Significance of Lysozyme in Heat-Induced Aggregation of Egg White

Chapter 6

Significance of Lysozyme in Heat-Induced Aggregation of Egg White Protein Naotoshi Matsudomi

Downloaded by YORK UNIV on July 1, 2012 | http://pubs.acs.org Publication Date: February 19, 1991 | doi: 10.1021/bk-1991-0454.ch006

Department of Agricultural Chemistry, Yamaguchi University, Yamaguchi 753, Japan

Heat coagulation is one of the important functional properties of egg white. The heat-induced interactions among the heterogeneous proteins in egg white were investigated. The factors contributing to heat-induced aggregation between ovalbumin and lysozyme were examined to elucidate the mechanism of aggregation. The heat-induced aggregation was due to an electrostatic attraction and SH-SS interchange between the heat-denatured protein molecules. It was found that the addition of native lysozyme into heat-denatured ovalbumin forms insoluble aggregates. These results indicated that lysozyme interacts electrostatically with the monomeric molecule of fully unfolded ovalbumin. Ovotransferrin is the most thermolabile protein in egg white protein. The heat-induced aggregation of ovotransferrin with lysozyme was examined. The aggregation was increased remarkably in the presence of lysozyme. The heat-induced aggregation was mainly due to electrostatic and hydrophobic interactions. In addition, lysozyme affected the rheological properties of ovalbumin gel. We discuss significance of lysozyme in the heat-induced aggregation of egg white. Egg w h i t e i s e x t e n s i v e l y u t i l i z e d as a f u n c t i o n a l food m a t e r i a l i n f o o d p r o c e s s i n g . Heat c o a g u l a b i l i t y i s one o f i m p o r t a n t f u n c t i o n a l p r o p e r t i e s o f egg w h i t e ; t h e r e f o r e , t h e h e a t d e n a t u r a t i o n o f egg w h i t e and i t s component p r o t e i n s has been s t u d i e d by many i n v e s t i g a t o r s (1-8). However, h e a t - i n d u c e d i n t e r a c t i o n among t h e h e t e r o g e n e o u s p r o t e i n s i n egg w h i t e has been l i t t l e s t u d i e d . I t has been r e p o r t e d (9,10) t h a t ovalbumin and lysozyme, t h e major p r o t e i n s i n egg w h i t e , can i n t e r a c t e l e c t r o s t a t i c a l l y i n n a t u r e . Cunningham and Lineweaver (11) have s t u d i e d t h e i n a c t i v a t i o n o f lysozyme by ovalbumin d u r i n g h e a t i n g , and r e p o r t e d t h a t lysozyme was r a p i d l y i n a c t i v a t e d and t h a t an i n s o l u b l e p r e c i p i t a t e was formed by h e a t t r e a t m e n t . They s u g g e s t e d t h a t t h e s u l f h y d r y l groups o f ovalbumin c o u l d be i m p l i c a t e d i n t h e

0097-6156/91/0454-0073$06.00A) © 1991 American Chemical Society In Interactions of Food Proteins; Parris, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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i n a c t i v a t i o n o f lysozyme; however, t h e mechanism f o r h e a t - i n d u c e d a g g r e g a t i o n between t h e s e p r o t e i n s has n o t been e l u c i d a t e d . Matsuda e t a l . (12) have s t u d i e d t h e h e a t - i n d u c e d a g g r e g a t i o n o f ovomucoid w i t h lysozyme, and presumed t h a t ovomucoid and lysozyme m o l e c u l e s were b r o u g h t c l o s e t o g e t h e r by t h e e l e c t r o s t a t i c a t t r a c t i v e f o r c e , u n f o l d e d by h e a t i n g , and then a g g r e g a t e d t h r o u g h i n t e r m o l e c u l a r f o r c e s . I n t h i s s t u d y , we examine t h e h e a t - i n d u c e d i n t e r a c t i o n between lysozyme and ovalbumin o r o v o t r a n s f e r r i n and d e s c r i b e t h e s i g n i f i c a n c e o f lysozyme i n t h e h e a t - i n d u c e d a g g r e g a t i o n o f egg w h i t e . T h i s i n f o r m a t i o n c a n be used i n p r e p a r i n g egg w h i t e p r o t e i n b l e n d s w i t h a range o f g e l l i n g p r o p e r t i e s t o s u p p l y t h e b u r g e o n i n g f o o d f o r m u l a t i o n s s e c t o r o f t h e f o o d i n d u s t r y , and p r e p a r i n g non t h e r m a l c o a g u l a t i n g egg w h i t e p r o t e i n t o s u p p l y many c u l t u r e mediums f o r m i c r o o r g a n i s m s and a n i m a l t i s s u e c e l l s (13,14). M a t e r i a l s and Methods Preparation of Proteins. Ovalbumin was p r e p a r e d from f r e s h egg w h i t e by a c r y s t a l l i z a t i o n method i n sodium s u l f a t e (15). Lysozyme was p r e p a r e d from f r e s h egg white by a d i r e c t c r y s t a l l i z a t i o n method (16). O v o t r a n s f e r r i n was o b t a i n e d from Sigma C h e m i c a l . The lysozymef r e e egg w h i t e p r o t e i n was p r e p a r e d by t r e a t m e n t w i t h D u o l i t e C-464, c a t i o n exchange r e s i n , a c c o r d i n g t o t h e method o f L i - C h a n e t a l . ( 1 7 ) Chemical M o d i f i c a t i o n o f P r o t e i n s . S u c c i n y l a t i o n o f p r o t e i n was c a r r i e d o u t a c c o r d i n g t o t h e p r o c e d u r e s o f Habeeb (18). Acetylation and c i t r a c o n y l a t i o n o f lysozyme were c a r r i e d o u t a c c o r d i n g t o t h e method o f Yamasaki e t a l . (19) and N i e t o and P a l a c i a n (20), r e s p e c t i v e l y . R e d u c t i o n and carboxyamide m e t h y l a t i o n (RCAM) o f ovalbumin was c a r r i e d o u t as d e s c r i b e d by C r e s t i f i e l d e t a l . (21), u s i n g i o d o acetamide. Measurement o f G e l Hardness. G e l s were made w i t h 10% p r o t e i n s o l u t i o n c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s o f lysozyme by h e a t t r e a t m e n t f o r 30 min a t 75°C o r 80°C. The g e l h a r d n e s s was d e t e r m i n e d on t h e g e l s e c t i o n s (6.0 mm d i a m e t e r χ 5.0 mm h e i g h t ) w i t h an I n s t r o n U n i v e r s a l T e s t i n g Instrument (Model 112, I n s t r o n C o . ) , a c c o r d i n g t o t h e method o f M u l v i h i l l and K i n s e l l a (22). The g e l s e c t i o n was com p r e s s e d a t 40% o f i t s o r i g i n a l h e i g h t (2.0 cm). The g e l hardness (gram) was c a l c u l a t e d from t h e h e i g h t o f t h e f o r c e peak on t h e f i r s t compression c y c l e . Measurement o f T u r b i d i t y . A t u r b i d o m e t r i c method was used t o study the i n t e r a c t i o n between lysozyme and ovalbumin o r o v o t r a n s f e r r i n . The t u r b i d i t y development i s used as a measure o f t h e e x t e n t o f i n t e r a c t i o n . I n a t y p i c a l experiment, 1.0 ml a l i q u o t s o f ovalbumin s o l u t i o n (0.2% i n 35 mM p o t a s s i u m phosphate b u f f e r a t pH 7.6) were t r e a t e d w i t h i n c r e a s i n g amounts o f lysozyme (0.2% i n t h e phosphate b u f f e r ) and t h e t o t a l volume was made up t o 2.0 ml by a d d i n g t h e b u f f e r . I n t h e case o f o v o t r a n s f e r r i n - l y s o z y m e i n t e r a c t i o n , 0.1% p r o t e i n s o l u t i o n s were used. Heat t r e a t m e n t was c a r r i e d o u t by h e a t i n g a t t h e r a t e o f 2°C p e r minute from 20°to 90°C, and t h e t u r b i d i t y was measured a t 540 nm. The h e a t d e n a t u r a t i o n o f ovalbumin was c a r r i e d o u t w i t h 2.0 ml p o r t i o n s o f t h e p r o t e i n s o l u t i o n ( 0 . 1 % ) .

In Interactions of Food Proteins; Parris, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

6.

MATSUDOMI

Heat-Induced Aggregation of Egg White Protein 75


Gel E l e c t r o p h o r e s i s . P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n sodium d o d e c y l s u l f a t e (SDS) was p e r f o r m e d i n a 15% g e l a c c o r d i n g t o the method o f Laemmli (23). The i n s o l u b l e a g g r e g a t e formed was c o l l e c t e d by c e n t r i f u g a t i o n . The p r e c i p i t a t e was washed t w i c e w i t h the phos phate b u f f e r and then d i s s o l v e d i n a g i v e n volume o f a 10 mM T r i s HC1 b u f f e r (pH 6.8) c o n t a i n i n g 1% SDS, 25% g l y c e r o l , 1% 2-mercaptoe t h a n o l (2-ME) and 0.025% bromophenol b l u e . The p r o t e i n bands were s t a i n e d w i t h 0.05% Coomassie b r i l l i a n t b l u e and d e s t a i n e d by d i f f u s i o n i n m e t h a n o l - a c e t i c a c i d - w a t e r (20:10:70, v / v ) . The c o n c e n t r a t i o n o f p r o t e i n s on the e l e c t r o p h o r e t i c p a t t e r n s was d e t e r m i n e d by measuring t h e i r c o l o r d e n s i t i e s a t 565 nm on a d e n s i t o m e t e r . The p r o t e i n c o n t e n t s o f ovalbumin and lysozyme were e s t i m a t e d from the s t a n d a r d c u r v e s o f t h e s e p r o t e i n s measured by the d e n s i t o m e t e r . Gel F i l t r a t i o n . G e l f i l t r a t i o n o f h e a t - d e n a t u r e d ovalbumin was c a r r i e d o u t by h i g h - p e r f o r m a n c e l i q u i d chromatography on a TSK G e l G3000SW column (Tosoh, M a n u f a c t u r i n g Co., Tokyo, 0.75 χ 30 cm). A 20 u l p o r t i o n o f 0.1% p r o t e i n s o l u t i o n was l o a d e d on the column a t a f l o w r a t e o f 0.8 ml/min, u s i n g 0.2 M p o t a s s i u m phosphate b u f f e r (pH 6.9) as an e l u e n t . A UV m o n i t o r was used t o m o n i t o r the e f f l u e n t a t 280 nm. The chromatogram was d e p i c t e d by u s i n g a Shimadzu C-R3A Chromatopac w i t h a c h a r t speed o f 5 mm/min. R e s u l t s and D i s c u s s i o n Heat S t a b i l i t y o f Lysozyme-free Egg White P r o t e i n . Lysozyme i s a h i g h l y b a s i c p r o t e i n which a c c o u n t s f o r 3.5% o f the p r o t e i n i n egg w h i t e (24). I t i s known t o form e l e c t r o s t a t i c complexes w i t h o t h e r p r o t e i n s i n egg w h i t e , such as ovomucin (25, 26), ovomucoid (12), o v o t r a n s f e r r i n (27) and ovalbumin (28, 2 9 ) . Thus, lysozyme i s thought t o p l a y i m p o r t a n t r o l e s i n the h e a t a g g r e g a t i o n o r c o a g u l a t i o n o f egg w h i t e . To e l u c i d a t e the r o l e o f lysozyme, l y s o z y m e - f r e e egg w h i t e p r o t e i n was p r e p a r e d by t r e a t m e n t w i t h D u o l i t e C-464, c a t i o n exchange r e s i n , a c c o r d i n g t o the method o f L i - C h a n e t a l . ( 1 7 ) . F i g u r e 1 shows SDS-PAGE p a t t e r n s o f D u o l i t e - t r e a t e d egg w h i t e protein. From the r e s u l t , lysozyme was c o n f i r m e d t o be r e d u c e d s p e c i f i c a l l y from egg w h i t e . F i g u r e 2A shows the e f f e c t o f h e a t i n g temperature on the a g g r e g a t i o n o f D u o l i t e - t r e a t e d egg w h i t e ( l y s o z y m e - f r e e egg w h i t e ) . The p r o t e i n s o l u t i o n s (0.2%) i n phosphate b u f f e r (pH 7.6, i o n i c s t r e n g t h 0.1) were h e a t e d , and t u r b i d i t y was measured a t 540 nm. The t u r b i d i t y o f u n t r e a t e d egg w h i t e p r o t e i n i n c r e a s e d p r o g r e s s i v e l y w i t h i n c r e a s i n g h e a t i n g t e m p e r a t u r e , w h i l e the l y s o z y m e - f r e e egg w h i t e p r o t e i n d i d n o t show s i g n i f i c a n t changes o f t u r b i d i t y , even a t 85 °C. T h i s i n d i c a t e s t h a t the l y s o z y m e - f r e e egg w h i t e p r o t e i n i s much more s t a b l e t o h e a t . To examine a s i g n i f i c a n c e o f lysozyme i n egg w h i t e , lysozyme was added t o the l y s o z y m e - f r e e egg w h i t e p r o t e i n , and then h e a t e d t o 70 °C. F i g u r e 2B shows the e f f e c t o f added lysozyme. As lysozyme c o n c e n t r a t i o n i n c r e a s e d , the t u r b i d i t y g r e a t l y i n c r e a s e d . One mg o f egg w h i t e p r o t e i n c o n t a i n s 34 ug o f lysozyme (30). As shown by an arrow i n F i g . 2B, the t u r b i d i t y a t t h i s p o i n t c o r r e s p o n d e d t o t h a t o f n a t i v e egg w h i t e p r o t e i n (see F i g . 2A) T h i s r e s u l t i n d i c a t e d t h a t the e x i s t e n c e o f lysozyme i n egg w h i t e i s v e r y i m p o r t a n t f o r h e a t c o a g u l a t i o n o f egg w h i t e .




F i g u r e 1. SDS-PAGE P a t t e r n s o f D u o l i t e - t r e a t e d Egg w h i t e . A, u n t r e a t e d egg w h i t e ; B, D u o l i t e - t r e a t e d egg w h i t e ; OA, ovalbumin; OT, o v o t r a n s f e r r i n ; LZ, lysozyme.

HEATING TEMP., °C

LYSOZYME C O N C M g

F i g u r e 2. E f f e c t s o f H e a t i n g Temperature and Added Lysozyme on A g g r e g a t i o n o f D u o l i t e - t r e a t e d Egg White ( l y s o z y m e - f r e e egg white). A, e f f e c t o f h e a t i n g temperature; B, e f f e c t o f added lysozyme; • , l y s o z y m e - f r e e egg w h i t e ; • , u n t r e a t e d egg w h i t e .



6.

MATSUDOMI


H e a t - i n d u c e d A g g r e g a t i o n between Ovalbumin and Lysozyme. When the d i l u t e ovalbumin and lysozyme s o l u t i o n s (0.1%) were h e a t e d s e p a r a t e l y , the p r o t e i n s o l u t i o n d i d n o t show any changes o f t u r b i d i t y , even a t 90 °C. However, when mixed and h e a t e d , an i n s o l u b l e a g g r e g a t e was formed by h e a t i n g t o 70°C. T h e r e f o r e , the h e a t - i n d u c e d a g g r e g a t e was examined by e l e c t r o p h o r e t i c a n a l y s i s . F o r the a n a l y s i s o f the a g g r e g a t e , a m i x t u r e o f ovalbumin and lysozyme (1:0.1 mg/mg) was d i s s o l v e d i n the phosphate b u f f e r c o n t a i n i n g v a r i o u s NaCl c o n c e n t r a t i o n s and then h e a t e d t o 75 °C. F i g u r e 3 shows SDS-slab g e l e l e c t r o p h o r e t i c p a t t e r n s o f the p r e c i p i t a t e s . The p r e c i p i t a t e o b t a i n e d from the h e a t e d s o l u t i o n was found t o c o n s i s t o f ovalbumin and lysozyme. The amount o f p r e c i p i t a t e was h i g h e r i n the case o f h e a t i n g i n the absence o f N a C l , and d e c r e a s e d w i t h an i n c r e a s e o f i o n i c s t r e n g t h . The weight r a t i o o f lysozyme t o ovalbumin i n the p r e c i p i t a t e s was assumed t o be 1 i n a l l c a s e s , and the molar r a t i o o f lysozyme (14,500) and ovalbumin (45,000) was e s t i m a t e d t o be about 3. The e f f e c t s o f i o n i c s t r e n g t h and h e a t i n g temperature on the a g g r e g a t i o n a r e shown i n F i g . 4. The t u r b i d i t y d e c r e a s e d w i t h an i n c r e a s e o f i o n i c s t r e n g t h , when compared a t the same h e a t i n g temperature. I t has been r e p o r t e d by some i n v e s t i g a t o r s (10,11) t h a t ovalbumin and lysozyme i n t e r a c t e d more e l e c t r o s t a t i c a l l y i n a s a l t f r e e s o l u t i o n a t n e u t r a l pH, and t h a t sodium and c h l o r i d e i o n s s h i e l d e d the c h a r g e d groups i n t h e s e p r o t e i n s . 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 between ovalbumin and lysozyme i s thus c o n s i d e r e d t o be weakened by the a d d i t i o n o f N a C l , and the f o r m a t i o n o f h e a t - i n d u c e d a g g r e g a t e s would be d e p r e s s e d as the r e s u l t o f a d e c r e a s e o f e l e c t r o static interaction. On the o t h e r hand, i t has been r e p o r t e d (1,3) t h a t the h e a t - i n d u c e d a g g r e g a t i o n o f ovalbumin a l o n e was a f f e c t e d by the n e t charge o f p r o t e i n m o l e c u l e s and i n h i b i t e d by e l e c t r o s t a t i c r e p u l s i o n among the p r o t e i n m o l e c u l e s i n the absence o f s a l t s . We have a l s o r e p o r t e d (31) t h a t , i n the p r e s e n c e o f N a C l , the s o l u b l e ovalbumin a g g r e g a t e s were r e a d i l y formed d u r i n g h e a t d e n a t u r a t i o n by h y d r o p h o b i c i n t e r a c t i o n and d i s u l f i d e bond f o r m a t i o n , whereas t h e f o r m a t i o n o f the s o l u b l e a g g r e g a t e s was i n h i b i t e d i n the absence o f NaCl. T h e r e f o r e , i n heterogeneous p r o t e i n systems such as the m i x t u r e o f ovalbumin and lysozyme, the h e a t - i n d u c e d a g g r e g a t i o n would be i n h i b i t e d by a l o s s o f the r e a c t i v i t y o f ovalbumin t o lysozyme due t o the f o r m a t i o n o f s o l u b l e a g g r e g a t e s o f ovalbumin i t s e l f as the c o n c e n t r a t i o n o f NaCl was i n c r e a s e d . The p r o t e i n s d i d n o t show any changes i n t u r b i d i t y u n t i l h e a t i n g t o 60 °C, b u t became t u r b i d when the h e a t i n g temperature exceeded 70 °C. The h e a t - i n d u c e d a g g r e g a t i o n was o b s e r v e d a t temperatures below the t h e r m a l t r a n s i t i o n p o i n t f o r h e a t d e n a t u r a t i o n o f ovalbumin (76°C) and lysozyme (78°C) when d e t e r m i n e d from the changes i n s u r f a c e h y d r o p h o b i c i t y and CD a n a l y s i s (32). T h i s i n d i c a t e s t h a t the a g g r e g a t i o n p r o c e e d e d w i t h the degree o f h e a t d e n a t u r a t i o n o f t h e s e p r o t e i n s . I t i s sugg e s t e d t h a t the h e a t - i n d u c e d a g g r e g a t i o n may have been i n i t i a t e d by 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 p r o t e i n m o l e c u l e s and subseq u e n t l y enhanced by h y d r o p h o b i c i n t e r a c t i o n w i t h h e a t d e n a t u r a t i o n . I t i s i n t e r e s t i n g t h a t the e x t e n t o f t u r b i d i t y l e v e l e d o f f when samples were h e a t e d t o 75 °C a t lower i o n i c s t r e n g t h . In o r d e r t o examine the r o l e o f amino a c i d r e s i d u e s i n the h e a t - i n d u c e d a g g r e g a t i o n , the e f f e c t o f c h e m i c a l m o d i f i c a t i o n s was studied. F i g u r e 5 shows the e f f e c t o f s u c c i n y l a t e d ovalbumin o r lysozyme on the h e a t - i n d u c e d a g g r e g a t i o n . The h e a t - i n d u c e d aggrega-




78

F i g u r e 3. SDS E l e c t r o p h o r e s i s and D e n s i t o m e t r i c Scanning o f a M i x t u r e o f Ovalbumin (0) and Lysozyme (L) Heated i n V a r i o u s I o n i c S t r e n g t h s a t 75°C. The numbers show N a C l c o n c e n t r a t i o n ( M ) . S, s u p e r n a t a n t ; P, p r e c i p i t a t e . (Reprinted with permission from ref. 9. Copyright 1986 Japan Society for Bioscience, Biotechnology, and Agrochemistry.)


MATSUDOMI



1.0

60

70 HEATING

80

80

T E M P . , °C

F i g u r e 4. E f f e c t s o f I o n i c S t r e n g t h and H e a t i n g Temperature on H e a t - i n d u c e d A g g r e g a t i o n between Ovalbumin and Lysozyme. The weight r a t i o o f lysozyme t o ovalbumin was 0.1. The c o n c e n t r a t i o n o f N a C l as f o l l o w s : O, no s a l t ; ·, 0.1 M; • , 0.2 M; • , 0.4 M. (Reprinted with permission from ref. 32. Copyright 1986 Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

10

A

60

70

80 60

HEATING

70

80

TEMP, °C

F i g u r e 5. E f f e c t o f S u c c i n y l a t i o n o f Amino Groups i n P r o t e i n on H e a t - i n d u c e d A g g r e g a t i o n between Ovalbumin (OA) and L y s o z y m e ( L Z ) . The weight r a t i o o f lysozyme t o ovalbumin was 0.1. A: O, u n t r e a t e d OA - u n t r e a t e d LZ; ·, 85% m o d i f i e d OA - u n t r e a t e d LZ; • , u n t r e a t e d OA - 85% m o d i f i e d L Z . Β: O, u n t r e a t e d OA - u n t r e a t e d LZ; ·, 35% m o d i f i e d OA - u n t r e a t e d LZ; • , 52% m o d i f i e d OA - u n t r e a t e d LZ; • , 85% m o d i f i e d OA untreated LZ. (Reprinted with permission from ref. 32. Copyright 1986 Japan Society for Bioscience, Biotechnology, and Agrochemistry.)



80


t i o n was d e p r e s s e d c o m p l e t e l y by t h e s u c c i n y l a t i o n o f ovalbumin o r lysozyme. S u c c i n y l a t i o n c o n v e r t e d c a t i o n i c amino groups t o a n i o n i c r e s i d u e s , and c o n s e q u e n t l y t h e n e t charge o f t h e s u c c i n y l a t e d ovalbumin became n e g a t i v e . T h e r e f o r e , i t was presumed t h a t s u c c i n y l a t e d ovalbumin c o u l d i n t e r a c t e l e c t r o s t a t i c a l l y t o a g r e a t e r e x t e n t w i t h p o s i t i v e l y charged lysozyme a t n e u t r a l pH. However, t h e s u c c i n y l a t e d ovalbumin d i d n o t form a g g r e g a t e s w i t h lysozyme d u r i n g heating. The h e a t - i n d u c e d a g g r e g a t i o n may t h e r e f o r e have been d e p r e s s e d by t h e b l o c k o f charged amino groups. The e f f e c t o f t h e degree o f s u c c i n y l a t i o n o f ovalbumin on t h e h e a t - i n d u c e d a g g r e g a t i o n i s shown i n F i g . 5B. As t h e e x t e n t o f s u c c i n y l a t i o n o f ovalbumin i n c r e a s e d , t h e h e a t - i n d u c e d a g g r e g a t i o n w i t h lysozyme reduced t o a g r e a t e r e x t e n t . I t i s s u g g e s t e d from t h e s e r e s u l t s t h a t t h e c h a r g e d amino groups ( t h e ε-amino group) o f ovalbumin and lysozyme p l a y e d an i m p o r t a n t r o l e i n t h e h e a t - i n d u c e d a g g r e g a t i o n between ovalbumin and lysozyme. F i g u r e 6 shows t h e e f f e c t o f RCAM o f ovalbumin on t h e h e a t - i n d u c e d a g g r e g a t i o n . The m i x t u r e o f RCAM-ovalbumin and lysozyme was h e a t e d a t 0.1 and 0.5 i o n i c s t r e n g t h s . The RCAM-ovalbumin s i g n i f i c a n t l y i n h i b i t e d t h e h e a t - i n d u c e d a g g r e g a t i o n w i t h lysozyme. However, t h e h e a t - i n d u c e d a g g r e g a t e s i n c r e a s e d g r a d u a l l y w i t h h e a t i n g temperature. T h i s r e s u l t s u g g e s t s t h a t i n a d d i t i o n t o s u l f h y d r y l groups, e l e c t r o s t a t i c and h y d r o p h o b i c i n t e r a c t i o n s a l s o p a r t i c i p a t e i n the heat-induced aggregation. I n a d d i t i o n , from t h e changes o f s u l f h y d r y l groups i n p r o t e i n s d u r i n g h e a t i n g , i t was s u g g e s t e d t h a t s u l f h y d r y l - d i s u l f i d e i n t e r c h a n g e r e a c t i o n between ovalbumin and lysozyme i s i n v o l v e d i n t h e h e a t - i n d u c e d a g g r e g a t i o n . In c o n c l u s i o n , t h e scheme f o r h e a t - i n d u c e d a g g r e g a t i o n between ovalbumin and lysozyme i s i l l u s t r a t e d i n F i g . 7. When h e a t e d a t a low i o n i c s t r e n g t h , ovalbumin and lysozyme i n t e r a c t e l e c t r o s t a t i c a l l y a t t h e molar r a t i o o f 1 t o 3 i n t h e i n i t i a l s t e p o f h e a t i n g , and i n t h e n e x t s t e p , t h e two p r o t e i n s a r e a g g r e g a t e d by h y d r o p h o b i c i n t e r a c t i o n and a s u l f h y d r y l - d i s u l f i d e i n t e r c h a n g e r e a c t i o n . On t h e o t h e r hand, when h e a t e d a t a h i g h i o n i c s t r e n g t h , ovalbumin i t s e l f forms t h e s o l u b l e a g g r e g a t e s t h r o u g h h y d r o p h o b i c i n t e r a c t i o n and d i s u l f i d e bonding i n t h e i n i t i a l s t a g e s o f h e a t i n g , and then lysozyme i s l i n k e d by a s u l f h y d r y l - d i s u l f i d e i n t e r c h a n g e r e a c t i o n t o the s o l u b l e ovalbumin a g g r e g a t e s a t t h e m o l a r r a t i o o f 3 t o 1, con s e q u e n t l y t h e complex becoming i n s o l u b l e . A g g r e g a t i o n between Lysozyme and H e a t - d e n a t u r e d Ovalbumin. During f u r t h e r experiments on t h e i n t e r a c t i o n between ovalbumin and lysozyme, we have found t h a t t h e a d d i t i o n o f n a t i v e lysozyme i n t o h e a t - d e n a t u r e d ovalbumin formed an i n s o l u b l e a g g r e g a t e , and t h a t t h e a g g r e g a t i o n w i t h lysozyme was d e p r e s s e d when ovalbumin was h e a t e d above 75 C. I n t h i s s e c t i o n , t h e f a c t o r s a f f e c t i n g t h e a g g r e g a t i o n were i n v e s t i g a t e d . The e f f e c t o f t h e r a t i o o f lysozyme t o h e a t - d e n a t u r e d ovalbumin on t h e a g g r e g a t i o n was i n v e s t i g a t e d by measuring t h e development o f turbidity. The ovalbumin s o l u t i o n (0.1%) was h e a t e d t o 75 C, and then lysozyme was added t o t h e h e a t - d e n a t u r e d ovalbumin s o l u t i o n , the r e s u l t s b e i n g shown i n F i g . 8A. When t h e weight r a t i o o f lysozyme t o ovalbumin was 0.02, t h e t u r b i d i t y began t o i n c r e a s e slightly. The t u r b i d i t y i n c r e a s e d s i g m o i d a l l y w i t h an i n c r e a s e o f lysozyme c o n c e n t r a t i o n . Then, t h e r e s u l t i n g i n s o l u b l e a g g r e g a t e s were examined by e l e c t r o p h o r e t i c a n a l y s i s ( F i g . 8 B ) . The o b t a i n e d


6. MATSUDOMI



10

50

60

70

HEATING

80

90

T E M P . , °C

F i g u r e 6. E f f e c t o f C a r b o x y m e t h y l a t i o n (RCAM) o f SH Groups i n Ovalbumin on H e a t - i n d u c e d A g g r e g a t i o n . The weight r a t i o o f lysozyme t o ovalbumin was 0.1. O h e a t e d w i t h o u t NaCl; ·, h e a t e d w i t h 0.4 M N a C l ; , u n t r e a t e d ovalbumin; , RCAM-ovalbumin. f

(Reprinted with permission from ref. 32. Copyright 1986 Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

F i g u r e 7. Scheme f o r A g g r e g a t i o n between Ovalbumin and Lysozyme d u r i n g H e a t i n g . O, ovalbumin; • , lysozyme; — , d i s u l f i d e bond.

(Reprinted with permission from ref. 32. Copyright 1986 Japan Society for Bioscience, Biotechnology, and Agrochemistry.)



82


p r e c i p i t a t e s were found t o c o n s i s t o f ovalbumin and lysozyme. The molar r a t i o o f lysozyme t o ovalbumin i n the p r e c i p i t a t e s was e s t i m a t e d t o be about 1.5 i n a l l c a s e s , r e g a r d l e s s o f the concent r a t i o n o f a d d i n g lysozyme. F i g u r e 9 shows the e f f e c t o f N a C l c o n c e n t r a t i o n on t h e a g g r e gation. A f t e r the ovalbumin s o l u t i o n had been h e a t e d t o 75 °C, s a l t and lysozyme were s u c c e s s i v e l y added t o the h e a t - d e n a t u r e d ovalbumin solution. The t u r b i d i t y g r e a t l y d e c r e a s e d as the c o n c e n t r a t i o n o f the s a l t was i n c r e a s e d . The f o r m a t i o n o f an i n s o l u b l e a g g r e g a t e was c o m p l e t e l y d e p r e s s e d by the a d d i t i o n o f NaCl t o a f i n a l concent r a t i o n o f 0.1 M. The i n t e r a c t i o n between the h e a t - d e n a t u r e d ovalbumin and lysozyme i s t h e r e f o r e c o n s i d e r e d t o be e l e c t r o s t a t i c , the f o r m a t i o n o f an i n s o l u b l e a g g r e g a t e b e i n g d e p r e s s e d as the r e s u l t of a decrease o f e l e c t r o s t a t i c i n t e r a c t i o n . The e f f e c t o f the degree o f h e a t d e n a t u r a t i o n o f the ovalbumin on the a g g r e g a t i o n w i t h lysozyme i s shown i n F i g . 10. The ovalbumin s o l u t i o n s were h e a t e d t o v a r i o u s t e m p e r a t u r e s from 60 °C t o 90 °C, and lysozyme was added t o the h e a t - d e n a t u r e d ovalbumin s o l u t i o n s . The t u r b i d i t y began t o i n c r e a s e when ovalbumin was h e a t e d t o 65 °C, and r e a c h e d i t s maximum a t 75 °C, b e f o r e d r a s t i c a l l y d e c r e a s i n g after that. T h i s phenomenon was a l s o o b s e r v e d , when the m i x t u r e o f ovalbumin and lysozyme was h e a t e d as d e s c r i b e d i n the p r e v i o u s s e c t i o n ( F i g . 4 ) . Thus, the a g g r e g a t i o n was found t o be i t s maximum a t the t h e r m a l t r a n s i t i o n temperature f o r the h e a t d e n a t u r a t i o n o f ovalbumin, when d e t e r m i n e d from changes i n s u r f a c e h y d r o p h o b i c i t y , CD a n a l y s i s and exposure o f s u l f h y d r y l groups as p r e v i o u s l y d e s c r i b e d (32). We have p r o p o s e d i n the p r e v i o u s paper (31) t h a t ovalbumin d u r i n g h e a t d e n a t u r a t i o n was p o l y m e r i z e d m a i n l y by h y d r o p h o b i c i n t e r a c t i o n and d i s u l f i d e bond f o r m a t i o n . The m o l e c u l a r p r o p e r t i e s o f ovalbumin m o l e c u l e s d u r i n g h e a t d e n a t u r a t i o n were t h e r e f o r e i n v e s t i g a t e d i n d e t a i l by HPLC a n a l y s i s . F i g u r e 11 shows the e l u t i o n p a t t e r n s o f the s u p e r n a t a n t s o b t a i n e d by c e n t r i f u g a t i o n b e f o r e and a f t e r the a d d i t i o n o f lysozyme. Ovalbumin t h a t was h e a t e d t o 70 °C o r 75 °C, as w e l l as the unheated ovalbumin, gave a s i n g l e peak w i t h a r e t e n t i o n time o f 14.5 min. On the o t h e r hand, ovalbumin h e a t e d t o 80 °C o r 85 °C gave t h r e e peaks. These peaks seem t o c o r respond t o the monomer, dimer and t r i m e r o f the ovalbumin m o l e c u l e . The f r a c t i o n o f the o l i g o m e r i n the ovalbumin i n c r e a s e d w i t h the r i s e i n h e a t i n g temperature. As a l r e a d y mentioned f o r F i g . 10, the i n t e r a c t i o n w i t h lysozyme d e c r e a s e d when ovalbumin was h e a t e d above 75 °C. T h e r e f o r e , the i n t e r a c t i o n w i t h lysozyme would be i n h i b i t e d by p o l y m e r i z a t i o n o f the ovalbumin m o l e c u l e . I t was s u g g e s t e d t h a t lysozyme m a i n l y i n t e r a c t e d w i t h t h e the monomeric m o l e c u l e o f f u l l y u n f o l d e d ovalbumin by h e a t i n g . The e l u t i o n p a t t e r n s o f s u p e r n a t a n t s o b t a i n e d by c e n t r i f u g a t i o n a f t e r t h e a d d i t i o n o f lysozyme t o the h e a t - d e n a t u r e d ovalbumin a r e shown i n F i g . 11B. Lysozyme gave a r e t e n t i o n time o f 20 min. The peak a r e a o f lysozyme i n t h e s u p e r n a t a n t was i n v e r s e l y p r o p o r t i o n a l t o t h e degree o f t u r b i d i t y . When ovalbumin was h e a t e d t o 75 °C, the added lysozyme seemed t o r e a c t almost e n t i r e l y w i t h the h e a t - d e n a t u r e d ovalbumin, because o f no e v i d e n c e o f lysozyme i n the s u p e r n a t a n t . When ovalbumin was h e a t e d t o 80 °C o r 85 °C, a peak h a v i n g a r e t e n t i o n time o f 8 min appeared i n the s u p e r n a t a n t by the a d d i t i o n o f lysozyme. The peak a r e a i n c r e a s e d i n p r o p o r t i o n t o the d e c r e a s e i n the o l i g o m e r i c f r a c t i o n o f the ovalbumin m o l e c u l e . T h i s peak was found t o c o n s i s t o f the



MATSUDOMI


0.05

0.1

0.2

0.3

F i g u r e 8. E f f e c t o f the Weight R a t i o o f Lysozyme t o Ovalbumin on A g g r e g a t i o n and D e n s i t o m e t e r Scans o f S D S - P o l y a c r y l a m i d e G e l s under 2-ME o f A g g r e g a t e s O b t a i n e d . To 2 ml o f a 0.1% ovalbumin s o l u t i o n (·) h e a t e d t o 75°C, i n c r e a s i n g amounts o f lysozyme were added. The numbers i n F i g . 8B show the weight r a t i o o f lysozyme (L) t o the h e a t - d e n a t u r e d ovalbumin. (Reprinted with permission from ref. 33. Copyright 1987 Japan Society for Bioscience, Biotechnology, and Agrochemistry.)


84



t=

0.2

0 0.01 0.02

NaCl

0.05 C0NCN.,

" 0.1

(M)

F i g u r e 9. E f f e c t o f N a C l C o n c e n t r a t i o n on the A g g r e g a t i o n between Ovalbumin and Lysozyme. To 2 ml o f a 0.1% ovalbumin s o l u t i o n h e a t e d t o 75°C, i n c r e a s i n g amounts o f N a C l were added, and t h e n lysozyme was added. The weight r a t i o o f lysozyme t o ovalbumin was was 0.1. (Reprinted with permission from ref. 33. Copyright 1987 Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

â

0.2

20

60

70

80

90

HEATING TEMP., °C F i g u r e 10. E f f e c t o f H e a t - d e n a t u r e d Ovalbumin on the A g g r e g a t i o n w i t h Lysozyme. To 2 ml o f a 0.1% ovalbumin s o l u t i o n h e a t e d t o t h e i n d i c a t e d t e m p e r a t u r e , lysozyme was added. The weight r a t i o o f lysozyme t o ovalbumin was 0.1. (Reprinted with permission from ref. 33. Copyright 1987 Japan Society for Bioscience, Biotechnology, and Agrochemistry.)



MATSUDOMI

Heat-Induced Aggregation of Egg White Protein

10

15

20

ELUTION

5

10

15

20

TIME (MIN)

F i g u r e 11. E l u t i o n P a t t e r n s o f S u p e r n a t a n t s O b t a i n e d by C e n t r i f u g a t i o n b e f o r e and a f t e r A d d i n g Lysozyme t o t h e H e a t - d e n a t u r e d Ovalbumin. (A) b e f o r e a d d i n g lysozyme; (B) a f t e r a d d i n g lysozyme; P, ovalbumin polymer; T, ovalbumin t r i m e r ; D, ovalbumin dimer; M, ovalbumin monomer; L, lysozyme.



86


ovalbumin m o l e c u l e a l o n e . T h e r e f o r e , the a d d i t i o n o f lysozyme seems t o have caused p o l y m e r i z a t i o n o f the o l i g o m e r i c ovalbumin m o l e c u l e s . T h i s phenomenon has been known t o be i n d u c e d by the a d d i t i o n o f N a C l . T h e r e f o r e , the a c t i o n o f lysozyme i s c o n s i d e r e d t o have weakened the e l e c t r o s t a t i c r e p u l s i v e f o r c e s between the d e n a t u r e d ovalbumin m o l e c u l e s and p o l y m e r i z e d the o l i g o m e r i c ovalbumin m o l e c u l e s by h y d r o p h o b i c i n t e r a c t i o n and d i s u l f i d e bond f o r m a t i o n . In c o n c l u s i o n , the a g g r e g a t i o n between lysozyme and heat-denat u r e d ovalbumin was caused by 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 a t the molar r a t i o o f 1.5. I t was found t h a t lysozyme i n t e r a c t e d more w i t h the monomeric m o l e c u l e o f the f u l l y u n f o l d e d ovalbumin by h e a t i n g , and formed i n s o l u b l e a g g r e g a t e s w i t h the ovalbumin. The added lysozyme a l s o p o l y m e r i z e d the o l i g o m e r i c ovalbumin m o l e c u l e s t o form the s o l u b l e ovalbumin a g g r e g a t e s . Thus, h e a t c o a g u l a t i o n o f egg white c o u l d be f a c i l i t a t e d by i n t e r a c t i o n s among such heterogeneous p r o t e i n s as ovalbumin and lysozyme. H e a t - i n d u c e d A g g r e g a t i o n between O v o t r a n s f e r r i n and Lysozyme. Among the c o n s t i t u e n t p r o t e i n s o f egg w h i t e , o v o t r a n s f e r r i n i s known t o be one o f the most h e a t - l a b i l e p r o t e i n , and i t i s more u n s t a b l e t o h e a t a t pH 7.5 than a t pH 9.0 (34). The pH o f egg white i s a l s o known t o change from 7.3 t o 9.7 d u r i n g s t o r a g e (35). T h e r e f o r e , t h e h e a t i n d u c e d a g g r e g a t i o n o f egg w h i t e may be dependent on the d e n a t u r a t i o n of o v o t r a n s f e r r i n . F i g u r e 12 shows the e f f e c t o f h e a t i n g temperature on the aggregat i o n between o v o t r a n s f e r r i n and lysozyme. The t u r b i d i t y o f ovot r a n s f e r r i n h e a t e d a t pH 7.5 i n c r e a s e d w i t h an i n c r e a s e o f h e a t i n g t e m p e r a t u r e , and the f o r m a t i o n o f an i n s o l u b l e aggregate was promoted by the a d d i t i o n o f lysozyme ( F i g . 12A). When h e a t e d a t pH 9.0, o v o t r a n s f e r r i n d i d n o t show any changes i n t u r b i d i t y , even a t 85 C. However, when lysozyme was mixed and h e a t e d , an i n s o l u b l e a g g r e g a t e was formed by h e a t i n g above 70 C ( F i g . 12B). I t i s known t h a t i r o n - b i n d i n g o v o t r a n s f e r r i n i s much more s t a b l e t o h e a t denat u r a t i o n than the m e t a l - f r e e one (36). However, the h e a t - i n d u c e d a g g r e g a t i o n o f i r o n - b i n d i n g o v o t r a n s f e r r i n was a l s o promoted remarka b l y by a d d i n g lysozyme, a t the b o t h pHs. F i g u r e 13 shows the e f f e c t o f NaCl c o n c e n t r a t i o n on the aggregation. In the case o f pH 7.5, samples were h e a t e d t o 60 C (Fig. 13A). The t u r b i d i t y g r e a t l y d e c r e a s e d as the c o n c e n t r a t i o n o f the s a l t was i n c r e a s e d . The f o r m a t i o n o f i n s o l u b l e a g g r e g a t e o f ovot r a n s f e r r i n i t s e l f o c c u r r e d by h e a t i n g t o 60 C a t pH 7.5, was a l s o i n h i b i t e d by the a d d i t i o n o f N a C l . A t pH 9.0, samples were h e a t e d t o 75 C ( F i g . 13B). The f o r m a t i o n o f an i n s o l u b l e a g g r e g a t e was c o m p l e t e l y d e p r e s s e d by the a d d i t i o n o f NaCl t o a f i n a l c o n c e n t r a t i o n o f 0.5 M. In a d d i t i o n , the i n s o l u b l e a g g r e g a t e s formed by a d d i n g lysozyme were d i s s o c i a t e d c o m p l e t e l y i n t o i t s components by SDS. Thus, the i n t e r a c t i o n between o v o t r a n s f e r r i n and lysozyme was c o n s i d e r e d t o be e l e c t r o s t a t i c and/or h y d r o p h o b i c f o r c e s . E f f e c t o f Lysozyme on H e a t - i n d u c e d G e l a t i o n o f Ovalbumin. F i g u r e 14 shows the e f f e c t o f added lysozyme on h a r d n e s s o f ovalbumin g e l . As lysozyme c o n c e n t r a t i o n i n c r e a s e d , the g e l h a r d n e s s d e c r e a s e d g r a d u a l l y , and the t r a n s p a r e n c y o f g e l became more t u r b i d . Gel propert i e s o f p r o t e i n a r e a f f e c t e d by t h e mode o f d e n a t u r a t i o n and aggregation of p r o t e i n . F o r the f o r m a t i o n o f the h i g h l y o r d e r e d g e l



MATSUDOMI

Heat-Induced Aggregation of Egg White Protein

50

60

70

HEATING

60

TEMP.,

70

80

°C

F i g u r e 12. E f f e c t o f H e a t i n g Temperature on A g g r e g a t i o n between O v o t r a n s f e r r i n and Lysozyme. A, h e a t e d a t pH 7.5; B, h e a t e d a t pH 9.0; •, m i x t u r e o f o v o t r a n s f e r r i n and lysozyme; •, o v o t r a n s f e r r i n o n l y .




501-

0.2 LYSOZYME

0.5 1.0 CONCN., %

F i g u r e 14. E f f e c t o f Lysozyme on G e l Hardness o f Ovalbumin. •, h e a t e d f o r 30 min a t 80°C; 0, h e a t e d f o r 30 min a t 75°C.


6. MATSUDOMI

Heat-Induced Aggregation ofEgg White Protein 89


matrix, i t i s imperative t h a t the aggregation step proceeds a t a slower r a t e than t h e u n f o l d i n g s t e p (37, 3 8 ) . Thus, i t was s u g g e s t e d t h a t an o r d e r e d network f o r m a t i o n o f ovalbumin may be i n h i b i t e d by the added lysozyme, because o f t h e r a p i d f o r m a t i o n o f d i s o r d e r e d a g g r e g a t e s o f ovalbumin w i t h lysozyme d u r i n g h e a t i n g . Conclusions. I n commercial f o o d p r o c e s s i n g , t h e i n t e r a c t i o n o f heterogeneous p r o t e i n s i s an i m p o r t a n t f a c t o r i n d e t e r m i n i n g t h e s t r u c t u r e and t e x t u r e o f end p r o d u c t s , and t h e c a p a c i t y o f p r o t e i n s t o a g g r e g a t e o r c o a g u l a t e under p r a c t i c a l c o n d i t i o n s i s a u s u a l f u n c t i o n a l p r o p e r t y i n many systems. We d e s c r i b e d t h e s i g n i f i c a n c e o f lysozyme i n h e a t - i n d u c e d a g g r e g a t i o n o f egg w h i t e p r o t e i n . I n the i n i t i a l s t e p o f h e a t i n g between lysozyme and ovalbumin, lysozyme i n t e r a c t s e l e c t r o s t a t i c a l l y w i t h t h e monomeric m o l e c u l e s o f f u l l y u n f o l d e d ovalbumin, and i n t h e next s t e p , t h e two p r o t e i n s a r e a g g r e g a t e d by h y d r o p h o b i c i n t e r a c t i o n and a s u l f h y d r y l - d i s u l f i d e i n t e r c h a n g e r e a c t i o n o r lysozyme i n t e r a c t s w i t h t h e s o l u b l e ovalbumin a g g r e g a t e s t h r o u g h i n t e r m o l e c u l a r f o r c e s such as d i s u l f i d e bonds and h y d r o p h o b i c f o r c e s . Thus, h e a t a g g r e g a t i o n o f egg w h i t e c o u l d be f a c i l i t a t e d by i n t e r a c t i o n s among such h e t e r o g e n e o u s p r o t e i n s as lysozyme and ovalbumin o r o v o t a n s f e r r i n . Such a i n t e r a c t i o n m i g h t a f f e c t g r e a t l y t h e g e l p r o p e r t i e s o f egg w h i t e .

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June 20, 1990


Significance of Lysozyme in Heat-Induced Aggregation of Egg White

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