l a r non-cova lent i n t e r a c t i o n s r e s pon s i b l e f o r g e l f o rmat i on . However, as t he re were no low mo lecu la r weight ovomucins produced du r ing n a t u r a l t h i n n i n g that were produced i n the presence of reduc ing agents, i t seems tha t i n t e rmo l e cu l a r d i s u l f i d e c leavage of ovomucin does not occur dur ing n a t u r a l t h i n n i n g . I t should be noted t ha t g e l f i l t r a t i o n of b lended egg wh i te i s o l a t e s a l l s o l ub l e ovomucins, i . e . , those which may be d i saggregated from the t h i c k wh i te g e l and are thus present i n the t h i n wh i te and those t ha t were present i n the g e l but were s o l u b i l i z e d by b l end i ng . Thus i t i s d i f f i c u l t , i f not impo s s i b l e , to d i s t i n g u i s h between these two c l a s s e s of ovomucins i n the present experiment. To show tha t d i s agg rega t i on of ovomucin molecules does occur dur ing n a t u r a l t h i n n i n g , the f o l l o w i n g experiments a re suggested. A f t e r s epa ra t i on of f r e s h t h i c k and t h i n wh i te s by u l t r a c e n t r i f u g a t i o n (59,000 x g f o r 60 min) acco rd ing to the method of Kato e t a l . (1971), determine the mo lecu la r weight and chemica l compos i t ion of the ovomucin f r a c t i o n s obta ined upon g e l f i l t r a t i o n of blended t h i c k wh i te versus unblended t h i n wh i t e . Repeat the experiment us ing n a t u r a l l y th inned egg wh i te ( i . e . , albumen s to red at 30°C f o r one week). Comparison of the r e s u l t s from these experiments may suggest that there i s an e q u i l i b r i u m between aggregat ion and d i s agg rega t i on of ovomucin molecu les which i s i n favour of the aggregated s t a t e a t the pH of f r e s h albumen and i s s h i f t e d i n favour of the d i saggregated s t a t e as the pH of the wh i te i nc rea se s dur ing s torage (Powrie, 1980). 46 GENERAL DISCUSSION Although many mechanisms have been proposed f o r the r o l e of ovomucin i n the t h i n n i n g of t h i c k wh i te (Feeney, 1955; Osuga and Feeney, 1977) those tha t have a t t r a c t e d the most study can be c l a s s i f i e d i n t o th ree genera l groups. One of these i s based on the fo rmat ion of a complex between lysozyme and ovomucin. Another i s based upon a p h y s i c a l or chemica l change i n ovomucin such as d i s a gg rega t i on or chemica l h y d r o l y s i s and the t h i r d i s e s s e n t i a l l y a combinat ion of the f i r s t two. The purpose of t h i s genera l d i s -cu s s i on i s to r e l a t e the r e s u l t s obta ined i n the present study of ovomucin to these th ree mechanisms of egg wh i te t h i n n i n g . 1. Lysozyme-Ovomucin Complex Accord ing to t h i s mechanism the r i g i d i t y or s t r u c t u r e of t h i c k wh i te i s due to the fo rmat ion of a complex between lysozyme and ovomucin. The non-cova lent d i s s o c i a t i o n of t h i s complex accompanying the i n c rea se i n pH, (from 7.8 to 9 .5) , dur ing storage of s h e l l eggs at room temperature, r e s u l t s i n t h i n n i n g . I t has been repor ted (Brooks and Ha le , 1959, 1961) that a s imple network of ovomucin cha ins w i l l not account f o r the mechanica l p r o p e r t i e s of t h i c k wh i te and tha t the ge l a t i nou s s t r u c t u r e i s b e t t e r exp la ined by assuming that cha ins of an ovomucin-lysozyme complex are c r o s s - l i n k e d i n t o a network, which i s i n accord w i t h accepted hypotheses concern ing the nature of g e l s t r u c t u r e ( Fe r r y , 1948). Moreover, mucin-albumin i n t e r a c t i o n s have been shown to a f f e c t the p r o p e r t i e s of mucin s o l u t i o n s no tab l y enhancement of v i s c o s i t y ( L i s t et a l . , 1978). Thus a requirement of t h i s mechanism f o r a p p l i c a t i o n to egg wh i te t h i n n i n g i s t ha t lysozyme and ovomucin i n t e r a c t under the c o n d i t i o n s ( i o n i c s t r eng th and pH) found i n f r e s h t h i c k wh i t e . A f u r t h e r requirement of t h i s 47 mechanism i s t ha t the extent of the p r o t e i n - p r o t e i n i n t e r a c t i o n decrease w i t h i n c r e a s i n g pH. In t h i s s tudy, d i r e c t evidence has been obta ined that the i n t e r a c t i o n between ovomucin and lysozyme i n v i t r o i s e l e c t r o s t a t i c , i n v o l v i n g the p o s i t i v e l y charged l y s y l ee.amino groups of lysozyme and the net negat i ve charge ( i . e . , not that c a r r i e d e x c l u s i v e l y by the s i a l i c a c i d re s idues ) of ovomucin. Moreover, i t was shown tha t the re i s an i n t e r a c t i o n between lysozyme and n a t i v e ovomucin a t i o n i c s t r eng th 0.10, which i s the approximate i o n i c s t r eng th of egg wh i te (Donovan et a l . , 1972; Sato et a l . , 1976). These r e s u l t s suggest but do not prove the e x i s t ence of an i n t e r a c t i o n between lysozyme and ovomucin, _in v i v o . A l though the extent of the i n t e r -a c t i o n as a f u n c t i o n of pH was not measured i n the present study, because of the tendency of ovomucin and lysozyme to p r e c i p i t a t e out of d i l u t e s a l t s o l u t i o n s above pH 7.4, i t can be concluded that i f the i n t e r a c t i o n between lysozyme and ovomucin i s e l e c t r o s t a t i c , an i n c rea se i n pH would cause a decrease i n the extent of i n t e r a c t i o n (assuming no con fo rmat iona l changes) due to a decrease i n the p o s i t i v e charge of lysozyme as i t s i s o i o n i c pH of 10.7 i s approached. However, t he re has been l i t t l e i n f o rma t i on on whether the lysozyme-ovomucin i n t e r a c t i o n e x i s t s _in v i v o i n t h i c k wh i t e . Kato et a l . (1978^a) s t ud i ed the changes i n lysozyme dur ing t h i n n i n g and found t ha t the r e l a t i v e concen t r a t i on of lysozyme i n the g e l f r a c t i o n was 1.4 t imes tha t of the l i q u i d f r a c t i o n i n t h i c k wh i t e . A f t e r s torage of t h i c k wh i te f o r 5 days, some d i f f e r e n c e s i n lysozyme content between the g e l and l i q u i d f r a c t i o n s were found. These r e s u l t s suggest tha t lysozyme has a s t rong a f f i n i t y f o r the t h i c k wh i te g e l (which i s main ly composed of ovomucin) and that lysozyme d i s s o c i a t e s from the g e l du r i ng s to rage. In a d d i t i o n to i n t e r a c t i n g w i t h ovomucin, b a s i c lysozyme has been shown to b ind w i t h some of the other p r o t e i n s i n egg wh i te - ovalbumin (Howlett and. N i c h o l , - 1973; Naka i and Kason, 1974) and conalbumin (Ehrenpre i s and Warner, 1956) both of which are present i n much l a r g e r amounts than ovomucin i n egg wh i t e . As w e l l , ovomucin has been shown to i n t e r a c t n o n - s p e c i f i c a l l y w i t h ovalbumin and conalbumin (Kato et a l . , 1976). The r e l a t i o n s of these i n t e r a c t i o n s to the r i g i d i t y of t h i c k wh i te and t h e i r p o s s i b l e r o l e i n t h i n n i n g i s unknown and needs i n v e s t i g a t i o n . 2. Chemical or P h y s i c a l Change i n Ovomucin Dur ing Th inn ing A c on s i s t en t ob se r va t i on on the d i f f e r e n c e s between t h i c k and t h i n wh i t e i n f r e s h eggs (a s ide from the obvious s t r u c t u r a l v i s c o s i t y ) has been tha t t h i c k wh i te conta in s approx imate ly four t imes as much ovomucin as t h i n wh i te (McNal ly , 1933; For sy the and F o s t e r , 1949; Lann i et a l . , 1949; Feeney et a l . , 1952; Feeney et a l . , 1955; Brooks and Ha le , 1961; B a l i g a et a l . , 1971). T h i s , i n a d d i t i o n to the g e l l i k e p roper ty of p u r i f i e d ovomucin, l e d many to b e l i e v e tha t the g l y c o p r o t e i n ovomucin p l ay s an important r o l e i n dete rmin ing the p h y s i c a l p r o p e r t i e s of t h i c k egg whi te and that t h i n n i n g i s caused by a chemica l change i n ovomucin. That the v i s c o s i t y of t h i c k wh i te or i s o l a t e d ovomucin g e l was r e a d i l y decreased upon a d d i t i o n of a sma l l amount of reduc ing agent such as 2-mercaptoethanol , d i t h i o t h r e i t o l , t h i o g l y c o l l i c a c i d e t c . (MacDonnell e t a l . , 1950, 1951; Robinson and Monsey, 1964; Dam, 1971; Kato e t a l . , 1971) suggested tha t t h i n n i n g was caused by a r e d u c t i o n 49 of d i s u l f i d e bonds ( d i s u l f i d e cleavage) of ovomucin and attempts were made to determine the presence and p o s s i b l e i d e n t i t y of a n a t u r a l reduc ing agent i n egg w h i t e . S ince attempts to f i n d s i g n i f i c a n t amounts of reduc ing agents f a i l e d (Ducay et a l . , 1960) and s i nce the t ime s c a l e f o r the n a t u r a l t h i n n i n g i s of a complete ly d i f f e r e n t order than the s i m u l a t i o n of n a t u r a l t h i n n i n g by reduc ing agents, i t was suggested and l a t e r shown that a l k a l i n e h y d r o l y s i s of d i s u l f i d e bonds caused a decrease i n v i s c o s i t y of egg wh i te and i s o l a t e d ovomucin g e l (Donovan et a l . , 1972). These experiments were c a r r i e d out a t pH 11.5 i n order to reduce the time necessary to observe a s i g n i f i c a n t amount of t h i n n i n g . As the r a t e of the t h i n n i n g r e a c t i o n was n e a r l y equal to the r a t e of a l k a l i n e h y d r o l y s i s of p r o t e i n d i s u l f i d e bonds (Donovan, 1967; Donovan and White, 1971) i t seemed p o s s i b l e tha t the t h i n n i n g of egg wh i te accompanying s torage of s h e l l eggs was due to a l k a l i n e h y d r o l y s i s of the d i s u l f i d e bonds of ovomucin. Determinat ion of the molecu la r weight of ovomucin was theree f o r e needed to s u b s t a n t i a t e these t h e o r i e s . In the presence of 6 M guan id ine -HC l and 0.2 M 2-mercaptoethanol an average molecu lar weight of 1.5 x 10~* was repo r ted (Donovan et a l . , 1970) w h i l e Robinson and Monsey (1971, 1975) repor ted mo lecu la r weights of 2.1 x 10^ and 7.2 x 10~* f o r ovomucin reduced w i t h 0.3 M 2-mercaptoethanol i n 6 M guan id ine -HC l . Tomimatsu and Donovan (1972) determined the weight average mo lecu la r weights of ovomucin under v a r i ou s c ond i t i o n s of pH and i o n i c s t r eng th and noted a l a r g e d i f f e r e n c e i n molecu la r weight of ovomucin as a f u n c t i o n of pH, be ing 210 - 270 x 10 f o r a pH 6.2 s o l u t i o n of ovomucin compared to 27 - 56 x 10 f o r a pH 7.9 50 s o l u t i o n . S ince the mo lecu la r weight a t n e u t r a l pH i n 6.5 M guan id ine -HC l was found to be 23 x 10 i t was concluded that ovomucin i s h i g h l y aggregated at pH 6.2 and on ly p a r t i a l l y aggregated at pH 7.9. Moreover, time-dependent changes i n the molecu la r weight of aggregated ovomucin a t pH 11.5 that cou ld be accounted f o r by a l k a l i n e h y d r o l y s i s of d i s u l f i d e bonds, y i e l d e d a low molecu la r weight ovomucin (2.2 x 10^). Thus two processes — d i s -aggregat ion and degradat ion of ovomucin occur at a l k a l i n e pH. S ince n a t u r a l t h i n n i n g occurs between pH 7.8 and 9.7 and s i n ce the a l k a l i n e degradat ion process i s ve ry slow at 9.7, these authors concluded tha t much of the t h i n n i n g accompanying s torage of s h e l l eggs might be produced by d i s agg rega t i on of g e l - l i k e aggregates of h i gh mo lecu la r weight ovomucin molecules r a t he r than by a l k a l i n e degradat ion of d i s u l f i d e bonds. To determine whether a chemica l change (degradat ion) or a p h y s i c a l change (d i saggregat ion) occurs dur ing n a t u r a l t h i n n i n g i t i s necessary to study the change i n mo lecu la r weight of unmodif ied or n a t i v e ovomucin accompanying the n a t u r a l t h i n n i n g of egg wh i t e . A requirement ,of the d i s u l f i d e c leavage mechanism be i t by reduc ing agents present i n the wh i te or by a l k a l i n e h y d r o l y s i s , i s tha t low molecu la r weight ovomucins be formed. That these were not detected du r i ng n a t u r a l t h i n n i n g i n the present study argues aga in s t t h i s mechanism. As l i t t l e change i n the mo lecu la r weight and chemica l compos i t ion of ovomucin from f r e s h versus n a t u r a l l y th inned wh i te cou ld be detected and s i nce ovomucin g e l i n f r e s h t h i c k wh i te cou ld be s o l u b i l i z e d by low speed b l end i ng , non-cova lent d i s agg rega t i on of h i gh 51 mo lecu la r weight ovomucin molecules might occur dur ing n a t u r a l t h i n n i n g . Th i s d i s agg rega t i on may be caused by a change i n conformat ion o f the ovomucin molecu le as the pH of the wh i te i nc reases (Jonathan et a l . , 1977) or may be due to charge cond i t i oned r e p u l s i v e f o r c e s ( B l u n d e l l jet a l . , 1972) and needs f u r t h e r i n v e s t i g a t i o n . Ovomucin has an asymmetric d i s t r i b u t i o n of carbohydrate s i de cha ins (Kato et_ al., 1973) and i t i s p o s s i b l e that ovomucin g e l may be p a r t i a l l y h e l d by hydrophobic i n t e r a c t i o n s as those carbohydrate m o i e t i e s may induce a s p e c i f i c f o l d i n g of the po l ypep t i de cha ins a l l o w i n g maximum exposore of t h e i r hydrophobic reg ions ( M i z r a h i et a l . , 1978) . The presence of hydrophobic b i nd i ng reg ions i n g l y c o -p r o t e i n s of the mucin type has been repor ted (Sachdev ejt a l . , 1979) . Th i s idea i s supported by the r e s u l t s of Adach i e± a l . . (1973) who repo r ted tha t the sed imentat ion c o e f f i c i e n t of ovomucin which was i s o l a t e d by g e l f i l t r a t i o n of egg wh i te s o l u b i l i z e d w i t h 0.5 - 1.0% sodium dodecy l s u l f a t e , remained unchanged before and a f t e r l i q u e f a c t i o n . F r a c t i o n a t i o n of a s o l u b l e ovomucin (without p r i o r b lend ing of egg w h i t e , i f p o s s i b l e ) and subsequent molecu la r weight de te rm ina t i on ( i f sed imentat ion 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 a t much lower running speed i s p o s s i b l e ) would be a f u r t h e r approach to the study of t h i s mechanism. 3. D i s s o c i a t i o n of an Ovomucin Complex S t a b i l i z e d by Lysozyme D e t a i l e d chemica l and p h y s i c a l s t ud i e s of ovomucin revea led that ovomucin has a subun i t s t r u c t u r e c o n s i s t i n g of carbohydrate r i c h (3-ovomucin) and carbohydrate poor (a-ovomucin) g l y c o p r o t e i n s (Kato et: a l . , 1970 a; Kato and Sato, 1971; Robinson and Monsey, 1971; 52 Hayakawa and Sato, 1976; Kato et a l . , 1977). Thus to e l u c i d a t e the mechanism of egg wh i te th inn ing , the chemica l or phys iochemica l changes which ovomucin undergoes dur ing t h i n n i n g were s t ud i ed . I t was found (Kato e t a l . , 1970 b; Kato et a l . , 1971; Kato and Sato, 1972; Kato et a l . , 1972; Robinson and Monsey, 1972 a) t ha t the carbohydrate contents of ovomucin g e l remarkably decreased du r i ng s torage and tha t the carbohydrate r i c h component a l s o d im in i shed . Accompanying the decrease i n c oncen t r a t i on of carbohydrate r i c h component from ovomucin g e l was a decrease i n lysozyme content . Thus a model of ovomucin g e l s t r u c t u r e and mechanism of t h i n n i n g were proposed (Kato e t a l . , 1971). Accord ing to t h i s model the swo l l en , r i g i d ge l a t i nou s s t r u c t u r e of t h i c k wh i te i s due to a s s o c i a t i o n of po lymer ized a-ovomucin subun i t s and po lymer ized g-ovomucin subun i t s supported by lysozyme and t h i n n i n g r e s u l t s from the d i s s o c i a t i o n of g-ovomucin and lysozyme. I t was assumed tha t a-ovomucin and/or g-ovomucin subun i t s were po lymer ized through d i s u l f i d e bonds because ovomucin g e l i s s o l u b i l i z e d by treatment w i t h 0.01 M 2-mercaptoethanol y i e l d i n g a - and g - subun i t s . Subsequently i t was shown that the i n t e r -a c t i o n between lysozyme and ovomucin was e l e c t r o s t a t i c i n v o l v i n g the n e g a t i v e l y charged t e r m i n a l s i a l i c a c i d re s i dues of ovomucin and the p o s i t i v e l y charged l y s y l E-amino groups of lysozyme, and tha t the i n t e r a c t i o n of lysozyme w i t h g-ovomucin was much s t ronger than that w i t h a-ovomucin (Kato e± a l . , 1975). As i t was l a t e r observed that the O - g l y c o s i d i c a l l y l i n k e d carbohydrate u n i t s of g-ovomucin (con-t a i n i n g the n e g a t i v e l y charged t e r m i n a l s i a l i c a c i d re s idues ) were l i b e r a t e d from s e r i n e or th reon ine re s idues by a l k a l i treatment (Kato ej: al., 1978 b, c) i t was suggested that the s p e c i f i c s o l u b i l i z a t i o n 53 of g-ovomucin which occurs du r i ng t h i nn i n g might be due to the n a t u r a l degradat ion of g - e l i m i n a t i o n of the O - g l y c o s i d i c a l l y l i n k e d carbohydrate u n i t s . g - e l i m i n a t i o n of O - g l y c o s i d i c a l l y l i n k e d carbohydrate u n i t s of egg wh i te p r o t e i n s was observed dur ing t h i n n i n g and seemed to be caused by g - e l i m i n a t i o n of the O - g l y c o s i d i c a l l y l i n k e d carbohydrate u n i t s of ovomucin because most of the O - g l y c o s i d i c l i n kage s i n egg wh i te are found i n ovomucin (Kato jat a l . , 1979), suppor t ing t h i s model. The data i n the present study lend support to t h i s mechanism i n t ha t a p r e f e r e n t i a l i n t e r a c t i o n between g-ovomucin and lysozyme was shown, as r equ i r ed by t h i s model, a l though the importance of the n e g a t i v e l y charged s i a l i c a c i d re s idues f o r the i n t e r a c t i o n i s d o u b t f u l . However, the carbohydrate s i de cha ins a l s o con ta i n a n e g a t i v e l y charged e s t e r s u l f a t e which was shown i n the present study to be important f o r the lysozyme-g-ovomucin i n t e r a c t i o n . I f g - e l i m i n a t i o n of O - g l y c o s i d i c l i n k a g e s does occur dur ing n a t u r a l t h i n n i n g , i t would decrease t h i s i n t e r a c t i o n . Whether or not the g - e l i m i n a t i o n r e a c t i o n i s s i g n i f i c a n t has yet to be proven. For example, a d i f f e r e n c e i n carbohydrate content (notab ly s i a l i c a c i d ) between ovomucin from f r e s h egg wh i te versus n a t u r a l l y th inned wh i t e s to red f o r 7 days cou ld not be detec ted i n the present study. Th i s may not be unreasonable as i t was repor ted tha t on l y 10% of the t o t a l g - e l i m i n a t i o n was shown to occur i n egg wh i te a f t e r 30 days of s torage (Kato ejt a l . , 1979) . That on ly 10% g - e l i m i n a t i o n was shown to occur may i n d i c a t e that t h i s r e a c t i o n i s not important i n terms of t h i n n i n g . I f t h i s i s so, a d i s s o c i a t i o n of 8-ovomucin cou ld s t i l l occur as the extent of the e l e c t r o s t a t i c i n t e r a c t i o n of lysozyme w i t h ovomucin would decrease w i t h the i n c r e a s i n g pH of egg wh i te dur ing s torage. Whether such changes i n the i n t e r a c t i o n of ovomucin w i t h lysozyme ( i n v iew of the f a c t that ovomucin comprises on ly a very sma l l p o r t i o n of t o t a l egg wh i te p r o t e i n ) can r e s u l t i n the d r a s t i c change i n the p h y s i c a l p r o p e r t i e s of t h i c k whi te observed dur ing t h i n n i n g remains i n doubt. The r o l e of lysozyme i n the p o s s i b l e s t a b i l i z a t i o n of a g e l formed by non-eova lent a s s o c i a t i o n of po lymer ized a-ovomucins and po lymer ized 8-ovomucins needs more a t t e n t i o n but may not be important . To t h i s end an i n t e r e s t i n g e xp l ana t i on f o r the p o s s i b l e d i s agg rega t i on of ovomucin du r i ng t h i n n i n g was r e c e n t l y proposed (Kato,.1980). Accord ing to t h i s mechanism the ovomucin of f r e s h t h i c k wh i te has a swo l len s t r u c t u r e because of i t s l a r g e water b i nd i n g c apac i t y (one gram of ovomucin b inds over 200 grams of wa te r ) , the water be ing he ld by the carbohydrate s i de cha in s . The l a r ge amount of water bound to ovomucin i s unusual (being about 1.5 g per g p r o t e i n f o r most p r o t e i n s ) and can account f o r i t s g e l l i k e p r o p e r t i e s a t low concen t r a t i on ( Fe r r y , 1948). D i sagg regat ion of ovomucin molecules cou ld thus be caused..under c o n d i t i o n s i n which the ordered s t ruc ture . : of bound water i s des t royed, such as b l end i ng , s o n i c a t i o n , or p a r t i a l degradat ion of the O - g l y c o s i d i c a l l y l i n k e d carbohydrate u n i t s of ovomucin. 55 CONCLUSION The i n t e r a c t i o n between lysozyme and ovomucin a t n e u t r a l pH i s e l e c t r o s t a t i c i n v o l v i n g p o s i t i v e l y charged l y s y l - am ino groups of lysozyme and the net nega t i ve charge of ovomucin, and a p r e f e r e n t i a l i n t e r a c t i o n between lysozyme and B-ovomucin was observed. A l a r g e d i f f e r e n c e i n the extent of i n t e r a c t i o n of lysozyme w i t h reduced and a l k y l a t e d ovomucin complex as compared to that w i t h n a t i v e ovomucin a t low i o n i c s t r eng th was observed, hence i t i s recommended tha t f u t u r e s t ud i e s on the p r o p e r t i e s of ovomucin be done us ing n a t i v e or unmodif ied ovomucin. The ex i s t ence of such a lysozyme-ovomucin i n t e r a c t i o n i n v i v o needs to be demonstrated and u n t i l then i t s importance i n c o n t r i b u t i n g to the ge l a t i nou s p roper ty of t h i c k wh i te remains d o u b t f u l . The ge l a t i n ou s s t r u c t u r e of t h i c k wh i te appears to be due to a s s o c i a t i o n of h i gh mo lecu la r weight ovomucin molecules he ld together by non-cova lent i n t e r a c t i o n s (hydrogen bonding, hydrophobic i n t e r a c t i o n s , e t c . ) . I t seems u n l i k e l y that chemica l degradat ion of ovomucin occurs du r i ng t h i nn i n g a l though a sma l l amount of a l k a l i n e h y d r o l y s i s of g l y c o s i d i c l i n kage s may occur . I n s tead , i t i s probable tha t d i s agg rega t i on of a s s oc i a ted h i gh molecu la r weight ovomucin molecules causes t h i n n i n g of t h i c k wh i t e . The cause of t h i s d i s a g g r e -ga t i on which accompanies the pH i nc rea se observed upon the storage of s h e l l eggs needs f u r t h e r i n v e s t i g a t i o n as does the •quaternary s t r u c t u r e of n a t i v e ovomucin. 56 REFERENCES Adach i , N., Azuma, J . , Janado, M. and Onodera, K. 1973. S o l u b i l i z a t i o n and c h a r a c t e r i z a t i o n of ovomucin w i thout chemica l m o d i f i c a t i o n . Agr. B i o l . Chem. 37: 2175. A l d e r t o n , A. and Fevo l d , M.C. 1946. D i r e c t c r y s t a l l i z a t i o n of lysozyme from egg wh i te and some c r y s t a l l i n e s a l t s of lysozyme. J . B i o l . Chem. 164: 1. B a l i g a , B.R., Kadko l , S.B. and L a h i r y , N.L. 1971. Th inn ing of t h i c k albumen i n s h e l l eggs-changes i n ovomucin. P o u l t r y S c i . 50: 466. Bever idge, T. and Naka i , S. 1975. E f f e c t s of s u l f h y d r y l b l o c k i n g on the t h i n n i n g of egg wh i t e . J . Food S c i . 40: 864. B l u n d e l l , T. , Dodson, G., Hodgkin, D. and Merco la , D. 1972. I n s u l i n : the s t r u c t u r e i n the c r y s t a l and i t s r e f l e c t i o n i n chemist ry and b i o l o g y . Adv. P r o t . Chem. 26: 279. B ran t , A.W., D i cke r son , G.E. and Feeney, R.E. 1955. Fac to r s a f f e c t i n g albumen. P o u l t r y S c i . 34: 1181. Brooks, J . and Ha l e , H.P. 1959. The mechanica l p r o p e r t i e s of the t h i c k wh i te of the hen ' s egg. B ioch im. B iophys. A c t a . 32: 237. Brooks, J . and Ha l e , H.P. 1961. The mechanica l p r o p e r t i e s of the t h i c k wh i te of the hen ' s egg. 2. The r e l a t i o n between r i g i d i t y and compos i t ion . B ioch im. B iophys. A c t a . 46: 289. C a n f i e l d , R.E. 1963 :a. Pept ides de r i ved from t r y p t i c d i g e s t i o n of egg wh i te lysozyme. J . B i o l . Chem. 238: 2691. C a n f i e l d , R.E. 1963 b. The amino a c i d sequence of egg wh i te lysozyme. J . B i o l . Chem. 238: 2698. Casassa, E.F. and E i senberg , H. 1964. Thermodynamic a n a l y s i s of multicomponent s o l u t i o n s . Advan. P r o t . Chem. 19: 287. C o t t e r i l l , O.J . and Win te r , A.R. 1955. Egg wh i te lysozyme. 3. The e f f e c t of pH on the lysozyme-ovomucin i n t e r a c t i o n . P o u l t r y S c i . 34: 679. Dam, R. 1971. In v i t r o s t ud i e s on the lysozyme-ovomucin complex. P o u l t r y S c i . 50: 1824. Dam, R. and Bennett , N.S. 1963. S tud ies on the lysozyme-ovomucin complexes of ch i cken and duck egg wh i t e . P o u l t r y S c i . 42: 1263. Deon ier , R.C. and W i l l i a m s , J.W. 1970. S e l f - a s s o c i a t i o n of muramidase o (lysozyme) i n s o l u t i o n at 25 C, pH 7.0 and 1/2=0.20. B iochemi s t ry 9: 4260. 57 D ixon, M. and Webb, E.C. 1961. Enzyme f r a c t i o n a t i o n by s a l t i n g out : a t h e o r e t i c a l no te . Adv. P r o t . Chern. 16: 197. Donovan, J.W. 1967. Spectrophotometr ic ob se r va t i on of the a l k a l i n e h y d r o l y s i s of p r o t e i n d i s u l f i d e bonds. Biochem. B iophys. Res. Commun. 29: 734. Donovan, J.W. and White, L.M. 1971. A l k a l i n e h y d r o l y s i s of the d i s u l f i d e bonds of ovomucoid and of low molecu la r weight a l i p h a t i c and aromat ic d i s u l f i d e s . B iochemi s t r y 10: 32. Donovan, J.W., Dav i s , J . G . and White, L.M. 1970. Chemical and p h y s i c a l c h a r a c t e r i z a t i o n of ovomucin, a s u l f a t e d g l y c o p r o t e i n complex from ch i cken eggs. B ioch im. B iophys. Ac ta 207: 190. Donovan, J.W., Dav i s , J .G . and W i e l e , M.B. 1972. V i s c o s i m e t r i c s t ud i e s of a l k a l i n e degradat ion of ovomucin. J . Agr. Food Chern. 20: 223. Douzou, P. and Ba lny , C. 1978. P r o t e i n f r a c t i o n a t i o n a t subzero temperatures. Adv. P r o t . Chem. 32: 77. Ducay, E.D., K l i n e , L. and Mandeles, S. 1960. Free amino a c i d content of i n f e r t i l e ch i cken eggs. P o u l t r y S c i . 39: 831. Eh renp re i s , S. and Warner, R.C. 1956. The i n t e r a c t i o n of conalbumin and lysozyme. A rch . Biochem. B iophys. 61: 38. Feeney, R.E. 1955. Fac to r s a f f e c t i n g albumen. P o u l t r y S c i . 34: 1181. Feeney, R.E., Ducay, E.D., S i l v a , R.B. and MacDonnel l , L.R. 1952. Chemistry of s h e l l egg d e t e r i o r a t i o n : the egg wh i te p r o t e i n s . P o u l t r y S c i . 31: 639. Feeney, R.E., Jones, J.R. and Weaver, J.M. 1955. I n f l uence of temperature on the d e t e r i o r a t i v e t h i n n i n g of egg wh i t e , ovomucin, and y o l k membrane. P o u l t r y S c i . 34: 1193. Feeney, R.E., Rhodes, M.B. and Anderson, J . S . 1960. The d i s t r i b u t i o n and r o l e of s i a l i c a c i d i n ch i cken egg wh i t e . J . B i o l . Chem. 235: 2633. Feeney, R.E., S i l v a , R.B. and MacDonnel l , L.R. 1951. Chemistry of s h e l l egg d e t e r i o r a t i o n : the d e t e r i o r a t i o n of separated components. P o u l t r y S c i . 30: 645. Feeney, R.E., Weaver, J .M . , Jones, J.R. and Rhodes, M.B. 1956. S tud ies of the k i n e t i c s and mechanisms of y o l k d e t e r i o r a t i o n i n s h e l l eggs. P o u l t r y S c i . 31: 1061. F e r r y , J.D. 1948. P r o t e i n g e l s . Adv. P r o t . Chem. 4: 1. Fo r s y the , R.H. and F o s t e r , J . F . 1949. Note on the e l e c t r o p h o r e t i c compos i t ion of egg wh i t e . A rch . Biochem. 20: 161. 58 G a r i b a l d i , J . A . , Donovan, J.W., Dav i s , J .G . and Cimino, S.L. 1968. Heat dena tu ra t i on of the ovomucin-lysozyme e l e c t r o s t a t i c complex-a source of damage to the whipping p r o p e r t i e s of pa s t eu r i z ed egg wh i t e . J . Food S c i . 33: 514. Go t t s cha l k , A. and L i n d , P.E. 1949 a. Ovomucin, a s ub s t r a te f o r the enzyme of i n f l u e n z a v i r u s . 1. Ovomucin as an i n h i b i t o r of haemagg lut inat ion by heated Lee v i r u s . B r i t . J . Exp. Pa th . 30: 85. Go t t s cha l k , A. and L i n d , P.E. 1949 b. Product of i n t e r a c t i o n between i n f l u e n z a v i r u s and ovomucin. Nature 164: 232. Go t t s cha l k , A. and Thomas, M. 1961. S tud ies on mucoproteins. 5. The s i g n i f i c a n c e of n -ace ty l neu ramin i c a c i d f o r the v i s c o s i t y of ov ine s ubmax i l l a r y g land mucoprote in. B ioch im. B iophys. A c t a . 46: 91. Hawthorne, J.R. 1950. The a c t i o n of egg wh i te lysozyme on ovomucoid and ovomucin. B ioch im. Biophys. A c t a . 6: 28. Hayakawa, S. and Sato, Y. 1976. S tud ies on the d i s s o c i a t i o n of the s o l u b l e ovomucin by s o n i c a t i o n . Agr. B i o l . Chem. 40: 2397. Haynes, R., Osuga, D.T. and Feeney, R.E. 1967. M o d i f i c a t i o n of amino groups i n i n h i b i t o r s of p r o t e o l y t i c enzymes. B iochemi s t r y 6: 541. H i l l , E.G., Bu r ton , G.R. and Charkey, L.W. 1949. The n u t r i e n t content of h i gh and low q u a l i t y f r e s h eggs. 3. Mucin i n r e l a t i o n to t ryptophan. P o u l t r y S c i . 28: 862. H i l l , H.D., Reynolds, J .A . and H i l l , R.L. 1977. P u r i f i c a t i o n , composition,...molecular weight and subunit s t r u c t u r e of ov ine submax i l l a r y mucin. J . B i o l . Chem. 252: 3791. Howlet t , C . J . and N i c h o l , L.W. 1973. A sed imentat ion e q u i l i b r i u m study of the i n t e r a c t i o n between ovalbumin and lysozyme. J . B i o l . Chem. 248: 619. J e f f r e y , P.D. and Coates, J . H . 1966. An 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 e study of the s e l f - a s s o c i a t i o n of bov ine i n s u l i n . B iochemi s t r y 5: 489. Jonathan, P., B u t l e r , G. and Durham, A.C. 1977. Tobacco mosaic v i r u s p r o t e i n aggregat ion and the v i r u s assembly. Adv. P r o t . Chem. 31: 187. Kato , A. 1980. Pe r sona l communication. Kato , A. and Sato, Y. 1971. The sepa ra t i on and c h a r a c t e r i z a t i o n of carbohydrate r i c h component from ovomucin i n ch icken eggs. Agr. B i o l . Chem. 35: 439. Ka to , A. and Sato, Y. 1972. The r e l e a s e of carbohydrate r i c h component from ovomucin g e l du r i ng s torage. Agr. B i o l . Chem. 36: 831. 59 Kato , A. , F i j i n a g a , K. and Yaga sh i t a , K. 1973. Nature of the carbohydrate s i de cha ins and t h e i r l i n kage to the p r o t e i n i n ch i cken egg wh i te ovomucin. Agr. B i o l . Chem. 37: 2479. Ka to , A . , Hayash i , H. and Yaga sh i t a , K. 1974. The i n h i b i t o r y p r o p e r t i e s of ch i cken egg wh i te ovomucin aga in s t aggregat ion of K - c a s e i n by r enn i n . Agr. B i o l . Chem. 38: 1137. Kato, A . , H i r a t a , S. and Kobayash i , K. 1978 c. S t r u c tu re of the s u l f a t e d o l i g o s a c c h a r i d e cha in of ovomucin. Agr. B i o l . Chem. 42: 1025. Kato , A. , H i r a t a , S., Sato, H. and Kobayash i , K. 1978 b. F r a c t i o n a t i o n and c h a r a c t e r i z a t i o n of the s u l f a t e d o l i g o s a c c h a r i d e cha ins of ovomucin. Agr. B i o l . Chem. 42: 835. Ka to , A. , Imoto, T. and Yaga sh i t a , K. 1975. The b i nd i ng groups i n ovomucin-lysozyme i n t e r a c t i o n . Agr. B i o l . Chem. 39: 541. Kato , A . , Nakamura, R. and Sato, Y. 1970 a. S tud ies on changes i n s to red s h e l l eggs. 5. The d i f f e r e n c e i n the chemica l and p h y s i c o -chemica l p r o p e r t i e s of ovomucin (B) between the t h i c k and t h i n wh i t e . Agr. B i o l ; . Chem. 34: 854. Kato , A., Nakamura, R. and Sato, Y. 1970 b. S tud ies on changes i n s to red s h e l l eggs. 6. Changes i n the chemica l compos i t ion of ovomucin du r i ng s torage. Agr. B i o l . Chem. 34: 1009. Kato , A. , Nakamura, R. and Sato, Y. 1971. S tud ies on changes i n s to red s h e l l eggs. 7. Changes i n the phys i cochemica l p r o p e r t i e s of ovomucin s o l u b i l i z e d by treatment w i t h mercaptoethanol dur ing s torage. Agr. B i o l . Chem. 35: 351. Kato, A. , Nakamura, R. and Sato, Y. 1972. E f f e c t s of the s torage i n an atmosphere of carbon d i o x i d e on ovomucin. Agr. B i o l . Chem.. 36: 947. Kato, A. , Ogino, K., Kuramoto, Y. and Kobayash i , K. 1979. Degradat ion of the 0 - g l y c o s i d i c a l l y l i n k e d carbohydrate u n i t s of ovomucin du r i ng egg wh i te t h i n n i n g . J . Food S c i . 44: 1341. Kato, A . , Ogino, K., Matsudomi, N. and Kobayash i , K. 1977. Separa t ion of ovomucin i n t o c a r b o h y d r a t e - r i c h and poor components by chromatography on lysozyme-Sepharose 4B. Agr. B i o l . Chem. 41: 1925. Ka to , A. , Wakinaga, T., Matsudomi, N. and Kobayash i , K. 1978 a. Changes i n lysozyme du r i ng egg wh i te t h i n n i n g . Agr. B i o l . Chem. 42: 175. Ka to , A. , Yo sh ida , K., Matsudomi, N. and Kobayash i , K.. 1976. The i n t e r a c t i o n between ovomucin and egg wh i te p r o t e i n s . Agr. B i o l . Chem. 40: 2361. Kauzmann, W. 1959. Some f a c t o r s i n the i n t e r p r e t a t i o n of p r o t e i n dena tu r a t i on . Adv. P r o t . Chem. 14: 1. K l o t z , I.M. and Walker, F.M. 1948. Complexes of lysozyme. A r ch . Biochem. 18: 319. L a n n i , F. and Beard, J.W. 1948. I n h i b i t i o n by egg-white of hem-a g g l u t i n a t i o n by swine i n f l u e n z a v i r u s . P roc . Soc. Exp. B i o l . Med. 68: 312. L a n n i , F., Sharp, D.G., E c ke r t , E., D i l l o n , E., Beard, 0 . , and Beard, J.W. 1949. The egg wh i te i n h i b i t o r of i n f l u e n z a v i r u s hemagg lu t ina t ion . 1. P r e p a r a t i o n and p r o p e r t i e s of s e m i - p u r i f i e d i n h i b i t o r . J . B i o l . Chem. 179: 1275. L i s t , S . J . , F i n d l a y , B.P., F o r s t n e r , G.G. and Fo r s t ne r , J . F . 1978. Enhancement of the v i s c o s i t y of mucin by serum albumin. Biochem. J . 175: 565. Lowry, O.H., Rosebrough, N . J . , F a r r , A.L. and R a n d a l l , R . J . 1951. P r o t e i n measurement w i t h the F o l i n phenol reagent. J . B i o l . Chem. 193: 265. MacDonnel l , L.R., L ineweaver, H. and Feeney, R.E. 1950. D e t e r i o r a t i o n of eggs caused by reduc ing s o l u t i o n s and gases. P o u l t r y S c i . 29: 769. MacDonnel l , L.R., L ineweaver, H. and Feeney, R.E. 1951. Chemistry of s h e l l egg d e t e r i o r a t i o n : e f f e c t of reduc ing agents. P o u l t r y S c i . 30: 856. McNa l l y , E. 1933. R e l a t i v e amount of ovomucin i n t h i c k and t h i n egg wh i t e . P roc . Soc. Expt. B i o l . Med. 30: 1254. M i z r a h i , A . , O 'Ma l l e y , J . , C a r t e r , W., Taka t s uk i , A . , Tamura, G. and Su lkowsk i , E. 1978. G l y c o s y l a t i o n of i n t e r f e r o n s . E f f e c t s of tun icamyc in on human immune i n t e r f e r o n . J . B i o l . Chem. 253: 7612. Moore, S. and S t e i n , W.H. 1954. Procedures for. the chromatographic de te rm ina t i on of amino a c i d s on. f ou r percent c r o s s - l i n k e d su l f ona ted po l y s t y r ene r e s i n s . J . B i o l . Chem. 211: 893. Morawieck i , A. 1964. D i s s o c i a t i o n of M- and N- group mucoproteins i n t o subun i t s i n detergent s o l u t i o n s . B ioch im. B iophys. A c t a . 83: 339. Naka i , S. and Van de Voo r t , F.R. 1979. A p p l i c a t i o n of m u l t i p l e r e g r e s s i o n a n a l y s i s to sed imentat ion e q u i l i b r i u m data of as^-and K - c a s e i n i n t e r a c t i o n s f o r c a l c u l a t i o n of molecu la r weight d i s t r i b u t i o n s . J . Da i r y Res. 46: 283. 61 Naka i , S. and Kason, C M . 1974. A f l uo re s cence study of the i n t e r -a c t i o n s between K - and as - c a s e i n and between lysozyme and ovalbumin. B ioch im. B iophys. A c t a . 351: 21. Nakamura, R., Ka to , A. and Sato, Y. 1969. S tud ies on ovomucin. E f f e c t of sodium t h i o g l y c o l i c a c i d and hydrogen perox ide treatment on ovomucin (B). J . Agr. Chem. Soc. Japan. 43: 105. Neuhaus, 0. and L e t z r i n g , M. 1957. Determinat ion of hexosamines i n con junc t i on w i t h e l e c t r o p h o r e s i s on s t a r c h . A n a l . Chem. 29: 1230. Osuga, D.T. and Feeney, R.E. 1968. B iochemi s t ry of the egg wh i te p r o t e i n s of the R a t i t e group. A rch . Biochem. B iophys. 124: 560. Osuga, D.T. and Feeney, R.E. 1977. Egg p r o t e i n s . In "Food P r o t e i n s " . Whi taker , J.R. and Tannenbaum, S.R. ed. A v i P u b l i s h i n g Co. Inc. Westport, CT. Powr ie , W.D. 1980. Pe r sona l communication. Rhodes, M.B, and Feeney, R.E. 1957. Mechanisms of s h e l l egg d e t e r i o r a t i o n : comparisons of ch i cken and duck eggs. P o u l t r y S c i . 36: 891. Robinson, D.S. 1972. Egg wh i te g l y cop ro t e i n s and the p h y s i c a l p r o p e r t i e s of egg wh i t e . In "Egg Formation and P r o d u c t i o n " . B.M. Freeman and P.E. Lake, ed. B r i t i s h P o u l t r y Sc ience L t d . , Edinburgh. Robinson, D.S. and Monsey, J . B . 1964. Reduct ion of ovomucin by mercaptoethanol . B ioch im. B iophys. A c t a . 83: 368. Robinson, D.S. and Monsey, J . B . 1966. Composit ion of ovomucin. Biochem. J . 100: 61p. Robinson, D.S..and Monsey, J . B . 1969 a. A reduced ovomucin-reduced lysozyme complex from egg wh i t e . Biochem. J . 115: 64p. Robinson, D.S. and Monsey, J . B . 1969 b. A q u a n t i t a t i v e study of a reduced ovomuc in-natura l lysozyme i n t e r a c t i o n . Biochem. J . 115: 65p. Robinson, D.S. and Monsey, J . B . 1971. S tud ies on the compos i t ion of egg wh i te ovomucin. Biochem. J . 121: 537. Robinson,. D.S. and Monsey, J . B . 1972 a. Changes i n the compos i t ion of ovomucin du r i n g l i q u e f a c t i o n of t h i c k egg wh i t e . J . S c i . Fd. A g r i c . 23: 29. Robinson, D.S. and Monsey, J . B . 1972 b. Changes i n the compos i t ion of ovomucin du r i ng l i q u e f a c t i o n of t h i c k egg wh i t e : The e f f e c t of i o n i c s t r eng th and magnesium s a l t s . J . S c i . Fd. A g r i c . 23: 893. 62 Robinson, D.S. and Monsey, J . B . 1975. The compos i t ion and proposed subuni t s t r u c t u r e of egg wh i te 3-ovomucin. Biochem. J . 147: 55. Robson, T. , A l l e n , A. and P a i n , R.H. 1975. Non-covalent f o r ce s tha t ho ld g l y c o p r o t e i n molecules together i n mucous g e l . Biochem. Soc. Trans. 3: 1105. Sachdev, G.P., Zodrow, J.M. and C a r u b e l l i , R. 1979. Hydrophobic i n t e r a c t i o n of f l u o r e s c e n t probes w i t h f e t u i n , ov ine sub-m a x i l l a r y mucin, and canine t r a c h e a l mucins. B ioch im. B iophys. A c t a . 580: 85. Sato, Y. and Hayakawa, S. 1977. Fu r the r i n s p e c t i o n f o r the s t r u c t u r e of t h i c k egg wh i t e . Nippon Nogeikagaku K a i s h i . 51: 47. Sato, Y. , Hayakawa, S. and Nakamura, R. 1976. S tud ies on f a c t o r s of s o l u b i l i z a t i o n of i n s o l u b l e ovomucin dur ing t h i c k wh i te t h i n n i n g . J . A g r i c . Food Chem. 24: 798. Schachman, H.K. 1959. U l t r a c e n t r i f u g a t i o n i n B i ochemi s t r y . Academic P re s s , New York, N.Y. Sharp, D.G., L ann i , F. and Beard, J.W. 1950. The egg wh i te i n h i b i t o r of i n f l u e n z a v i r u s hemagg lu t ina t ion . 2. E l e c t r o n microscopy of the i n h i b i t o r . J . B i o l . Chem. 185: 681. Sharp, D.G., L ann i , F., L ann i , Y .T . , Csaky, T.Z. and Beard, J.W. 1951. The egg wh i te i n h i b i t o r of i n f l u e n z a v i r u s hemagg lu t ina t ion . 5. E l e c t r o p h o r e t i c s t u d i e s . A r ch . Biochem. 30: 251. S l e i g h , R.W., Me l rose , G . J . and Smith, M.B. 1973. I s o l a t i o n and c h a r a c t e r i z a t i o n of hen egg wh i te ovomucin. B ioch im. B iophys. A c t a . 310: 453. Smith, M.B., Reynolds, T.M., Buckingham, C P . and Back, J . F . 1974. S tud ies on the carbohydrate of egg wh i te ovomucin. Aust . J . B i o l . S c i . 27:. 349. S t e i n e r , R.F. 1953. R e v e r s i b l e a s s o c i a t i o n processes of g l obu l a r p r o t e i n s . I I . E l e c t r o s t a t i c complexes of plasma albumin and lysozyme. A rch . Biochem. B iophys. 47: 56. Tomimatsu, Y. and Donovan, J.W. 1972. L i g h t s c a t t e r i n g of ovomucin. J . Agr. Food Chem. 20: 1068. Van de Voo r t , F. and Naka i , S. 1978. An automated system f o r c a l c u l a t i o n of mo lecu la r weight u s ing a p r e p a r a t i v e u l t r a c e n t r i f u g e w i t h a U.V. Scanner. Can. I n s t . Food S c i . Tech. J . 11: 162. Van de Voo r t , F., Ma, C.Y. and Naka i , S. 1979. Mo lecu la r weight d i s t r i b u t i o n of i n t e r a c t i n g p r o t e i n s c a l c u l a t e d by m u l t i p l e r e -g re s s i on a n a l y s i s from sed imentat ion e q u i l i b r i u m data : An i n t e r -p r e t a t i o n of as - K - c a s e i n i n t e r a c t i o n . A rch . Biochem. B iophys. 195: 596. 63 Van Holde, K.E. and Ba ldwin , R.L. 1958. Rapid atta inment of sed imentat ion e q u i l i b r i u m . J . Phys. Chem. 62: 734. Warren, L. 1959. The t h i o b a r b i t u r i c a c i d assay of s i a l i c a c i d s . J . B i o l . Chem.. 234: 1971. Waugh, D.F. 1954. P r o t e i n - p r o t e i n i n t e r a c t i o n s . Adv. P r o t . Chem. 9: 325. W i l c ox , F.H. 1955. Ev idence f o r an a s s o c i a t i o n between the lysozyme l e v e l and the q u a l i t y of egg wh i t e . P o u l t r y S c i . 34: 1170. W i n z l e r , R .J . 1955. In "Methods of B i ochemica l A n a l y s i s " , v o l . 2. D. G l i c k , ed. I n t e r s c i e n c e P u b l i s h e r s , New York. Yamasaki, N., Hayash i , K. and Funatsu, M. 1968. A c e t y l a t i o n of lysozyme. 1. P r e p a r a t i o n , f r a c t i o n a t i o n , and p r o p e r t i e s of a c e t y l a t e d lysozyme. Agr. B i o l . Chem. 32: 55. Young, G.E. 1937. On the s epa ra t i on and c h a r a c t e r i z a t i o n of the p r o t e i n s of egg wh i t e . J . B i o l . Chem. 120: 1. Young, L.L. and Gardner, F.A. 1972. P r e p a r a t i o n of egg wh i te ovomucin by g e l f i l t r a t i o n . J . Food S c i . 37: 8. TABLE 1 Amino Acid Composition of Whole Ovomucin. Values expressed as g amino acid per 100 g of protein. Amino Acids (1) (2) (3) (4) (5) (6) (7) (8) (9) Aspartic Acid 9. 77 9. 31 12. ,29 9. ,18 Threonine 7. 02 8. ,50 7. ,68 7. ,51 Serine 7. .11 7. ,76 5. ,97 7. .11 Glutamic Acid 11. 94 11. .98 11. .94 11. ,49 Proline 5. .62 5. ,82 4. ,44 5. .51 Glycine 3. ,85 3. .67 4. .27 2. ,96 Alanine 4. ,32 4. .00 3. .58 2. ,97 Valine 5. .88 5. .92 5. ,63 5. ,38 Methionine 2. ,49 0. .78 2. .39 2. ,36 Isoleucine 4. ,78 4. .71 4. .95 4. .33 Leucine 7. ,28 7. .45 7. .34 6. ,63 Tyrosine 4. .30 4. ,12 5. .46 4. .19 Phenylalanine 5. .17 4, .86 5. .63 5. .07 Lysine 6. .18 6, .38 7, .34 7. .13 Histidine 2. .70 2, .43 2, .73 2. .81 Tryptophan ND* ND* ND* 1, .36 Arginine 4. .21 4, .54 5. .12 4. .35 Cysteine 4. .04 7 .75 3 .24 6, .56 10.11 9. 76 11.66 10.98 12.39 6.53 5. 76 5.93 6.48 7.40 6.54 6. .79 7.22 6.36 7.00 12.83 12. .76 10.81 11.87 13.63 4.89 • 6. .79 1.99 4.15 5.65 3.58 3. ,07 4.40 3.71 3.59 3.78 4. ,11 3.96 4.62 5.66 5.93 6. .68 6.89 5.03 5.38 2.27 3. .07 1.95 1.98 2.31 4.76 5. ,22 5.78 5.84 4.50 7.15 7. ,83 6.55 8.46 6.49 6.12 4, .64 5.00 5.09 4.95 5.28 5. .83 6.74 5.37 5.82 6.94 6, .20 7.66 7.59 7.88 2.56 2, .40 2.40 2.20 2.79 ND* 2, .72 ND* ND* ND* 4.94 4, .76 4.84 4.76 4.55 5.80 3, .93 6.37 5.51 ND* *Not determined. (1) This study. (4) Robinson and Monsey, 1971. (7) Adachi ejc a l . , 1973. (2) Young and Gardner, 1972. (5) Osuga and Feeney, 1968. (8) Sato and Hayakawa, 1977. (3) Kato et a l . , 1973. (6) Donovan et a l . , 1970. (9) Smith et a l . , 1974. TABLE 2 Amino Acid Composition of Ovomucins from Native and Stored Egg White Amino Acid Ovomucin from fresh Ovomucin from thick thick egg white egg white held at 30 ± 2 C for 166 h Aspartic acid 9.77 9.96 Threonine 7.02 7.14 Serine 7.11 7.67 Glutamic acid 11.94 11.72 P r o l i n e 5.62 5.51 Glycine 3.85 3.74 Alanine 4.32 4.19 Valine 5.88 5.95 Methionine 2.49 2.26 Isoleucine 4.78 4.60 Leucine 7.28 7.42 Tyrosine 4.30 4.45 Phenylalanine 5.17 5.38 Lysine 6.18 6.06 H i s t i d i n e 2.70 2.74 Tryptophan ND* ND* Arginine 4.21 4.13 Cysteine 4.04 4.22 ^Values for amino acids are averages of duplicate analyses on 22 h acid hydrolysates. *Not determined. 66 TABLE 3 Carbohydrate Composition of Ovomucins vfrom Fresh arid Stored Egg White, g per 100 g dry weight. Component fresh stored S i a l i c acid Hexose Hexosamine , 4.65 9.77 10.14 4.63 9.74 10.23 i, 67 TABLE 4 M of Native Ovomucin as a Function of Protein Concentration w app )_ >  , - * < -6 Protein concentration, mg/ml M X 10 < w app O-^O 5 . 1 5 3 °-169 ' , 4.865 0-!99 • • 4.611 0.226 3.987 0.336 , 4.334 0.369 ' 3.944 °-453 ; 3.014 0.671 ^ 2.426 ^Calculated from molar extinction coefficient, E 1 % = 7 0 280 nm, 1cm TABLE 5 M of Stored Ovomucin as a Function of Protein Concentration w app > , * -6 Protein concentration, mg/ml M X 10 w app 0.144 4.433 0.149 '' 4.579 0.179 4.758 0.364 3.234 0.387 3.046 0.446 2.852 0.457 3.045 0.521 2.830 0.. 600 2.574 17 *Calculated from molar extinction coefficients E ° „ , =7.0 280 nm, 1cm ) TABLE 6 Molecular Weight of Soluble Ovomucins' S o l u b i l i z a t i o n method sMolecular weight (million) Determination Reference 0.1 S NaOH 0.3 M 2-mercaptoethanol Sonication Blending in mild a l k a l i n e buffer ^ pH 8.6 Blending in mild a l k a l i n e buffer, pH 8.6 0.06 M phosphate^ buffer, pH 7.2 ) Blending i n mild alkal i n e buffer, pH 9.0 -6.5 M guanidine-HCl 1 M KC1, pH 7.9, 1 M KC1, pH 6.2 0.22 0.21 i 0.72 1.1 3.0 i 5.5-7.5 7.6 3.3 11.5 40.0 240.0 r i Light scattering (1) Sedimentation (2) equilibrium Light scattering (3) Molecular exclusion (4) on Sepharose 4B Sedimentation (5) equilibrium Sedimentation v e l o c i t y (6) Light scattering (3) Sedimentation v e l o c i t y (7) Light scattering (1) Light scattering • (1) (1) Tomimatsu and Donovan, 1972. (2) Robinson and Monsey, 1971. (3) Hayakawa 'and Sato, 1976. (4) Young and Gardner, 1972. (5) This study. (6) Lanni et a l . , 1949. (7) Robinson and Monsey, 1975. 70 ' FIGURE 1 Mo lecu la r weight d i s t r i b u t i o n of an RA ovomucin complex-lysozyme mix tu re at i o n i c s t rengths 0.13 and 0.07. (A) Lysozyme at i o n i c s t r eng th 0.13. I n i t i a l absorbance, 0.298; r o t o r speed, 19,329 rpm. (B) RA ovomucin complex-lysozyme (1:4) at i o n i c s t r eng th 0.13. I n i t i a l absorbance, 0.298; r o t o r speed, 19,329 rpm. (C) RA ovomucin complex-lysozyme mixture (1:4) at i o n i c s t r eng th 0.07. I n i t i a l absorbance, 0.300; r o t o r speed, 19,944 rpm. For a l l f i g u r e s , t r ue va l ue f o r A^ can be obta ined by d i v i d i n g by 4. MOLECULAR WEIGHT 72 FIGURE 2 Mo lecu la r weight d i s t r i b u t i o n of an RA ovomucin complex-lysozyme mix ture at i o n i c s t r eng th 0.13. (A) Lysozyme. I n i t i a l absorbance, 0.300; r o t o r speed, 19,939 rpm. (B) RA ovomucin complex. I n i t i a l absorbance, 0.300; r o t o r speed, 9,866. (C) RA ovomucin complex-lysozyme mix tu re (1 :4 ) . I n i t i a l absorbance 0.298; r o t o r speed, 9,866 rpm. 73 MOLECULAR WEIGHT 74 FIGURE 3 Mo lecu l a r weight d i s t r i b u t i o n of an RA ovomucin complex-lysozyme mix tu re at i o n i c s t r eng th 0.07. (A) Lysozyme. I n i t i a l absorbance, 0.300; r o t o r speed, 19,944 rpm. (B) RA ovomucin complex. I n i t i a l absorbance, 0.303; r o t o r speed, 9,974 rpm. (C) RA ovomucin complex-lysozyme m ix tu re (1:4). I n i t i a l absorbance, 0.305; r o t o r speed, 9,974 rpm. MOLECULAR WEIGHT 76 FIGURE 4 Molecular weight distribution of a native ovomucin-lysozyme mixture at ionic strengths 0.13 and 0.07. (A) Lysozyme at ionic strength 0.13. Initial absorbance, 0.305. (B) Native ovomucin-lysozyme mixture (1:4) at ionic strength 0.13. Initial absorbance, 0.300. (C) Native ovomucin-lysozyme mixture (1:4) at ionic strength 0.07. Initial absorbance, 0.298. Rotor speed, 31,908 rpm. 78 FIGURE 5 Mo lecu la r weight d i s t r i b u t i o n s of ovomucin-acety lated lysozyme mixtures at i o n i c s t r eng th 0.07. (A) A c e t y l a t e d lysozyme. I n i t i a l absorbance, 0.298; r o t o r speed, 33,570 rpm. (B) A c e t y l a t e d l y sozyme-nat i ve ovocumin (4:1). I n i t i a l absorbance, 0.300; r o t o r speed, 33,570 rpm. (C) A c e t y l a t e d lysozyme. I n i t i a l absorbance, 0.302; r o t o r speed, 34,224 rpm. (D) A c e t y l a t e d lysozyme-RA ovomucin complex (4:1). I n i t i a l absorbance, 0.300; r o t o r speed, 34,224 rpm. 7 9 80 FIGURE 6 Mo lecu la r weight d i s t r i b u t i o n of an a s i a l o , RA ovomucin complex-lysozyme mix tu re a t i o n i c s t r eng th 0.07. (A) Lysozyme. I n i t i a l absorbance, 0.297; r o t o r speed, 35,235 rpm. (B) RA ovomucin complex-lysozyme m ix tu re (1 :4 ) . I n i t i a l absorbance, 0.298; r o t o r speed, 9,263 rpm. (C) A s i a l o , RA ovomucin complex-lysozyme mix tu re (1 :4 ) . I n i t i a l absorbance, 0.300; r o t o r speed, 9,263 rpm. 81 82 FIGURE 7 Mo lecu la r weight d i s t r i b u t i o n of an a s i a l o , n a t i v e ovomucin-lysozyme m ix tu re a t i o n i c s t r eng th 0.07. (A) Lysozyme. I n i t i a l absorbance, 0.300. (B) Na t i v e ovomucin-lysozyme mix ture (1 :4 ) . I n i t i a l absorbance, 0.300. (C) A s i a l o , n a t i v e ovomucin-lysozyme mix tu re (1 :4 ) . I n i t i a l absorbance, 0.300. Rotor speed, 35,798 rpm. 83 84 FIGURE 8 Mo lecu la r weight d i s t r i b u t i o n s of RA ovomucin complex-lysozyme mixtures (1:4) (-0-) and lysozyme (-•-) at 20° and 3°C. I n i t i a l absorbances. of a l l s o l u t i o n s , 0.250. (A) , (B) . Temperature, 20 ± 5°C. Rotor speed, .33,696 rpm. (C) , (D). Temperature, 3 ± 5°C. Rotor speed, 34,416 rpm. I on i c s t rengths of s o l u t i o n s : 0.13 (A) , (C) ; 0.07 (B) , (D). MOLECULAR WEIGHT 86 FIGURE 9 Ge l f i l t r a t i o n of egg wh i te on Sepharose 4B. 10 ml of egg wh i te (prepared as desc r ibed i n " M a t e r i a l s and Methods") was app l i ed to a column (2.5 X 40 cm) of Sepharose 4B e q u i l i b r a t e d a 20°C w i t h 0.04 M T r i s - H C l b u f f e r , pH 8.6, c on ta i n i n g 0.85% NaC l , and e l u t ed w i t h the same b u f f e r . The e f f l u a n t was c o l l e c t e d i n 3.5 ml f r a c t i o n s and analyzed f o r p r o t e i n (-•-) and s i a l i c a c i d (• A. Na t i ve egg wh i t e . ( F r a c t i o n s 23-28 combined f o r chemica l a n a l y s i s . ) B. Stored (166 h) egg wh i t e . ( F r a c t i on s 23-28 combined f o r chemica l a n a l y s i s . ) C. Egg wh i te reduced w i t h 0.02% 2-mercaptoethanol. 87 0 10 20 30 40 50 60 70 FRACTION NUMBER 88 FIGURE 10 Sedimentat ion e q u i l i b r i u m pa t te rn s of ovomucins, In A 2 vs r . A l l samples were d i a l y z e d aga ins t 0.07 M sodium phosphate, pH 6.95, c o n t a i n i n g 0.02% sodium az i de w i t h a column he ight of 3 mm. The p r epa r a t i o n s , i n i t i a l p r o t e i n concent ra t i on s and r o t o r speeds were as f o l l o w s : A, n a t i v e ovomucin, 0.45 mg/ml, 4,085 rpm; B, n a t i v e ovomucin, 0.23 mg/ml, 4,048 rpm; C, s to red ovomucin, 0.52 mg/ml, 4,014 rpm; D, s to red ovomucin, 0.36 mg/ml, 4,034 rpm. 90 FIGURE 11 Apparent molecular weight (M ) of ovomucins as a w app function of protein concentration in 0.07 M sodium phosphate containing 0.02% sodium azide, pH 6.95, 20°C. (a) Native ovomucin. (b) Stored ovomucin. 91 92 FIGURE 12 Sedimentat ion 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 of f r a c t i o n s 24 and 37 from g e l f i l t r a t i o n of egg wh i te reduced w i t h 0.02% 2-mercaptoethanol . Samples were d i a l y z e d aga ins t 0.07 M sodium phosphate, pH 6.95, c on t a i n i n g 0.02% sodium a z i d e . F i n a l concen t ra t i on s were 0.09 and 0.11 mg/ml f o r f r a c t i o n s 24 and 37, r e s p e c t i v e l y . Rotor speed, 9,891 rpm. (a) F r a c t i o n 24. (b) F r a c t i o n 37. a 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0095244/manifest

Comment

Related Items