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Isolation of avidin and lysozyme from egg albumen Durance, Timothy Douglas 1987

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ISOLATION OF AVIDIN AND LYSOZYME FROM EGG ALBUMEN. by TIMOTHY DOUGLAS DURANCE A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN 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 e q u i r e d s t a n d a r d The U n i v e r s i t y o f B r i t i s h Columbia Augus t , 1987 © Timothy Douglas Durance , 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) i i ABSTRACT A s i n g l e co lumn c a t i o n exchange method was d e v e l o p e d w h i c h a l l o w e d s i m u l t a n e o u s r e c o v e r y o f lysozyme and a v i d i n from u n d i l u t e d egg w h i t e u s i n g a u n i q u e e l u t i o n sequence w h i c h i n v o l v e d a c c u m u l a t i o n o f a v i d i n on t h e co lumn t h r o u g h s e v e r a l c y c l e s o f egg w h i t e a p p l i c a t i o n and lysozyme e l u t i o n . Lysozyme was r e c o v e r e d w i t h h i g h e r y i e l d s t h a n r e p o r t e d f o r t h e i s o e l e c t r i c p r e c i p i t a t i o n methods o f t e n u s e d i n t h e i n d u s t r y (86% vs 60 - 80%) and i n h i g h p u r i t y . A v i d i n r e c o v e r y was a l s o as good o r b e t t e r t h a n t h a t o f p r e v i o u s l y r e p o r t e d i o n exchange methods (74 - 80% vs 17 -80%). The p u r i t y o f t h e a v i d i n f r a c t i o n (up t o 40.9%) was s u p e r i o r t o o t h e r r e p o r t e d p r i m a r y a v i d i n f r a c t i o n s . A v i d i n was shown t o have a g r e a t e r p o t e n t i a l f o r b o t h 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 w i t h D u o l i t e C-464 t h a n lysozyme b u t under t h e c o n d i t i o n s o f t h i s s e p a r a t i o n , 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 were d o m i n a n t . S e c o n d a r y p u r i f i c a t i o n o f a v i d i n by c a r b o x y m e t h y l c e l l u o s e c a t i o n exchange (CMC), g e l f i l t r a t i o n , m e t a l c h e l a t e i n t e r a c t i o n chromatography ( M C I C ) , a l i p h a t i c h y d r o p h o b i c i n t e r a c t i o n chromatography ( H I C ) , and P h e n y l -Sepharose i n t e r a c t i o n chromatography (PSIC) each r e s u l t e d i n c o n s i d e r a b l e i n c r e a s e i n a v i d i n p u r i t y . I n terms o f r e s i n c a p a c i t y , y i e l d s , and a v i d i n p u r i t y however , CMC i o n exchange was s u p e r i o r . A c o m p a r i s o n o f sodium d o d e c y l s u l f a t e 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 (SDS-PAGE) and n a t i v e p r o t e i n e l e c t r o p h o r e s i s p r o f i l e s gave c l e a r e v i d e n c e o f 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 between a v i d i n and lysozyme i n p a r t i a l l y p u r i f i e d a v i d i n p r e p a r a t i o n s . T h i s i n t e r a c t i o n may a l s o o c c u r between t h e n a t i v e p r o t e i n s i n t h e egg w h i t e , b u t has n o t been d e m o n s t r a t e d w i t h c e r t a i n t y . The m o l a r r a t i o o f a v i d i n t o a v a i l a b l e b i o t i n b i n d i n g s i t e s was e s t i m a t e d by 5 methods . F o r h i g h l y p u r i f i e d a v i d i n samples t h e h y d r o x y azo b e n z o i c a c i d (HABA) method p r o p o s e d by G r e e n (1965) was s u p e r i o r . A new method, u t i l i z i n g an i m m o b i l i z e d b i o t i n c o l u m n , w h i c h d i d n o t r e q u i r e e x t e n s i v e p u r i f i c a t i o n o f a v i d i n was found t o g i v e s i m i l a r r e s u l t s . F i n a l l y , a h i g h l y s e n s i t i v e a s s a y o f p r o t e i n s bound t o n i t r o c e l l u l o s e membranes was d e v e l o p e d w h i c h was c a p a b l e o f q u a n t i f y i n g as l i t t l e as 0 .12 fig o f p r o t e i n . Membrane bound p r o t e i n s were l a b e l e d w i t h p e r o x i d a s e v i a a b i o t i n - a v i d i n l i n k a g e as p r e v i o u s l y r e p o r t e d and bound p e r o x i d a s e a c t i v i t y was r e l a t e d t o i n i t i a l p r o t e i n c o n t e n t . The method was a p p l i c a b l e t o d e t e r m i n a t i o n o f t h e r e l a t i v e c o n c e n t r a t i o n s o f d i f f e r e n t p r o t e i n bands on W e s t e r n b l o t s o f e l e c t r o p h o r e s i s g e l s . TABLE OF CONTENTS A b s t r a c t T a b l e o f C o n t e n t s L i s t o f T a b l e s . L i s t o f F i g u r e s . Ac knowl edgements I . L i t e r a t u r e Review A v i d i n D i s t r i b u t i o n and B i o l o g i c a l F u n c t i o n . P h y s i c a l P r o p e r t i e s o f A v i d i n . B i o t i n - A v i d i n I n t e r a c t i o n . A v i d i n A s s a y s . A v i d i n A p p l i c a t i o n s . B i o t i n y l a t i o n . A v i d i n C o n j u g a t e d M a r k e r s . L o c a l i z a t i o n o f S p e c i f i c Groups w i t h A v i d i n . Immunoassays. Lysozyme Lysozyme Types Lysozyme A s s a y s A p p l i c a t i o n s o f Lysozyme i n t h e Food I n d u s t r y I s o l a t i o n Methods f o r Lysozyme E l e c t r o s t a t i c I n t e r a c t i o n Chromatography (EIC) I n f l u e n c e o f S a l t on E I C . H y d r o p h o b i c I n t e r a c t i o n Chromatography . C o n s o l i d a t i o n o f E I C and HIC E f f e c t s i n Chromatography . I I . S i m u l t a n e o u s I s o l a t i o n o f A v i d i n and Lysozyme from Egg Albumen. I n t r o d u c t i o n M a t e r i a l s and Methods R e a g e n t s . Egg Whi te P r e t r e a t m e n t . Enzyme A s s a y s . P r o t e i n A s s a y s E l e c t r o p h o r e s i s . I o n exchange I s o l a t i o n o f Lysozyme and A v i d i n S i m p l e x O p t i m i z a t i o n . A v i d i n B i n d i n g C a p a c i t y o f D u o l i t e C - 4 6 4 . Chromatography i n H i g h Ammonium S u l p h a t e C o n c e n t r a t i o n s . RESULTS S i m p l e x O p t i m i z a t i o n o f L y s o z y m e - A v i d i n S e p a r a t i o n . S t e p w i s e E l u t i o n . E l u t i o n L o o p i n g S m a l l P i l o t S c a l e T r i a l . C a p a c i t y o f D u o l i t e C-464 . P r o t e i n - D u o l i t e C-464 I n t e r a c t i o n . C o n c l u s i o n s I I I . S e c o n d a r y P u r i f i c a t i o n o f A v i d i n . I n t r o d u c t i o n . - V M a t e r i a l s and Methods . 68 R e a g e n t s . 68 CMC I o n Exchange . 6 8 G e l F i l t r a t i o n . 69 M e t a l C h e l a t e I n t e r a c t i o n Chromatography . 69 H y d r o p h o b i c I n t e r a c t i o n Chromatography . 70 C o m p a r i s o n o f Z e t a P o t e n t i a l . 70 R e s u l t s 71 CMC I o n E x c h a n g e . 71 G e l F i l t r a t i o n . 77 M e t a l C h e l a t e I n t e r a c t i o n Chromatography (MCIC) . 80 H y d r o p h o b i c I n t e r a c t i o n Chromatography ( H I C ) . 84 P h e n y l Sepharose I n t e r a c t i o n Chromotography 86 L y s o z y m e - A v i d i n I n t e r a c t i o n . 86 C o n c l u s i o n s 94 I V . S t o i c h i o m e t r y o f t h e B i o t i n - A v i d i n I n t e r a c t i o n 95 I n t r o d u c t i o n 95 M a t e r i a l and Methods 97 R e a g e n t s . 97 P r o t e i n A s s a y s 97 A v i d i n A s s a y s . 97 B i o t i n A s s a y . 98 B i o t i n - A v i d i n I n t e r a c t i o n on an I m m o b i l i z e d B i o t i n Column 99 P h e n y l a l a n i n e a s s a y . 100 R e s u l t s . 101 ANS D i s p l a c e m e n t by B i o t i n 101 P h e n y l a l a n i n e C o n t e n t 102 Column Method 106 D i s c u s s i o n . 106 C o n c l u s i o n s . 108 V . Q u a n t i t a t i o n o f P r o t e i n s I m m o b i l i z e d on N i t r o c e l l u l o s e Membranes u s i n g A v i d i n M e d i a t e d P e r o x i d a s e L a b e l l i n g . 109 I n t r o d u c t i o n 109 M a t e r i a l s and Methods . I l l R e a g e n t s . I l l M i c r o d o t t i n g " . 113 D i f f u s i o n B l o t t i n g o f PHAST G e l s . 113 W e s t e r n B l o t t i n g . 114 P e r o x i d a s e L a b e l l i n g o f I m m o b i l i z e d P r o t e i n s . 115 P e r o x i d a s e A c t i v i t y o f I m m o b i l i z e d , L a b e l l e d P r o t e i n s . 115 R e s u l t s and D i s c u s s i o n . 116 C h o i c e o f E l e c t r o n D o n o r . 116 M i c r o d o t P r o t e i n A s s a y . 117 A s s a y o f Wes tern B l o t P r o t e i n Bands . 120 B l o t t i n g o f PHAST G e l s . 123 C o n c l u s i o n s 125 R e f e r e n c e s 128 v i LIST OF TABLES. Table 1.1. Some important characteristics of avidin.. Table 1.2. Influence of various ions on the retention of cytochrome C and lysozyme on a strong cation exchange column. Chromatography was performed at pH 6.0 (Kopaciewicz et a l . , 1983). 23 Table 1.3. Influence of various ions on the retention of ovalbumin and soybean trypsin i n h i b i t o r on a strong anion exchange column. Chromatography was performed at ph 8.0 (Kopaciewicz et a l . , 1983) . 24 Table 2.1. Preliminary separations of lysozyme and avidin from egg white on a 7 mL column of Duolite C-464. 43 Table 2.2. Factor levels for simplex optimization and resulting avidin recovery and purity for lysozyme-avidin separation from egg white by Duolite C-464 chromatography. 45 Table 2.3. Results of 2-step elution for resolution of lysozyme and avidin fractions from egg white by Duolite C-464 chromatography on a 7 mL (1, 2) or a 170 mL (3) column. 48 Table 2.4. Results of lysozyme-avidin separation from egg white by Duolite C-464 chromatography with multi-cycle elution looping. 52 Table 2.5. E l e c t r o s t a t i c (B) and hydrophobic (C) interaction parameters for retention of avidin and lysozyme on Duolite C-464. Intercept (A) and R of multiple regression are also included. \ 61 Table 2.6. Retention volumes of lysozyme and avidin adsorbed from egg white onto Duolite C-46 4 and eluted with linear gradients of 4 d i f f e r e n t s a l t s . Columns were equilibrated with 0.1M NaP, pH 7.5 and a l l gradients were in the same buffer. 63 Table 3.1. Secondary chromatography of egg white protein fractions containing avidin and lysozyme on CMC. Avidin y i e l d and purity are reported as percentages. 73 Table 3.2. Retention of protein, avidin and lysozyme on hydrophobic chromatography columns with 0, 2, v i i 4, 6 , 8 or 10 c a r b o n a l i p h a t i c s i d e c h a i n s . 85 T a b l e 3 . 3 . Comparison o f f o u r c h r o m a t g r a p h i c methods f o r the secondary p u r i f i c a t i o n o f c r u d e a v i d i n . 88 T a b l e 4.1 S t o i c h i o m e t r y o f b i o t i n - a v i d i n i n t e r a c t i o n as de termined by t h r e e methods. 105 T a b l e 5.1 P e r o x i d a s e a c t i v i t y o f enzyme l a b e l e d n i t o c e l l u l o s e b l o t s o f p r o t e i n samples c o n t a i n i n g IgG heavy c h a i n , s e p a r a t e d on PHAST SDS e l e c t r o p h o r e s i s g e l s . O n l y the the t o t a l p r o t e i n c o n c e n t r a t i o n was v a r i e d between samples : 124 v i i i LIST OF FIGURES. Figure 1.1. The molecular structure of b i o t i n . Figure 1.2. Lysozyme retention data f i t t e d to an ion exchange model (Equation 3). Lysozyme was eluted from a cationic HPIEC column (Parente and Wetlaufer ,1984) . 22 Figure 2.1. Diagram of a p i l o t plant apparatus for i s o l a t i o n of avidin and lysozyme from egg white by Duolite C-46 4 chromatography. 39 Figure 2.2. Elution p r o f i l e of adsorbed proteins from Duolite C-464 column (170 mL) by i s o c r a t i c elution using 0.5M sodium phosphate buffer at pH 8. 42 Figure 2.3. Elution p r o f i l e of adsorbed egg white proteins from Duolite C-464 (7 mL resin) using a 5 step sodium chloride elution gradient. 46 Figure 2.4. Elution p r o f i l e of adsorbed egg white proteins from Duolite C-464 (170 mL resin) using a two step elution process. 49 Figure 2.5. P r o f i l e of adsorbed egg white proteins eluted from a Duolite C-464 column (170 mL) by the elution looping process. Five cycles of egg white application and lysozyme elution were followed by a single elution of avidin. 51 Figure 2.6. SDS-PAGE gel of lysozyme and avidin fractions recovered from egg white with 8 cycles of egg white application and lysozyme elution followed by a single avidin elution: A/ untreated egg white; B/ 1st cycle eluted egg white; C/ 4th cycle eluted egg white; D 8th cycle eluted egg white; E/ 1st cycle lysozyme; F/ 3rd cycle lysozyme; G/ 6th cycle lysozyme; H/ 8th cycle lysozyme; 1/ Avidin fraction. 54 Figure 2.7. SDS-PAGE gel of lysozyme and avidin fractions recovered from egg white with 16 cycles of egg white application and lysozyme elution followed by a single avidin elution: A/ untreated egg white; B/ 1st cycle eluted egg white; C/ 8th cycle eluted egg white; D/ 16th cycle eluted egg white; / 1st cycle lysozyme; F/ 8th cycle lysozyme; G/ 16th cycle lysozyme; H/ avidin fraction. 5 6 Figure 2.8. Relationship between the purity of the ix recovered a v i d i n f r a c t i o n s and the concentration of accumulated a v i d i n on the D u o l i t e C-464 column. 57 Figure 2.9. P l o t of logarithmic c a p a c i t y f a c t o r s of lysozyme (A) and a v i d i n (•) against the logarithm of ammonium s u l f a t e m o l a l i t y . E l u t i o n was i s o c r a t i c with 25 mM sodium phosphate b u f f e r , pH 7.3, at d i f f e r e n t ammonium s u l f a t e concentrations. 60 Figure 3.1. Chromatography of a v i d i n and lysozyme r i c h egg white f r a c t i o n s on CMC i n 0.02M T r i s - H C l , pH 9.0: A280 ( — ) ; ammonium carbonate m o l a r i t y x 100 ( ) ; a v i d i n a c t i v i t y ( ) ; and lysozyme a c t i v i t y (• • ••.). 72 Figure 3.2. CMC chromatography of an egg white p r o t e i n f r a c t i o n r i c h i n a v i d i n and lysozyme. 75 Figure 3.3. Adsorption of a v i d i n to CMC at d i f f e r e n t pH's. 76 Figure 3.4. Gel f i l t r a t i o n of egg white f r a c t i o n s r i c h i n a v i d i n and lysozyme on Sephadex G-75. 78 Figure 3.5. T e r t i a r y p u r i f i c a t i o n of a v i d i n on Sephadex G-75. A v i d i n f r a c t i o n s from a primary separation of egg white on D u o l i t e C-464 were chromatographed next on CMC before a p p l i c a t i o n to the g e l f i l t r a t i o n column. 79 Figure 3.6. SDS-PAGE of a v i d i n f r a c t i o n before g e l f i l t r a t i o n (A), o v o t r a n s f e r i n standard (B,C), and the a v i d i n f r a c t i o n from the g e l f i l t r a t i o n s eparation (D,). 81 Figure 3.7. MCIC separation of egg white f r a c t i o n s c o n t a i n i n g lysozyme and a v i d i n : A280 ( — ) ; pH ( ) ; a v i d i n (-•••0. 82 Figure 3.8. SDS-PAGE p r o f i l e s of f r a c t i o n s separated from egg white by D u o l i t e C-464 chromatography and MCIC chromatography: A,B/ untreated egg white; C,H/ a v i d i n f r a c t i o n from D u o l i t e C-464; D,E/ peak 2 from MCIC; F,G/ peak 1 from MCIC._83 Figure 3.9. Phenyl-Sepharose separation of a v i d i n and lysozyme: A280 ( — ) ; s a l t ( ); a v i d i n (...). 87 Figure 3.10. D u o l i t e C-464 chromatography of p u r i f i e d lysozyme i n sodium phosphate b u f f e r . pH 7.9: A280 (—); sodium c h l o r i d e ( . . . ) . 90 F i g u r e 3 . 1 1 . A c o m p a r i s o n o f t h e r e l a t i v e c h a r g e o f o i l d r o p l e t s c o a t e d w i t h p r o t e i n from lysozme peak I (o) and lysozyme peak I I (•) a t d i f f e r e n t p H ' s . 91 F i g u r e 3 . 1 2 . E l e c t r o p h o r e s i s o f a v i d i n and ly sozyme: A / a v i d i n f r a c t i o n from CMC ch romat ograp h y on n a t i v e p r o t e i n PAGE; B / lysozyme on n a t i v e p r o t e i n PAGE; C , E / h i g h l y p u r i f i e d a v i d i n on n a t i v e PAGE; D / egg w h i t e w i t h added a v i d i n on n a t i v e PAGE and F / sample i d e n t i c a l t o A on SDS-PAGE. 9 3 F i g u r e 4 . 1 . D e c r e a s e i n f l u o r e s c e n c e o f 1 , 8 - A N S - a v i d i n complex when t i t r a t e d w i t h b i o t i n 103 F i g u r e 4 . 2 . Dependence o f t h e 256-259nm i n t e r p e a k d i s t a n c e o f t h e second d e r i v a t i v e s p e c t r u m o f a v i d i n on t h e c o n c e n t r a t i o n o f added NAPhe 104 F i g u r e 5 . 1 . D i f f u s i o n b l o t t i n g o f p r o t e i n bands from PHAST e l e c t r o p h o r e s i s g e l s onto n i t r o c e l l u l o s e membranes. 113 F i g u r e 5 . 2 . A s s a y o f enzyme l a b e l e d IgG " m i c r o d o t s " on n i t r o c e l l u l o s e by p e r o x i d a s e a c t i v i t y : means ± s t a n d a r d d e v i a t i o n o f t r i p l i c a t e d e t e r m i n a t i o n s . 118 F i g u r e 5 . 3 . A s s a y o f enzyme l a b e l e d B - L g on n i t r o c e l l u l o s e by p e r o x i d a s e a c t i v i t y : means ± s t a n d a r d d e v i a t i o n o f t r i p l i c a t e d e t e r m i n a t i o n s . 119 F i g u r e 5 . 4 . P e r o x i d a s e a c t i v i t y o f enzyme l a b e l e d . W e s t e r n b l o t s o f B - L g e l e c t r o p h o r e s i s b a n d s . AA420 was d e t e r m i n e d o v e r 30s , 60s o r 90s p e r i o d s o f i n c u b a t i o n . 121 F i g u r e 5 . 5 . A s s a y o f r e l a t i v e c o n c e n t r a t i o n o f B - L g and IgG bands on Wes tern b l o t s (12 cm x 12 cm g e l s ) by a c t i v i t y o f p e r o x i d a s e l a b e l s . 122 F i g u r e 5 . 6 . A s s a y o f r e l a t i v e c o n c e n t r a t i o n s o f B - L g and IgG bands on PHAST d i f f u s i o n b l o t s (4 cm x 4 cm g e l s ) by a c t i v i t y o f p e r o x i d a s e l a b e l s . 126 x i ACKNOWLEDGEMENTS I wish to thank my s u p e r v i s o r , D r . S. Nakai f o r h i s i n v a l u a b l e guidance and support throughout my P h . D . program. I would a l s o l i k e to thank the o ther members o f my r e s e a r c h committee , D r . W.D. Powr ie , D r . B . J . S k u r a , D r . J . S im, and D r . K . M . Cheng f o r t h e i r h e l p f u l a d v i c e and comments on my r e s e a r c h program and t h e s i s . T h i s r e s e a r c h was supported i n p a r t by grant s from the B r i t i s h Columbia S c i e n c e C o u n c i l and the Canadian Egg M a r k e t i n g Agency. In a d d i t i o n I wish to acknowledge the k i n d i n t e r e s t and support of B r o o k s i d e L a b o r a t o r i e s , A b b o t s f o r d , B . C . 1 I . L i t e r a t u r e Review  AVIDIN A v i d i n i s a p r o t e i n w h i c h e x h i b i t s h i g h a f f i n i t y f o r t h e v i t a m i n b i o t i n . I n f a c t , t h e a v i d i n - b i o t i n i n t e r a c t i o n i s c h a r a c t e r i z e d by one o f t h e lowes t d i s s o c i a t i o n c o n s t a n t s known f o r a n o n - c o v a l e n t l i g a n d -p r o t e i n i n t e r a c t i o n ( G r e e n , 1963a) . T h i s h i g h l y s p e c i f i c a f f i n i t y i s t h e b a s i s f o r a h e t e r o g e n e o u s group o f b i o c h e m i c a l t e c h n i q u e s w h i c h u t i l i z e a v i d i n . These i n c l u d e h i s t o c h e m i c a l l o c a l i z a t i o n t e c h n i q u e s f o r use w i t h e l e c t r o n and l i g h t m i c r o s c o p y , s p e c i f i c immunoassays and even p o t e n t i a l p h a r m a c o l o g i c a l a p p l i c a t i o n s ( W i l c h e k and B a y e r , 1984) . D i s t r i b u t i o n and B i o l o g i c a l F u n c t i o n . A v i d i n was f i r s t d e t e c t e d as a f a c t o r i n raw hen egg albumen w h i c h c a u s e d an u n u s u a l d e r m a t i t i s i n r a t s when f e d as t h e i r s o l e p r o t e i n s o u r c e (Boas , 1927) . Subsequent s t u d i e s e s t a b l i s h e d t h a t the symptoms were due t o d e p r i v a t i o n o f a v i t a m i n , t h e n c a l l e d v i t a m i n ' H ' b u t now known as b i o t i n (Gyorgy e t a l . , 1940) . A v i d i n , i t was f o u n d , bound b i o t i n and made i t u n a v a i l a b l e t o t h e o r g a n i s m . S i n c e i t s d i s c o v e r y , a v i d i n has been found t o o c c u r w i d e l y i n the eggs o f a v i a n and o t h e r o v i p a r o u s 2 v e r t e b r a t e s p e c i e s . F r e q u e n t l y i t i s a s e c r e t o r y o v i d u c t p r o t e i n i n d u c e d by s t e r i o d hormones ( H e r t z e t a l . , 1943 ; K o r p e l a e t a l . , 1981) . I n c h i c k e n s , a v i d i n has a l s o been found i n c o n n e c t i o n w i t h t i s s u e damage and has been termed a p r o g e s t e r o n e independent s e c r e t o r y p r o d u c t i n a r e a s o f i n f l a m m a t i o n . The s y s t e m a t i c appearance o f a v i d i n i n r e s p o n s e t o t i s s u e damage re sembles an a c u t e phase r e s p o n s e i n t h a t t h e l e v e l s d e c l i n e and d i s a p p e a r w i t h i n a few days ( E l o e t a l . , 1979a) . The a c t u a l r o l e and c e l l u l a r o r i g i n o f t h e i n f l a m m a t i o n i n d u c e d a v i d i n i n t i s s u e s have n o t been d e t e r m i n e d , a l t h o u g h a n t i m i c r o b i a l and enzyme i n h i b i t o r r o l e s have been s u g g e s t e d ( K o r p e l a , 1984; E l o e t a l . , 1979b) . The b i o l o g i c a l r o l e o f a v i d i n i n t h e a v i a n egg has l i k e w i s e no t been c l e a r l y e s t a b l i s h e d . An a n t i m i c r o b i a l r o l e has been s u g g e s t e d by s e v e r a l a u t h o r s ( G r e e n , 1975; Banks e t a l . , 1986) . More r e c e n t l y some e x p e r i m e n t a l e v i d e n c e has become a v a i l a b l e w h i c h t ends t o s u p p o r t t h i s v i e w . A v i d i n was found t o b i n d t o the o u t e r s u r f a c e o f many b a c t e r i a , b o t h Gram n e g a t i v e s and some Gram p o s i t i v e s , w i t h o u t i n h i b i t i n g t h e a v i d i n ' s a f f i n i t y f o r b i o t i n . I n t h e case o f E s c h e r i c h i a c o l i K12 a major p o r i n p r o t e i n (OmpF a n d / o r OmpC) o f t h e o u t e r membrane was found t o be t h e p r i n c i p a l r e c e p t o r . The s u g g e s t i o n has been made t h a t a v i d i n so p l a c e d would be i d e a l l y s i t u a t e d t o d e p r i v e t h e b a c t e r i a l c e l l o f b i o t i n and t h u s i n h i b i t growth (Banks e t a l . , 1986) . 3 The d i s c o v e r y o f a b i o t i n b i n d i n g p r o t e i n o f s e v e r a l S t r e p t o m y c e s s p e c i e s i n d i r e c t l y r e i n f o r c e d t h e i d e a o f a n t i m i c r o b i a l a v i d i n . S t r e p t a v i d i n has a c l e a r l y e s t a b l i s h e d a n t i m i c r o b i a l a c t i v i t y and i s p r o d u c e d c o n c u r r e n t l y w i t h a p r o t e i n ( s t r a v i d i n ) w h i c h i n h i b i t s b i o t i n s y n t h e s i s ( C h a i e t e t a l . , 1963) P h y s i c a l P r o p e r t i e s o f A v i d i n . Some o f t h e i m p o r t a n t p h y s i c a l p r o p e r t i e s o f a v i d i n a r e p r e s e n t e d i n T a b l e 1 . 1 . A v i d i n i s a b a s i c g l y c o p r o t e i n w i t h an i s o e l e c t r i c p o i n t o f 10 .0 ( G r e e n , 1975; W o o l l e y and L o n g s w o r t h , 1942) . I n i t s n a t i v e form i t i s a t e t r a m e r w i t h s u b u n i t s o f 15,600 d a l t o n s . S u b u n i t s c a n be d i s a s s o c i a t e d i n 0 .1M H C 1 , 0 .1M sodium d o d e c y l s u l f a t e , o r 6M g u a n i d i n e - H C l , w i t h c o m p l e t e l o s s o f b i o t i n b i n d i n g a c t i v i t y ( G r e e n , 1963c) . The p r i m a r y s t r u c t u r e o f a v i d i n has been r e p o r t e d (DeLange and Huang, 1971) . S u b u n i t s c o n t a i n 128 amino a c i d r e s i d u e s e a c h . T r y p t o p h a n c o n t e n t i s h i g h (4 r e s i d u e s / s u b u n i t ) b u t t h r e o n i n e c o n t e n t i s e x c e p t i o n a l l y h i g h a t 19-20 r e s i d u e s / s u b u n i t . T h e r e i s a s i n g l e i n t r a m o l e c u l a r d i s u l f i d e bond between c y s - 5 and c y s - 8 3 . The c a r b o h y d r a t e m o i e t y o f a v i d i n i s a s i n g l e o l i g o s a c c h a r i d e p e r s u b u n i t l i n k e d by asp-17 on t h e p r o t e i n t o an a c e t y l g l u c o s a m i n e r e s i d u e o f t h e o l i g o s a c c h a r i d e . A t one t i m e t h e s u g g e s t i o n was made t h a t a v i d i n and lysozyme might be c l o s e l y r e l a t e d i n t h e e v o l u t i o n a r y 4 T a b l e 1 .1 . Some important c h a r a c t e r i s t i c s o f a v i d i n . m o l e c u l a r weight 68,000 d a l t o n s s u b u n i t M. W. 15,600 d a l t o n s K D ( a v i d i n - b i o t i n complex) 1 0 ~ 1 5 E 1 % @ 280nm 15.4 mannose/subunit 4-5 g l u c o s a m i n e / s u b u n i t 3 t r y p t o p h a n / s u b u n i t 4 c o n c e n t r a t i o n i n egg white 0.05 g / L i s o e l e c t r i c p o i n t 10. 0 5 sense of having arisen from a common ancestor, based on the similar amino acid compositions of lysozyme and avidin and a weak binding of b i o t i n by lysozyme (Green, 1968). However, comparison of the amino acid sequences showed only 12% homology, only s l i g h t l y more than would be expected on a random basis. Avidin i s very soluble i n water and s a l t solutions, although i t can be c r y s t a l l i z e d from strong s a l t solutions between pH 5 and 7 (Gatti et a l . , 1984). It is also remarkably stable over a wide range of pH and temperature (Wei and Wright, 1964). B i o t i n binding a c t i v i t y i s not lost between pH 2 and 13. Donovan and Ross (1973) demonstrated by calorimetry, that avidin was inactivated i n an endothermic t r a n s i t i o n at 85°C. In contrast, the comparable t r a n s i t i o n of the avidin-biotin complex was at 132°C. Resistance of avidin to proteolytic hydrolysis has also been noted. Green (1975) reported that avidin was not inactivated by trypsin or Pronase. Furthermore, b i o t i n bound to avidin i s not released by the proteolytic enzymes of the mammalian digestive t r a c t . Biotin-Avidin Interaction. The binding of avidin by b i o t i n i s so strong as to make determination of the disassociation constant (K D) d i f f i c u l t (Launer and Fraenkel-Conrat, 1951). Green (1963a) eventually calculated a K D of approximately 10~ 1 5 M based on the rate of exchange of bound [ 1 4C] - labelled b i o t i n with an excess of unlabelled b i o t i n . This constant was for the exchange of the f i r s t of 4 b i o t i n molecules bound per avidin molecule but subsequent studies indicated that the binding of each b i o t i n molecule was energetically independent (Green, 1975). The involvement of tryptophan in the b i o t i n binding s i t e was f i r s t suggested by u l t r a v i o l e t spectroscopy studies which showed a red s h i f t of 7nm for the 226nm tryptophan peak of avidin when avidin was bound (Green, 1963b). This involvement was later confirmed by studies in which N-bromosuccinimide was used to oxidize tryptophan. When an average of 2 tryptophans out of four were oxidized, b i o t i n binding a c t i v i t y was completely lost. Also, i f b i o t i n was complexed with avidin, the tryptophan residues were almost e n t i r e l y resistant to oxidation by N-bromosuccinimide (Green and Ross, 1968). Apparently an amino group i s also essential to the binding of b i o t i n - to avidin. Treatment of avidin with 1-fluoro-2,4 dinitrobenzene in which an average of one dinitrobenzene group per subunit was added resulted in complete loss of b i o l o g i c a l a c t i v i t y (Green, 1975). The carbohydrate moiety on the other hand does not play a role in b i o t i n binding, since oxidation of mannose residues with periodate had l i t t l e effect on a c t i v i t y . As was mentioned above, the binding of b i o t i n to avidin i s very strong. In fact, r e-equilibration of bound and free b i o t i n i s low enough to be ignored in a l l but the most long term experiments. In energy terms, the association o f a v i d i n and b i o t i n i s spontaneous and e x o t h e r m i c w i t h a AH o f - 2 1 . 5 k c a l / m o l e and a AG o f - 2 0 . 4 k c a l / m o l e (Green , 1963a, 1963b; G r e e n and Toms, 1973) . E x t e n s i v e s t u d i e s w i t h b i o t i n a n a l o g s have y i e l d e d q u i t e a b i t o f i n f o r m a t i o n about which s t r u c t u r a l d e t a i l s o f b i o t i n a r e i m p o r t a n t f o r b i n d i n g ( F i g u r e 1.1) ( G r e e n , 1975) . To summarize a g r e a t d e a l o f work i n a few l i n e s , the f o l l o w i n g a r e t h e g e n e r a l c o n c l u s i o n s o f t h e s e s t u d i e s . 1. The c a r b o n y l group o f b i o t i n i s no t i n v o l v e d i n b i n d i n g . 2. The i m i d a z o l i d o n e r i n g i s much more i m p o r t a n t t h a n t h e t h i o p h a n r i n g f o r b i n d i n g t o t h e a c t i v e s i t e o f a v i d i n . 3. A l t h o u g h b r e a k i n g t h e i m i d a z o l i d o n e r i n g s i g n i f i c a n t l y d e c r e a s e s the b i n d i n g s t r e n g t h , some b i n d i n g s t i l l o c c u r s as l o n g as the amino groups a r e no t removed. T h e r e i s a d e c r e a s e i n AG o f about 5.6 k c a l / m o l e i f e i t h e r n i t r o g e n i s r e p l a c e d w i t h oxygen . A p p a r e n t l y n i t r o g e n s e r v e s as a h y d r o g e n bond donor t o a c c e p t o r s i n t h e a c t i v e s i t e o f a v i d i n . 4. The l e n g t h o f t h e a l k y l c h a i n a f f e c t s the b i n d i n g s t r e n g t h . AG d e c r e a s e s as the c h a i n i s s h o r t e n e d , by about 0 .7 k c a l / m o l e / m e t h y l e n e g r o u p . The h i g h energy o f t h e b i o t i n - a v i d i n bond might sugges t a c o v a l e n t bond . The e v i d e n c e a g a i n s t t h i s i s o f two t y p e s . F i r s t , b i o t i n can be r e l e a s e d from a v i d i n by 6M 8 H ^ ^ *NH ( C H 2 ) 4 C 0 0 H Figure 1 . 1 . The molecular structure of b i o t i n . 9 guanidinium-HCl, 0. 1M sodium dodecyl sulfate, or by autoclaving. Secondly, analog binding studies have shown extensive non-covalent interactions and the sum of these interactions approach the strength of the avidin-biotin bond. Avidin Assays. The e a r l i e s t methods of avidin determination were microbiolgical, based on the binding of b i o t i n i n b a c t e r i a l nutrient medium and the subsequent growth i n h i b i t i o n of Lactobacillus arabinosus, a species with a s t r i c t requirement for b i o t i n (Hertz, 1943). This remains the most sensitive method available, measuring avidin concentrations as low as 2 ng/mL, but i s extremely time consuming. Methods u t i l i z i n g the binding of [ 1 4C] b i o t i n are also very sensitive but expensive i n terms of reagents and time (Green, 1963a). Green (1965) described a spectrophotometry assay based on the binding of an anionic dye, 2-(4'hydroxyazobenzene) benzoic acid. Binding of the dye to avidin resulted in a new absorbance band at 500nm. Furthermore, dye binding was reversed by binding of b i o t i n to the avidin. Back-titration of the avidin-dye complex with b i o t i n allowed determination of avidin concentrations down to 20 fig/mL. One of the fluroescent bands of avidin can also be used for assay purposes. The 290nm/350nm exitation/emission band due to tryptophan i s largely quenched by the binding of 10 b i o t i n . T i t r a t i o n with b i o t i n to an endpoint of minimum fluorescence gives a measure of avidin concentration (Lin and Kirsh, 1977). This method i s reportedly capable of estimating b i o t i n concentrations as low as 1.5 fig/mL. A drawback of the method i s the fact that the presence of other fluorescing compounds such as tryptophan containing proteins w i l l interfere with the resolution of the method. A fluorescent dye binding method of assaying avidin has also been described (Mock et a l . , 1985). 2-Anilino naphthalene-6-sulfonate exhibited a large increase in fluorescence in the presence of avidin. Addition of b i o t i n resulted i n a proportional reduction i n fluorescence. Avidin Applications. The avidin-biotin interaction has proved useful because of the twin facts of a highly s p e c i f i c interaction over a wide range of conditions and the r e l a t i v e ease with which b i o t i n can be covalently attached to most important macromolecules. Before considering the types of applications for which avidin has been used, the reactions by which b i o t i n may be attached to various functional groups and the means of conjugating avidin to other proteins w i l l be considered b r i e f l y . B i o t i n y l a t i o n . B i o t i n y l derivatives have been developed for selective b i o t i n y l a t i o n of a variety of functional groups found i n proteins and other b i o l o g i c a l macromolecules. This 11 variety allows a certain amount of f l e x i b i l i t y i n the b i o t i n y l a t i o n of many molecules. Thus destruction of functional groups known to be important to b i o l o g i c a l a c t i v i t y can be avoided. A l l of the derivatives described below attach b i o t i n to the respective functional group by way of the b i o t i n carboxylic acid group. The following are some examples of commonly used reagents. Amino reagents. Biotinyl-p-nitrophenyl ester and biotinyl-N-hydroxysuccinimide ester both react with free amino groups such as the epsilon amino group of lysine or the alpha amino terminus of a protein. These reagents have been used very frequently for the b i o t i n y l a t i o n of immunoglobulins (Bayer and Wilchek, 1974; 1977). Carboxyl and sugar reagents. Sugars have been biotinyl a t e d by oxidizing hydroxyl groups to aldehydes with periodate or with s p e c i f i c oxidases, followed by reaction with b i o t i n hydrazide (Bayer and Wilchek, 1980). In the case of carbonyls, carbodiimides may be added to the above reaction to act as coupling agents. Methods are also available for the b i o t i n y l a t i o n of t h i o l s , imidazoles, and phenols (Wilchek and Givol, 1977; Bayer and Wilchek, 1980). Avidin Conjugated Markers. Avidin conjugated to f e r r i t i n , horseradish peroxidase, fluorescein, etc. i s commercially available. Protein conjugates can be synthesized r e l a t i v e l y easily by reaction with glutaraldehyde or by reductive alkylation 12 (Hei tzman and R i c h a r d s , 1974; Bayer e t a l . , 1976) . F l u o r e s c e i n c o n j u g a t i o n may be a c h i e v e d by r e a c t i o n w i t h f l u o r e s c e i n i s o c y a n a t e (Heggeness and A s h , 1977) . F l u o r e s c e i n c o n j u g a t e s were s e p a r a t e d from r e a c t a n t s by u l t r a c e n t r i f u g a t i o n o r by g e l f i l t r a t i o n . L o c a l i z a t i o n o f S p e c i f i c Groups w i t h A v i d i n . The f i r s t r e p o r t e d use o f t h e a v i d i n - b i o t i n complex i n b i o c h e m i c a l s t u d i e s i n v o l v e d t h e b i n d i n g o f a v i d i n - f e r r i t i n c o n j u g a t e t o b i o t i n y l a t e d a n t i b o d i e s f o r the l o c a l i z a t i o n o f a n t i g e n s on t h e s u r f a c e o f e r y t h r o c y t e s (He i t zman and R i c h a r d s , 1974) . F e r r i t i n was t h e n l o c a t e d by means o f e l e c t r o n m i c r o s c o p y . T h e r e were two major advantages t o t h i s p r o c e d u r e o v e r d i r e c t c o n j u g a t i o n o f f e r r i t i n t o a n t i b o d i e s . The b i o t i n y l a t i o n r e a c t i o n was m i l d enough n o t t o i n t e r f e r e w i t h t h e b i o l o g i c a l a c t i v i t y o f t h e a n t i b o d y . A l s o , b i o t i n i s much l e s s b u l k y t h a n f e r r i t i n and a t t a c h m e n t o f s e v e r a l b i o t i n r e s i d u e s p e r a n t i b o d y was f e a s i b l e , r e s u l t i n g i n an a m p l i f i c a t i o n e f f e c t . Immunoassays. A v i d i n immunoassays have been b a s e d on the b i o t i n y l a t i o n o f m o n o c l o n a l a n t i b o d i e s w h i c h a r e i n t u r n r e a c t e d w i t h s p e c i f i c a n t i g e n s , f o l l o w e d by b i n d i n g o f a v i d i n c o n j u g a t e d m a r k e r s . The t e c h n i q u e s a r e o f t e n s u p e r i o r t o c o n v e n t i o n a l f l u o r e s c e i n o r enzyme l i n k e d immunoassays because t h e r e i s g e n e r a l l y l e s s l o s s o f a n t i b o d y a c t i v i t y t h a n i n methods r e q u i r i n g d i r e c t i m m u n o g l o b u l i n - p r o t e i n 13 c o n j u g a t i o n and because o f i n c r e a s e d s e n s i t i v i t y . S e n s i t i v i t y a m p l i f i c a t i o n was a t t r i b u t e d t o t h e f a c t t h a t e a c h i m m u n o g l o b u l i n may b i n d s e v e r a l b i o t i n s and t h e r e f o r e s e v e r a l marker m o l e c u l e s . The a v i d i n a m p l i f i e d immunoassay c a n be u s e d t o a s s a y any compound f o r w h i c h a s p e c i f i c a n t i b o d y i s a v a i l a b l e . LYSOZYME Lysozymes (EC 3 . 2 . 1 . 1 7 ) a r e d e f i n e d as 1,4 b e t a - N -a c e t y l - m u r a m i d a s e s c l e a v i n g t h e g l y c o s i d i c bond between t h e C - l o f t h e N - a c e t y l m u r a m i c a c i d and t h e C-4 o f N -a c e t y l g l u c o s a m i n e i n t h e b a c t e r i a l p e p t i d o g l y c a n ( J o l i e s and J o l l e s , 1984) . They have been c a l l e d u b i q u i t o u s because t h e y have been found t o be p r o d u c e d by v a r i o u s a n i m a l , p l a n t , i n s e c t and f u n g a l c e l l s . C e r t a i n o t h e r c h a r a c t e r i s t i c s have a l s o l o n g been a s s o c i a t e d w i t h l y s o z y m e s . Lysozymes a r e g e n e r a l l y (1) b a s i c p r o t e i n s ; (2) o f low m o l e c u l a r we ight (~15000 d a l t o n s ) ; (3) s t a b i l e a t a c i d i c pH a t f a i r l y h i g h t e m p e r a t u r e ; (4) l a b i l e a t a l k a l i n e p H ; (5) a b l e t o l y s e a s u s p e n s i o n o f M i c r o c o c c u s l y s o d e i k t i c u s : and (6) c a p a b l e o f a c t i n g on s u b s t r a t e t o l i b e r a t e r e d u c i n g s u g a r s and amino s u g a r s ( J o l l e s , 1964) . These a n c i l l i a r y c h a r a c t e r i s t i c s a r e however ,no l o n g e r i n c l u d e d i n t h e f o r m a l d e f i n i t i o n o f l y sozymes because o f t h e subsequent d i s c o v e r y o f enzymes h a v i n g t r u e lysozyme a c t i v i t y b u t o t h e r a t y p i c a l c h a r a c t e r i s t i c s ( L i e and S y e d , 1986) . 14 Lysozyme Types The most commonly encountered lysozyme i s t h a t o f hen egg w h i t e . T h i s i s the o r i g i n a l o f the c - t y p e lysozymes. I t i s one o f the most h i g h l y s t u d i e d o f p r o t e i n s and has the d i s t i n c t i o n o f be ing the f i r s t p r o t e i n f o r which complete x -ray c r y s t a l l o g r a p h i c a n a l y s i s o f 3 - d i m e n s i o n a l s t r u c t u r e was completed (Blake et a l , 1965). A l though c - t y p e lysozymes have been i s o l a t e d from other a v i a n eggs (Kaneda, et a l . , 1969 ; L a Rue and Speck, 1970), human m i l k ( J o l l e s and J o l l e s , 1971), and bovine stomach (Dobson et a l . , 1984) to name a few, s e v e r a l lysozymes o f v e r y d i f f e r e n t p h y s i c a l c h a r a c t e r i s t i c s have a l s o been d e s c r i b e d . The f i r s t o f these unusua l lysozymes to be r e p o r t e d was the goose egg white lysozyme (g-type) (Dianoux and J o l l e s , 1967). G-type lysozymes have a m o l e c u l a r weight around 21,000 d a l t o n s and do not c r o s s r e a c t i m m u n o l o g i c a l l y w i t h c - t y p e lysozymes. S t r u c t u r a l l y u n r e l a t e d bac ter iophage (T4) lysozymes , p l a n t lysozymes , and a funga l lysozyme have a l s o been d e s c r i b e d (Inouye et a l . , 1970; Smith et a l . , 1955 . , Fouche and Hash, 1978) Lysozyme Assays By f a r the most p o p u l a r method f o r d e t e r m i n a t i o n of lysozyme a c t i v i t y i s the t u r b i d i m e t r i c assay of M.  l v s o d e i k t i c u s l y s i s ( J o l l e s et a l . , 1965). Much more s e n s i t i v e radioimmunoassays ( Y u z u r i h a et a l . , 1978, Thomas et a l . , 1981) and a f l u o r o m e t r i c method w i t h a f l u o r e s c e n t p e p t i d o g l y c a n (Maeda, 1980) have a l s o been d e s c r i b e d . 15 A p p l i c a t i o n s o f Lysozyme i n the Food I n d u s t r y . The a n t i m i c r o b i a l a c t i v i t y o f lysozyme (a lmost e x c l u s i v e l y c h i c k e n egg lysozyme) has been e x p l o i t e d somewhat by t h e f o o d i n d u s t r y and may have p o t e n t i a l f o r much w i d e r u s e . I n E u r o p e , lysozyme has been e x t e n s i v e l y s t u d i e d and u s e d t o p r e v e n t a d e f e c t known as " l a t e b l o w i n g " o r " l a t e gas" i n h a r d c h e e s e s , p a r t i c u l a r i l y o f Edam and Gouda t y p e s . L a t e b l o w i n g i s c a u s e d by t h e g e r m i n a t i o n and growth o f c l o s t r i d i a l s p o r e s , e s p e c i a l l y o f t h e s p e c i e s C l o s t r i d i u m t y r o b u t y r i c u m , w h i c h a p p a r e n t l y o r i g i n a t e i n s i l a g e f e d t o d a i r y c a t t l e ( W a s s e r f a l l and T e u b e r , 1979) . Lysozyme c o n c e n t r a t i o n s o f 500 U / m l (~10 fig egg w h i t e lysozyme/mL) k i l l e d 99% o f 5 x 1 0 5 r e s t i n g v e g e t a t i v e c e l l s o f C . t y r o b u t r y c i u m w i t h i n 24 h o u r s a t 2 5 ° C . Spores were c o m p l e t e l y r e s i s t a n t b u t s p o r e o u t g r o w t h was d e l a y e d 24 h o u r s i n t h e p r e s e n c e o f l y s o z y m e , a p p a r e n t l y due t o d e s t r u c t i o n o f newly formed v e g e t a t i v e c e l l s . I n a c t u a l h a r d cheese p r o d u c t i o n lysozyme may be u s e d a t a l e v e l o f 1-2 g / l O O L o f m i l k ( B r i k k j a e r e t a l . , 1982) . More t h a n 99% o f t h e lysozyme added t o t h e m i l k was p r e c i p i t a t e d w i t h the c a s e i n and removed w i t h the c u r d f r a c t i o n . Lysozyme a t t h e s e l e v e l s d i d n o t a d v e r s e l y a f f e c t cheese q u a l i t y . A l s o lysozyme was no t i n a c t i v a t e d by the h e a t t r e a t m e n t o f m i l k ( 5 0 ° C f o r 20 m i n ; 8 0 ° C f o r 10 m i n ; o r 9 0 ° C f o r 1 m i n ) , n o r was i t i n f l u e n c e d by 1-5% N a C l ( L o d i e t a l . , 1983) . 16 Lysozyme has been shown to be an effective substitute for nitr a t e in prevention of late gas. The major obstacle to further use for this purpose has been reported to be the high cost of lysozyme (Banks et a l . , 1986). There have been numerous references to lysozyme use as a food preservative in the food science l i t e r a t u r e in Japan, where lysozyme usage i s much more extensive than in North America. For example, lysozyme has been used experimentally to extend the shelf l i f e of potato salad (Nakagawa and Maeshige, 1980), cured sausages (Akashi, 1970, 1971), and sake (Yajima et a l . , 1971). Japanese patents have been granted on processes u t i l i z i n g lysozyme to increase shelf l i f e of fresh vegetables, f r u i t , f i s h , meat, marine products, sake, and a dried milk product (Kanebo Ltd., 1973; E i s a i Co. Ltd., 1971, 1972; Morinaga Milk Industry, 1970). Lysozyme has been reported to effect major reduction in the microbial load of a variety of spices when added at a concentration of 0.1% and incubated for 2 hours at room temperature. For example, the t o t a l plate count of black pepper on nutrient agar was decreased from 40xl0 9 to 5.0xl0 3 cfu/mL (Roberts and Kruger, 1984). Isolation Methods for Lysozyme The c l a s s i c procedure for the i s o l a t i o n of lysozyme from egg albumin begins with dire c t p r e c i p i t a t i o n at pH 9.5 i n the presence of 5% NaCl (Alderton and Fevold, 1964). Lysozyme recoveries ranged from 60-80%. Further p u r i f i c a t i o n was often achieved by s e r i a l r e s o l u b i l i z a t i o n 17 and r e - c r y s t a l l i z a t i o n s t e p s . As w e l l as b e i n g v e r y t i m e c o n s u m i n g , t h e s e methods g r e a t l y d e c r e a s e t h e v a l u e o f the r e s i d u a l egg w h i t e due t o t h e a d d i t i o n o f t h e s a l t s . S e v e r a l a f f i n i t y and immunoadsorbant a f f i n i t y methods have been s u g g e s t e d f o r i s o l a t i o n o f lysozyme ( B a i l o n and N i s h i k a w a , 1977; MacKay e t a l . , 1982; Szewczyk e t a l . , 1982) . However, i n g e n e r a l t h e s e methods may n o t be s u i t e d t o l a r g e s c a l e a p p l i c a t i o n s because o f t h e h i g h c o s t o f t h e a f f i n i t y s u p p o r t s . S e p a r a t i o n o f lysozyme w i t h c a t i o n exchange r e s i n s has been i n v e s t i g a t e d . A h v e n a i n e n e t a l . (1980) d e s c r i b e d t h e s u c c e s s f u l use o f t h e c a t i o n exchanger D u o l i t e C-464 f o r lysozyme r e c o v e r y i n a b a t c h p r o c e d u r e . A v e r y e f f e c t i v e co lumn c h r o m o t o g r a p h i c method u s i n g c a t i o n exchange was d e s c r i b e d by L i - C h a n e t a l . (1986) . These a u t h o r s compared seven d i f f e r e n t r e s i n s i n terms o f lysozyme c a p a c i t y , p e r c e n t r e c o v e r y o f lysozyme from egg w h i t e and f low c h a r a c t e r i s t i c s and u l t i m a t e l y chose D u o l i t e C-464 c a t i o n exchange r e s i n as t h e b e s t s u i t e d f o r t h i s a p p l i c a t i o n . These a u t h o r s examined v a r i o u s means o f h o m o g e n i z i n g egg w h i t e t o reduce v i s c o s i t y p r i o r t o l o a d i n g an i o n exchange co lumn. A l t h o u g h s e v e r a l methods were e f f e c t i v e t h e most p r a c t i c a l l a r g e s c a l e method i n v o l v e d pas sage t h r o u g h a M a n t o n - G a u l i n l a b o r a t o r y m i l k homogenizer ( s i n g l e s t a g e , 6 .9 MPa) . They f u r t h e r d e m o n s t r a t e d t h a t n e i t h e r t h i s p r e - t r e a t m e n t , nor t h e remova l o f lysozyme and a v i d i n by i o n exchange t r e a t m e n t a d v e r s e l y a f f e c t e d t h e whip 18 volume o f raw egg w h i t e o r t h e g e l s t r e n g t h o f h e a t - s e t egg w h i t e . ELECTROSTATIC INTERACTION CHROMATOGRAPHY (EIC) I o n exchange o r e l e c t r o s t a t i c i n t e r a c t i o n c h r o m o t o g r a p h y (EIC) i s i n p r i n c i p l e dependent upon the a t t r a c t i v e f o r c e s o f o p p o s i t e l y c h a r g e d p a r t i c l e s as d e f i n e d by C o u l o m b ' s Law: F = ( q ^ J / d r 2 (1) where F = t h e a t t r a c t i v e f o r c e , q-L and q 2 a r e p o i n t c h a r g e s o f o p p o s i t e c h a r g e , r i s t h e d i s t a n c e between q± and q 2 and d i s t h e d i e l e c t r i c c o n s t a n t o f t h e medium. E I C o f a m p h o t e r i c m a c r o m o l e c u l e s s u c h as p r o t e i n s i s c o m p l i c a t e d b o t h by t h e f a c t t h a t t h e c h a r g e v a r i e s w i t h pH and t h a t c h a r g e s a r e n o t p o i n t c h a r g e s but a r e d i s t r i b u t e d o v e r t h e s u r f a c e o f t h e m o l e c u l e . None t h e l e s s t h e g e n e r a l p r o p o r t i o n a l i t y o f t h e r e l a t i o n s h i p remains c o r r e c t . P r o t e i n s a r e commonly c h a r a c t e r i z e d by t h e i r i s o e l e c t r i c p o i n t ( p i ) , t h e pH a t w h i c h t h e y have z e r o ne t c h a r g e . T h i s n e t c h a r g e has commonly been u s e d t o p r e d i c t t h e b e h a v i o r o f p r o t e i n s on i o n exchange r e s i n s b a s e d on the a s s u m p t i o n s t h a t p r o t e i n s w i l l n o t be r e t a i n e d a t t h e i r p i , t h a t t h e y w i l l be r e t a i n e d by a n i o n exchangers a t p H ' s above t h e i r p i o r by c a t i o n exchangers below t h e i r p i and w i l l 19 show a c o r r e l a t i o n between n e t c h a r g e and r e t e n t i o n t ime (Anon. 1980) . S t u d i e s have now shown t h a t t h i s i s an o v e r s i m p l i f i e d v iew and t h a t t h e n e t c h a r g e c o n c e p t i s no t adequate t o e x p l a i n t h e 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 o f p r o t e i n s . I n a s t u d y d i r e c t e d towards t h i s q u e s t i o n , K o p a c i e w i c z e t a l . (1983) d e m o n s t r a t e d t h a t n a t i v e p r o t e i n s ( a p p r o x i m a t e l y t h r e e f o u r t h s o f t h e f o u r t e e n p r o t e i n s s t u d i e d ) d e v i a t e d s i g n i f i c a n t l y from p r e d i c t e d b e h a v i o r , e i t h e r by b i n d i n g t o i o n exchange r e s i n s a t t h e i r p i , o r by f a i l i n g t o b i n d i n p r o p o r t i o n t o n e t c h a r g e when not a t t h e i r p i . The p o o r c o r r e l a t i o n between r e t e n t i o n t i m e s and n e t c h a r g e o f many p r o t e i n s might c o n c e i v a b l y be e x p l a i n e d as due t o n o n - e l e c t r o s t a t i c c o n t r i b u t i o n s t o a d s o r p t i o n , s u c h as h y d r o p h o b i c i n t e r a c t i o n s , o r t o i n t r a m o l e c u l a r c h a r g e asymmetry on t h e s u r f a c e o f t h e p r o t e i n . I n t h e s t u d y o f K o p a c i e w i c z e t a l . (1983) h y d r o p h o b i c i n t e r a c t i o n s were judged u n i m p o r t a n t because t h e a d d i t i o n o f 1% 2 - p r o p a n o l t o t h e m o b i l e phase was shown t o have a m i n i m a l e f f e c t on r e t e n t i o n t i m e s . The a u t h o r s c o n c l u d e d t h a t r e g i o n s o f l o c a l i z e d c h a r g e on p r o t e i n m o l e c u l e s , p r e s e n t even a t the p i o f t h e p r o t e i n , made n e t c h a r g e a p o o r measure o f p r o t e i n b i n d i n g on E I C . I n a s e p a r a t e s t u d y , t h e above h y p o t h e s i s r e c e i v e d s u p p o r t from a g e n e r a l i z e d computer s i m u l a t i o n o f p r o t e i n a d s o r p t i o n and chromatography (Gorbanov e t a l , 1986) . The t h e s p h e r i c a l s u r f a c e o f a h y p o t h e t i c a l p r o t e i n upon the d i s t r i b u t i o n c o n s t a n t o f t h e p r o t e i n was c a l c u l a t e d . P a r t i c u l a r l y a t h i g h a d s o r p t i o n e n e r g i e s , t h e d i s t r i b u t i o n o f b i n d i n g s i t e s had a marked e f f e c t on t h e d i s t r i b u t i o n c o n s t a n t between s t a t i o n a r y and m o b i l e phases and t h e r e f o r e on c h r o m a t o g r a p h i c r e t e n t i o n . I n f l u e n c e o f S a l t on E I C . The s t r e n g t h 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 s between p r o t e i n s and i o n exchangers d e c r e a s e s w i t h i n c r e a s i n g c o n c e n t r a t i o n o f n e u t r a l s a l t s . T h i s has been a t t r i b u t e d t o c o m p e t i t i o n f o r c h a r g e d s i t e s on t h e exchanger from s m a l l s a l t i o n s ( F r e i f e l d e r , 1976) . I n E I C t h e f a c t t h a t the l o g a r i t h m o f r e t e n t i o n f a c t o r s (k) ' d e c r e a s e s l i n e a r l y w i t h the l o g a r i t h m o f s a l t c o n c e n t r a t i o n has now been w e l l e s t a b l i s h e d ( V e l a y u d h a n and H o r v a t h , 1986; P a r e n t e and W e t l a u f e r , 1984; Boardman and P a r t r i d g e , 1955) . The r e t e n t i o n f a c t o r i s d e f i n e d a s : k = ( t r - t Q ) / t Q (2) where t r and t Q a r e r e t e n t i o n t i m e s o f a r e t a i n e d s o l u t e and o f an u n r e t a r d e d component r e s p e c t i v e l y . The r e l a t i o n s h i p between k and s a l t c o n c e n t r a t i o n i s r e p r e s e n t e d by the e q u a t i o n : 21 l o g k = z l o g a + l o g c (3) where a i s t h e i o n i c s t r e n g t h o f t h e m o b i l e p h a s e , c i s a c o n s t a n t , and z i s t h e s l o p e o f t h e l i n e ( P a r e n t e and W e t l a u f e r , 1984) . R e c e n t l y , t h e v a l u e s o f z and c have been shown t o v a r y d e p e n d i n g upon t h e n a t u r e o f t h e s a l t used f o r e l u t i o n . B o t h t h e c o u n t e r i o n and t h e c o - i o n a f f e c t r e t e n t i o n t i m e s . P a r e n t e and W e t f a u f e r (1984) showed t h a t t h e r e t e n t i o n o f lysozyme on a c a t i o n exchanger a t d i f f e r e n t i o n i c s t r e n g t h s v a r i e d w i t h t h e n a t u r e o f t h e c o u n t e r i o n ( F i g u r e 1 . 2 ) . K o p a c i e w i c z e t a l . (1983) r e p o r t e d t h a t t h e p r o t e i n r e t e n t i o n o f s t r o n g c a t i o n and a n i o n e x c h a n g e r s was i n f l u e n c e d s u b s t a n t i a l l y by b o t h t h e c o u n t e r - i o n and c o - i o n o f t h e e l u t i n g s a l t ( T a b l e 1.2 and 1 . 3 ) . The number o f p r o t e i n c h a r g e s i n t e r a c t i n g w i t h the i o n exchange r e s i n was e s t i m a t e d i n some c a s e s from t h e f o l l o w i n g r e l a t i o n s h i p : l o g k = 2 Z l o g ( l / [ N a C l ] ) + l o g K z (4) where k i s t h e r e t e n t i o n c o n s t a n t , l o g K z i s t h e i n t e r c e p t 22 Figure 1.2. Lysozyme retention data f i t t e d to an ion exchange model (Equation 3). Lysozyme was eluted from a cationic HPIEC column (Parente and Wetlaufer,1984). Table 1.2. Influence of various ions on the retention of cytochrome C and.lysozyme on a strong cation exchange column. Chromatography was performed at pH 6.0 (Kopaciewicz et a l . , 1983). r e l a t i v e retention* cytochrome C lysozyme anion (sodium salt) fluoride 0.79 0.88 chloride 0.68 0.64 bromide 0.58 0.51 phosphate 0.54 0.5 7 sulfate 0.90 0.85 acetate 0.74 0.74 ta r t r a t e 0.98 0.99 c i t r a t e 1.00 1.00 cation (chloride salt) lithium 1.00 1.00 sodium 0.58 0.55 potassium 0.57 0.55 ammonium 0.59 0.5 8 magnesium 0.56 0.54 *Unity refers to the longest retention time obtained with a 20 min gradient. T a b l e 1 .3 . I n f l u e n c e o f v a r i o u s i o n s on t h e r e t e n t i o n o f o v a l b u m i n and soybean t r y p s i n i n h i b i t o r on a s t r o n g a n i o n exchange c o l u m n . Chromatography was p e r f o r m e d a t pH 8.0 ( K o p a c i e w i c z e t a l . , 1983) . r e l a t i v e r e t e n t i o n t i m e o v a l b u m i n t r y p s i n i n h i b i t o r a n i o n (sodium s a l t ) c h l o r i d e 0.83 0.64 bromide 0. 72 0.56 p e r c h l o r a t e 0. 62 0 .64 b i c a r b o n a t e 0 .82 0.71 f o r m a t e 1.00 0. 72 a c e t a t e 1.00 0. 95 p r o p i o n a t e 1.00 1.00 s u l f a t e 0.68 0. 79 c i t r a t e 0.50 0 .69 c a t i o n ( c h l o r i d e s a l t ) l i t h i u m 1.00 0 . 82 sodium 0. 83 0.65 p o t a s s i u m 0. 85 0. 68 ammonium 0. 81 0. 64 magnesium 0. 68 0. 50 c a l c i u m 0. 68 0. 47 * U n i t y r e f e r s t o t h e l o n g e s t r e t e n t i o n t i m e o b t a i n e d w i t h 20 min g r a d i e n t . 25 and 2Z i s the slope of the lin e (Kopaciewicz et a l . , 1983). Z i s believed to be closely related to the number of charged sit e s on the protein which interact with the resin. The data suggested that the number of charges interacting with the resin may be greater or less than the net charge of the protein. In another study, Z values were seen to vary depending upon the nature of the counter-ion {i.e. Z (magnesium chloride) was greater than Z (sodium chloride)}, even though pH was constant (Rounds and Regnier, 1984). This may be due to enhanced ionization of protein functional groups by magnesium ions. Equation (3) was o r i g i n a l l y developed and v e r i f i e d for monovalent s a l t s , although i t may be modified to accommadate multivalent s a l t s . Velayudhan and Horvath (1986) proposed another form of the equation which they claim to be rigorously v a l i d for multivalent s a l t s . log k = log «p.Q) - ( Z p / Z g ) l o g [S] (5) where <p i s the phase r a t i o , Q i s a constant cha r a c t e r i s t i c of the exchanger, s a l t , solvent system, Zp i s the interaction charge on the proteins, and Z s i s the valence of the counterion. Clearly, equations (3), (4) and (5) are very similar. In each case the slope of log k vs log s a l t concentration (or log 1/salt concentration) in EIC i s closely related to the number of charges on the protein which interact with the ion exchange group of the stationary phase. 26 HYDROPHOBIC INTERACTION CHROMATOGRAPHY. Hydrophobic interaction chromatography (HIC) refers to the chromatography of compounds on inert matrices, such as s i l i c a or agarose, to which non-polar hydrocarbons, such as a l k y l or ar y l groups have been covalently attached. Elution i s generally achieved with decreasing s a l t gradients in aqueous buffers. HIC i s conceptually similar to reverse phase chromatography (RPC), the main d i s t i n c t i o n being that RPC stationary phases t y p i c a l l y have 10 to lOOx the density of hydrophobic groups of HIC stationary phases (Goheen and Engelhorn, 1984). Also, RPC elution i s commonly achieved by increasing the concentration of miscible non-polar solvent in the mobile phase (Nakai and Li-Chan, 1987). The theoretical framework for explaining hydrophobic interactions of proteins in solution i s somewhat controversial at thi s time. There exists two very d i f f e r e n t and occasionally contradictory schools of thought concerning the driving force behind the phenomena. One approach, the entropic approach, i s to explain the interaction between hydrophobic molecules in terms of entropy changes. The introduction of hydrophobic molecules into water i s said to induce increased l o c a l structure of water corresponding to a decrease i n entropy. The change in free energy associated with the interaction i s therefore attributed to a positive AS, and the hydrophobic effect i s seen as a repulsion by the 27 solvent rather than an attraction between the hydrophobic groups (Tanford, 1980). The second general approach considers hydrophobic interaction i n terms of the r e l a t i v e a ttraction between hydrophobic regions of the solute molecules, as well as solvent - solvent attractions and solute - solvent attractions (Van Oss et a l . , 1986, Nakai and Li-Chan, 1987). Thus the hydrophobic effect i s attributed to the interplay of a l l three intermolecular interactions. A variation of the solvent effect approach, c a l l e d the solvophobic theory, has been applied to the s a l t i n g out of proteins, HIC and RPC by Melander, Horvath, and their co-workers at Yale University. The s a l t mediated retention of proteins in HIC was related to the solvophobic theory by Melander et a l . (1984). The free energy change ( AG") associated with transfer of a molecule from a hypothetical gas phase into solution was described by: AG° = A G c a v + A G e s > + A G v d w + RT In (RT 7 PV) (6) where A G c a v i s the free energy change for formation of a cavity i n the solvent, AG e s i s the free energy associated with the e l e c t r o s t a t i c interactions between the solute and the solvent, A G v d w i s the free energy change due to Van der Waals interaction between the solute and the solvent, R is 28 t h e gas c o n s t a n t , T i s the t e m p e r a t u r e , P i s t h e p r e s s u r e , and V i s t h e mean m o l a r volume o f t h e s o l v e n t . A G c a v c a n be shown t o be dependant upon t h e molar s u r f a c e a r e a o f t h e s o l u t e and t h e s u r f a c e t e n s i o n o f t h e s o l v e n t . S u r f a c e t e n s i o n i s i n t u r n dependant upon s a l t c o n c e n t r a t i o n and t h e m o l a l s u r f a c e t e n s i o n i n c r e m e n t o f the s a l t (Melander and H o r v a t h , 1977) . A p p l i c a t i o n o f t h e above r e l a t i o n s h i p t o HIC g i v e s t h e f o l l o w i n g e q u a t i o n f o r t h e i s o c r a t i c r e t e n t i o n f a c t o r k o f a s o l u t e : i n k = ( - l - r R T ) { A G c a v + A G e s > + A G v d w + A G a s s o c . + A G r a d ) + i n (RT-rPV) + 0 (7) where A G a s s o c i s t h e f r e e energy change f o r l i g a t e - e l u a t e a s s o c i a t i o n i n t h e absence o f s o l v e n t ( i e . gas p h a s e ) , A G r a d ^ s t t i e f r e e energy change due t o s o l v e n t e l u a t e i n t e r a c t i o n s n o t i n c l u d e d i n the f i r s t t h r e e terms and <j> i s a c o n s t a n t r e l a t e d t o t h e c o n c e n t r a t i o n o f a c c e s s i b l e l i g a t e s on t h e co lumn T h i s e q u a t i o n c a n be s i m p l i f i e d i f one c o n s i d e r s In ( k / k Q ) i n s t e a d o f In k , where k Q i s t h e r e t e n t i o n f a c t o r a t z e r o s a l t c o n c e n t r a t i o n . A l s o , A G v d w i s e x p e c t e d t o be n e a r l y l i n e a r w i t h s a l t c o n c e n t r a t i o n (Melander e t a l . , 1984) , and on t h i s a s s u m p t i o n one i s l e f t w i t h : In k / k Q = A G c a v + A G e s + c o n s t a n t (8) 29 A G C A V c a n be shown t o be l i n e a r w i t h s a l t c o n c e n t r a t i o n (Melander and H o r v a t h , 1977) a n d ; A G C A V = - A A G arm. + c o n s t a n t (9) where A A s i s t h e d i f f e r e n c e i n s u r f a c e a r e a o f l i g a t e and p r o t e i n exposed t o m o b i l e phase between t h e bound and unbound s i t e s o r t h e m o l e c u l a r c o n t a c t a r e a o f b i n d i n g , 0~, t h e m o l a l s u r f a c e i n c r e m e n t o f t h e s a l t and m i s t h e m o l a l i t y o f t h e s a l t . A t a s u f f i c i e n t l y h i g h s a l t c o n c e n t r a t i o n the A G e . s . w i H a p p r o a c h a c o n s t a n t and t h e l o g a r i t h m i c r e t e n t i o n f a c t o r becomes l i n e a r as s a l t m o l a l i t y (m): l o g ( k / k Q ) = 5m + c o n s t a n t (10) where 5 i s a p a r a m e t e r w h i c h measures t h e r e t e n t i o n e f f e c t o f t h e s a l t on t h e p r o t e i n i n q u e s t i o n . C o n s o l i d a t i o n o f E I C and HIC E f f e c t s i n C h r o m a t o g r a p h y . The h y d r o p h o b i c and e l e c t r o s t a t i c c o n t r i b u t i o n s t o i o n exchange and m e t a l c h e l a t e i n t e r a c t i o n chromatography ( M C I C ) , have r e c e n t l y been examined w i t h r e f e r e n c e t o s o l v o p h o b i c t h e o r y ( E l R a s s i and H o r v a t h , 1986; H o r v a t h e t a l . , 1985) . An e q u a t i o n o f t h e f o l l o w i n g form has been p r o p o s e d : 30 l o g k = A + B l o g m + C m (11) where k i s the r e t e n t i o n f a c t o r , m i s the s a l t m o l a l i t y and A , B and C are c o n s t a n t s . At a s u f f i c i e n t l y low s a l t c o n c e n t r a t i o n , C m approaches z e r o , the hydrophobic i n t e r a c t i o n e f f e c t approaches a c o n s t a n t , and the r e l a t i o n s h i p i s governed by the l o g k vs l og m l i n e a r r e l a t i o n s h i p 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 . At a s u f f i c e n t l y h i g h s a l t c o n c e n t r a t i o n , 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 w i l l approach a cons tant and the r e l a t i o n s h i p i s governed by l og k vs m l i n e a r r e l a t i o n s h i p o f hydrophobic i n t e r a c t i o n s . The cons tant A i s t h e r e f o r e a composite o f the l i m i t i n g va lues o f hydrophobic and 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 and i s d i f f i c u l t to i n t e r p r e t m e a n i n g f u l l y . On the o ther hand, B i s the s lope o f the l o g k vs l o g m r e l a t i o n s h i p a t low s a l t c o n c e n t r a t i o n s and a measure o f the e f f e c t o f s a l t on the magnitude o f 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 the s o l u t e and the s t a t i o n a r y phase. Comparison between equat ions (3) and (11) a l s o l eads one to the c o n c l u s i o n t h a t B i s r e l a t e d to Z and t h e r e f o r e i s a measure o f the number o f charge i n t e r a c t i o n s between the p r o t e i n and the r e s i n . C i s the s l o p e o f the l o g k vs m r e l a t i o n s h i p at h i g h s a l t c o n c e n t r a t i o n and a measure o f the e f f e c t o f s a l t on the hydrophobic i n t e r a c t i o n between the p r o t e i n and the s t a t i o n a r y phase. 31 I I . S i m u l t a n e o u s I s o l a t i o n o f A v i d i n and Lysozyme from Egg Albumen. INTRODUCTION Lysozyme and a v i d i n a r e two p r o t e i n s found i n r a t h e r s m a l l q u a n t i t i e s i n egg w h i t e (3.5% d r y b a s i s and 0.05% d r y b a s i s r e s p e c t i v e l y ) . E a c h commands a s i g n i f i c a n t c o m m e r c i a l market and b o t h c a n be removed from egg w h i t e w i t h o u t s u b s t a n t i a l l y a l t e r i n g i t s f u n c t i o n a l o r n u t r i t i o n a l p r o p e r t i e s . I n f a c t , t h e U n i t e d S t a t e s Food and Drug A d m i n i s t r a t i o n s t a n d a r d o f i d e n t i t y f o r d r i e d egg w h i t e has r e c e n t l y been a l t e r e d t o a l l o w t h e s a l e o f t h i s p r o d u c t w i t h t h e lysozyme and a v i d i n r e d u c e d by i o n exchange t r e a t m e n t ( A n o n . , 1986a) . S e v e r a l methods f o r i s o l a t i n g and p u r i f y i n g lysozyme have a p p e a r e d i n t h e s c i e n t i f i c l i t e r a t u r e . The c l a s s i c method , i n v o l v i n g d i r e c t p r e c i p i t a t i o n from egg albumen a t pH 9.5 by a d d i t i o n o f 5% sodium c h l o r i d e i s s t i l l u s e d i n i n d u s t r y ( A l d e r t o n and F e v o l d , 1964) . Recent m o d i f i c a t i o n s o f t h i s p r o c e d u r e have been b r i e f l y r e v i e w e d by A h v e n a i n e n e t a l . (1980) . Y i e l d s o f 60-80% o f egg w h i t e lysozyme have been a c h i e v e d w i t h d i r e c t c r y s t a l l i z a t i o n but a major d i s a d v a n t a g e o f t h e s e methods i s t h a t t h e y r e q u i r e t h e a d d i t i o n o f s a l t s o r o t h e r a d d i t i v e s w h i c h c o n t a m i n a t e and d e c r e a s e t h e u s e f u l n e s s o f the t r e a t e d egg w h i t e . 32 Currently the most e f f i c i e n t and probabily the most widely used method for lysozyme i s o l a t i o n i s a two step ion exchange - i s o e l e c t r i c p r e c i p i t a t i o n process. Egg albumen i s mixed with a suitable ion exchange resin, commonly a modified carboxymethyl ce l l u l o s e , then f i l t e r e d o f f . The resin i s washed free of unadsorbed proteins, then placed i n 5% sodium chloride, which causes release of the lysozyme into solution. The lysozyme solution i s then adjusted to pH 9.5, causing the lysozyme to precipitate. Lysozyme i s redissolved, desalted, concentrated and spray dried. Many a f f i n i t y chromatography methods have been described for lysozyme i s o l a t i o n (Cherkasov and Kravchenko, 1967; 1969; Weaver et a l . , 1977; Imoto and Yagishita, 1973; Muzzarelli et a l . , 1978; Yoshimoto and Tsuru, 1974). Although good yields and purity have been achieved with these methods, they required specialized and expensive a f f i n i t y supports which generally have not been widely used or proven in industry. Furthermore, d i l u t i o n of the egg white was generally required. Ahvenainen et a l . , (1980) reported the use of the commercially available cation exchange resin Duolite C-464 with good y i e l d and purity of lysozyme. Their method however was most successful as a batch procedure and required a somewhat complex regeneration sequence between batches. The e a r l i e s t p u r i f i c a t i o n method for avidin involved alcohol p r e c i p i t a t i o n from egg white (Eakin et a l . , 33 1941; D h y s e , 1954) . M o d i f i c a t i o n s o f t h i s a p p r o a c h have been w i d e l y u s e d c o m m e r c i a l l y b u t w i t h c o m p l e t e d e s t r u c t i o n o f t h e f u n c t i o n a l i t y o f the r e m a i n i n g egg w h i t e . Melamed and G r e e n (1963) a c h i e v e d h i g h p u r i t y w i t h t h r e e s u c c e s s i v e p u r i f i c a t i o n s t e p s on c a r b o x y m e t h y l c e l l u l o s e and A m b e r l i t e CG-50 i o n exchange r e s i n , an e f f e c t i v e b u t v e r y t ime consuming method. The p r o d u c t o f t h e f i r s t s t e p o f t h e i r method c o n t a i n e d ~50% o f i n i t i a l a v i d i n b u t p u r i t y was o n l y a p p r o x i m a t e l y 1%. Y i e l d and p u r i t y f i g u r e s f o r o t h e r p r i m a r y i s o l a t i o n p r o c e d u r e s were 17% y i e l d and 10-20% p u r i t y ( F r a e n k e l - C o n r a t e t a l . , 1952) , 38% y i e l d and 10% p u r i t y (Dhyse , 1954) , 39% y i e l d (Rhodes e t a l . , 1958) and 80% y i e l d w i t h 24% p u r i t y (Green and Toms, 1970) . Two s u c c e s s f u l a f f i n i t y methods f o r a v i d i n have been d e s c r i b e d . C u a t r e c a s u s and W i l c h e k (1968) bound a v i d i n t o b i o c y t i n - S e p h a r o s e columns and e l u t e d w i t h 6M g u a n i d i n e -HC1 a t pH 1 .5 . A v i d i n was c o m p l e t e l y d e n a t u r e d by t h i s t r e a t m e n t b u t c o u l d be r e n a t u r e d by d i l u t i o n , w i t h a 90% y i e l d . I m i n o b i o t i n , w h i c h b i n d s a v i d i n a t pH 11 b u t no t a t pH 4, was a l s o employed as an a f f i n i t y l i g a n d f o r a v i d i n . W i t h t h i s a p p r o a c h , y i e l d s o f 95% and 99% a v i d i n p u r i t y have been r e p o r t e d (Heney and O r r , 1981) . B o t h a f f i n i t y methods u t i l i z e e x p e n s i v e a f f i n i t y s u p p o r t s w i t h l i m i t e d u s e f u l l i f e t i m e s . A l s o , a f f i n i t y p u r i f i e d a v i d i n may be o n l y 75% as a c t i v e as i o n exchange p u r i f i e d a v i d i n (Mock e t a l . , 1985) . C o n s e q u e n t l y the a f f i n i t y p r o d u c t has a lower v a l u e t h a n i o n exchange p u r i f i e d a v i d i n . F u r t h e r m o r e , t o da te 34 only ion exchange processes have been approved by the US FDA for treatment of egg albumen. Therefore at present, economic factors strongly favour the ion exchange processes. A convenient and p r a c t i c a l column chromatography method for recovery of lysozyme has recently been reported (Li-Chan et a l . , 1986). These authors indicated that a s i g n i f i c a n t amount of avidin co-eluted with the lysozyme. The main purpose of t h i s study was to develop a procedure whereby these proteins could be resolved and isolated in separate fractions by a single cation exchange column. The sequence i n which lysozyme and avidin were eluted from the Duolite C-464 column with increasing sodium chloride gradients was the reverse of the order expected from th e i r respective i s o e l e c t r i c points. In order to elucidate t h i s point, the interaction of these two proteins with t h i s resin was examined more closely. Duolite C-464, a copolymer of methacrylic acid and d i v i n y l benzene, has extensive aromatic ring structure and therefore might be expected to interact hydrophobically as well as e l e c t r o s t a t i c a l l y with proteins. A model of protein retention on ion exchangers has recently been proposed which allows estimation of the contributions of e l e c t r o s t a t i c and hydrophobic interactions to the binding of the protein at di f f e r e n t s a l t concentrations (Horvath et a l . , 1985; E l Rassi and Horvath, 1986). This model, as well as the theoretical treatment of ion exchange retention put forward by Kopaciewicz et a l . , (1983) were employed to determine the 35 r e l a t i v e i m p o r t a n c e o f h y d r o p h o b i c and 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 between lysozyme and a v i d i n and D u o l i t e C - 4 6 4 . MATERIALS AND METHODS R e a g e n t s . D u o l i t e C-464 was s u p p l i e d by t h e Diamond Shamrock C o . ( C l e v e l a n d , O H ) . 2 ( 4 ' - H y d r o x y a z o b e n z e n e ) - b e n z o i c a c i d . (HABA), i o n excahange p u r i f i e d a v i d i n s t a n d a r d s and 3X c r y s t a l l i z e d g r a d e 1 lysozyme were o b t a i n e d from Sigma C h e m i c a l C o . ( S t . L o u i s , MO). Egg Whi te P r e t r e a t m e n t . Egg w h i t e s were s e p a r a t e d from eggs o b t a i n e d from t h e 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 p o u l t r y farm o r s u p p l i e d f r o z e n by V a n d e r p o l ' s Eggs L t d . ( S u r r e y , B . C . ) . B e f o r e co lumn a p p l i c a t i o n , egg albumen was p a s s e d t h r o u g h a M a n t o n -G a u l i n l a b o r a t o r y homogenizer ( s i n g l e s t a g e , 6 .9 MPa) t o d e c r e a s e v i s c o s i t y . A c i t i v i t y A s s a y s . Lysozyme a c t i v i t y was d e t e r m i n e d by the t u r b i d i m e t r i c a s s a y method d e s c r i b e d by Sigma C h e m i c a l C o . , b u l l e t i n 11-77 (Sigma C h e m i c a l C o . , S t . L o u i s , M O . ) , w i t h m o d i f i c a t i o n s as r e p o r t e d by L i - C h a n e t a l . (1986) . A v i d i n a c t i v i t y was measured by t h e HABA s p e c t r o p h o t o m e t r y method o f G r e e n (1965) , w i t h minor m o d i f i c a t i o n s . To 2 mL o f sample s o l u t i o n , 1 mL o f 0 .2M sodium phosphate b u f f e r , pH 7 . 2 , was a d d e d , f o l l o w e d by 0.1 mL o f 2mM HABA. The a b s o r b a n c e o f t h e a v i d i n - H A B A complex was r e a d a t 500 run. Then 0 . 1 - m L o f 400mM b i o t i n s o l u t i o n was added t o d i s p l a c e HABA and A 5 0 Q was r e a d a g a i n . The c o n c e n t r a t i o n o f a v i d i n was c a l c u l a t e d as f o l l o w s : V o l A s s a y x MW a v i d i n x A 5 0 0 A v i d i n ( g / L ) = (1) V o l Sample x E x 4 where MW i s t h e m o l e c u l a r w e i g h t o f a v i d i n (68,000 d a l t o n s ) , 4 i s t h e number o f b i o t i n b i n d i n g s i t e s p e r a v i d i n m o l e c u l e and E i s 3 4 , 0 0 0 , t h e e x t i n c t i o n c o e f f i c i e n t o f t h e a v i d i n HABA complex . P r o t e i n A s s a y s P r o t e i n c o n c e n t r a t i o n s o f samples were r o u t i n e l y d e t e r m i n e d from t h e i r a b s o r b a n c e a t 280nm. P r o t e i n c o n t e n t was c a l c u l a t e d b a s e d on a E x ° v a l u e s o f 26.4 f o r lysozyme and 15.4 f o r a v i d i n ( A n o n . , 1981; G r e e n , 1975) . S i n c e lysozyme i s t h e main c o n t a m i n a n t o f t h e a v i d i n c o n t a i n i n g f r a c t i o n s , a v i d i n p u r i t y c a n more a c c u r a t e l y be e s t i m a t e d as f o l l o w s : A s = a v i d i n s p e c i f i c a c t i v i t y (mg/mL) A 2 3 Q = a b s o r b a n c e o f f r a c t i o n a t 280nm. A v i d i n p u r i t y (%) = A s . a . A s . a . + A 2 8 0 X 100% (2) ( A s a x 1.54) 37 E l e c t r o p h o r e s i s . Sodium d o d e c y l s u l f a t e 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 (SDS-PAGE) was p e r f o r m e d e s s e n t i a l l y a c c o r d i n g t o Laemmli (1970) , u s i n g a 10% a c r y l a m i d e s e p a r a t i o n g e l and a 3% s t a c k i n g g e l c o n t a i n i n g 0.2% SDS. Samples c o n t a i n i n g 1-2 mg p r o t e i n / m L were p r e p a r e d i n 0.01M sodium p h o s p h a t e b u f f e r (NaP) a t pH 7 . 0 , c o n t a i n i n g 2% SDS and 2% b e t a -mere a p t o e t h a n o l ; a f t e r h e a t i n g a t 1 0 0 ° C f o r 10 m i n , g l y c e r o l ( to 10%) was added and -25 uL was a p p l i e d t o t h e sample s l o t s . E l e c t r o p h o r e s i s was c a r r i e d out a t 100 v o l t s f o r 4-5 h . G e l s were s t a i n e d w i t h Coomass ie B r i l l i a n t B l u e R-250 f o r 1.5 h as d e s c r i b e d by Weber and O s b o r n (1969) . A v i d i n and lysozyme were i d e n t i f i e d by c o m p a r i s o n w i t h c o m m e r c i a l a v i d i n and lysozyme s t a n d a r d s . I o n Exchange I s o l a t i o n o f Lysozyme and A v i d i n . P r e l i m i n a r y e x p e r i m e n t s and o p t i m i z a t i o n o f e l u t i o n c o n d i t i o n s f o r s e p a r a t i o n o f lysozyme and a v i d i n were c a r r i e d out u s i n g a 7 mL (1 .4 cm i . d . ) co lumn o f D u o l i t e C-464 r e s i n . Subsequent l a b o r a t o r y e x p e r i m e n t s were p e r f o r m e d w i t h 25 mL (1.5 cm i . d . ) columns o r 170 mL (2 .5 cm i . d . ) co lumns o f D u o l i t e C-464 . Homogenized egg w h i t e was a p p l i e d t o columns 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 0. 1M NaP, a t a f l ow r a t e s u c h as t o a l l o w a c o n t a c t t ime between the r e s i n and egg w h i t e o f 20-30 m i n . I m m e d i a t e l y a f t e r egg w h i t e a p p l i c a t i o n , u n a d s o r b e d p r o t e i n s were washed o f f the co lumn w i t h 5-10 co lumn volumes o f d i s t i l l e d water o r s t a r t i n g b u f f e r . A d s o r b e d p r o t e i n s were e l u t e d w i t h b u f f e r 3 8 o f i n c r e a s i n g i o n i c s t r e n g t h , u s i n g e i t h e r s t e p w i s e o r g r a d i e n t e l u t i o n . D e t a i l s o f e l u t i o n c o n d i t i o n s a r e p r e s e n t e d i n t h e R e s u l t s s e c t i o n . A s m a l l p i l o t s c a l e e x p e r i m e n t was p e r f o r m e d w i t h a 470 mL (5 cm x 18 cm) D u o l i t e C-464 co lumn. The p r o c e s s was p a r t i a l l y automated w i t h a P h a r m a c i a C3 P r o c e s s C o n t r o l l e r ( P h a r m a c i a Canada I n c . , D o r v a l , Que . ) w h i c h c o n t r o l l e d a s e r i e s o f e l e c t r o m a g n e t i c v a l v e s and p e r i s t a l t i c pumps as diagrammed i n F i g u r e 2 . 1 . S i m p l e x O p t i m i z a t i o n . The mapping s i m p l e x o p t i m i z a t i o n t e c h n i q u e (Nakai e t a l . , 1984; N a k a i and Kaneko 1985) was employed t o i n c r e a s e t h e e f f i c i e n c y o f t h e s e a r c h f o r t h e b e s t c o n d i t i o n s f o r t h e s e p a r a t i o n o f lysozyme and a v i d i n from egg w h i t e . T h r e e e x p e r i m e n t a l f a c t o r s known t o have a b e a r i n g on t h e s e p a r a t i o n were s e l e c t e d f o r t h e o p t i m i z a t i o n ; ( l ) p H , (2) sodium c h l o r i d e c o n c e n t r a t i o n o f t h e l i m i t b u f f e r and (3) t h e shape o f t h e e l u t i o n g r a d i e n t . G r a d i e n t shape was d e t e r m i n e d by t h e g r a d i e n t power v a l u e P , s u c h t h a t Y = X p where Y was t h e volume r a t i o o f t h e l i m i t b u f f e r and X was t h e c o r r e s p o n d i n g e l u t i o n volume f r a c t i o n . G r a d i e n t s had a t o t a l volume o f 50 mL. S t e p w i s e changes i n t h e p r o p o r t i o n s o f t h e s t a r t i n g b u f f e r ( d i s t i l l e d water) and t h e l i m i t b u f f e r (as d e f i n e d by f a c t o r s 2 and 3) were used t o s i m p l i f y t h e f o r m a t i o n o f t h e g r a d i e n t . By v a r y i n g the v a l u e o f P , l i n e a r (P = 1 ) , convex (P < 1 ) , o r concave (P > 1) g r a d i e n t s were p o s s i b l e . To s i m p l i f y t h e o p t i m i z a t i o n EGG WHITE WATER L Y S O Z Y M E B U F F E R A V I D I N BUFFER UV MONITOR R E S I D U A L \ E C C WHITE WASTE A V I D I N L Y S O Z Y M E Figure 2.1. Diagram of a p i l o t plant apparatus for isolation of avidin and lysozyme from egg white by Duolite C-464 chromatography. 40 and d e c r e a s e t h e number o f e x p e r i m e n t s , o n l y the c h a r a c t e r i s t i c s o f t h e a v i d i n peak ( i . e . a v i d i n y i e l d and a v i d i n p u r i t y ) were o p t i m i z e d . T h i s a p p r o a c h was j u s t i f i e d because a p r e v i o u s s t u d y ( L i - C h a n e t a l . , 1986) had shown t h a t ly sozyme r e c o v e r y was a c c e p t a b l e w i t h i n t h e range o f c o n d i t i o n s u s e d i n t h i s s t u d y . The r e s p o n s e v a l u e t o be o p t i m i z e d was a f u n c t i o n o f b o t h t h e p u r i t y o f a v i d i n i n t h e a v i d i n f r a c t i o n and the y i e l d o f a v i d i n as a p e r c e n t a g e o f t h e a v i d i n a p p l i e d i n the egg w h i t e { i . e . r e s p o n s e i n d e x = a v i d i n p u r i t y (%) x a v i d i n y i e l d (%)}. A v i d i n B i n d i n g C a p a c i t y o f D u o l i t e C - 4 6 4 . A 2 mL column (3 .1 cm x 0.5 cm) o f D u o l i t e C-464 was e q u i l i b r a t e d w i t h 0.1 M NaP, pH 7 . 9 . A s o l u t i o n o f 1.0 mg/mL a v i d i n i n NaP (50 mL) was a p p l i e d a t a f l o w r a t e o f 0 .17 mL/min and c o l l e c t e d i n 2 mL f r a c t i o n s . E l u a n t f r a c t i o n s were a s s a y e d f o r a v i d i n by t h e HABA method. Chromatography i n H i g h Ammonium S u l p h a t e C o n c e n t r a t i o n s . A 1.5 cm x 18.0 cm column o f D u o l i t e C-464 was e q u i l i b r a t e d w i t h 0.025M NaP, pH 7.3 c o n t a i n i n g from 0.15 m o l a l t o 2.25 m o l a l ammonium s u l p h a t e . P r o t e i n samples (5mg o f a v i d i n o r lysozyme) were d i s s o l v e d i n 3mL o f the same b u f f e r and a p p l i e d t o t h e column a t a f l ow r a t e o f 0 . 3 m L / m i n . E l u t i o n volume was t a k e n as t h e p o i n t o f maximum A 2 8 0 f o r lysozywe o r t h e p o i n t o f maximum a v i d i n a c t i v i t y f o r a v i d i n . The e l u t i o n volume o f t h e s o l v e n t f r o n t 41 was determined with an inj e c t i o n of d i s t i l l e d water and detected as a decrease in eluant conductivity. The capacity factor, k was calculated as: k = (ev - ev Q) / ev Q ( 3) where ev = elution volume of a protein and ev Q = elution volume of the solvent front (ie. water). The values of log k and of m (m = molality of ammonium sulphate) were f i t t e d to the model of E l Rassi and Horvath (1986), by the multiple regression analysis performed with P C - S t a t i s t i c a l A n a l y t i c a l System (SAS Institute , Cary, N.Y.) RESULTS Simplex Optimization of Lysozyme-Avidin Separation. Li-Chan et- a l . (1986) found that avidin co-eluted with lysozyme when egg white was passed through a column of Duolite C-464 equilibrated at pH 8.0, washed with 0. 1M NaP and i s o c r a t i c a l l y eluted with 0.5M NaP (Figure 2.2). However, conditions for separation of avidin and lysozyme were not determined. In the present study, preliminary experiments in which these proteins were eluted with ionic strength gradients at different pH's resulted i n incomplete resolution and rather low y i e l d s . These results are shown in Table 2.1. In general responses were best in the pH range of 7 to 8. The purity of the avidin fracti o n was a useful 42 20 120 220 320 420 520 620 720 Elution Volume (mL) F i g u r e 2 .2 . E l u t i o n p r o f i l e o f adsorbed p r o t e i n s from D u o l i t e C-464 column (170 mL) by i s o c r a t i c e l u t i o n us ing 0.5M sodium phosphate b u f f e r a t pH 8. 43 Table 2.1. Preliminary separations of lysozyme and avidin from egg white on a 7 mL column of Duolite C-464. elution conditions recovered proteins lysozyme avidin y i e l d y i e l d purity ( %) ( %) ( %) NaP , pH 7.5, NaCl gradient. 66 60 0.8 NaP, pH 8.00, NaP gradient. 43 69 3.8 NaP, pH 5.0, NaP gradient. 67 84 1.3 pH 9.0, sodium carbonate gradient 51 59 3.3 44 i n d i c a t o r o f peak s e p a r a t i o n s i n c e a v i d i n was l e s s abundant than lysozyme. Unless the peaks were w e l l s e p a r a t e d , the a v i d i n peak tended to be h e a v i l y contaminated w i t h lysozyme. Simplex o p t i m i z a t i o n was used to a s s i s t i n f i n d i n g the bes t pH and s a l t g r a d i e n t c o n d i t i o n s . In an attempt to improve y i e l d o f a v i d i n , water r a t h e r than NaP was used to wash the unadsorbed p r o t e i n s from the column. The parameters and r e s u l t s o f seven experiments are shown i n T a b l e 2 .2 . The bes t y i e l d v a l u e was o b t a i n e d w i t h the e l u t i o n c o n d i t i o n s of v e r t e x 2 ( F i g u r e 2 . 3 ) . A l though 72% y i e l d was o b t a i n e d under these c o n d i t i o n s , the a v i d i n f r a c t i o n was e l u t e d w i t h o n l y f a i r r e s o l u t i o n from the l a r g e r A 2 8 o peak, r e s u l t i n g i n o n l y 4.4% p u r i t y . T a b l e 2.2 i n d i c a t e s t h a t o n l y moderate improvement i n the a v i d i n response v a l u e was a c h e i v e d by the o p t i m i z a t i o n p r o c e d u r e . .The p u r i t y o f a v i d i n was improved o n l y 15-33 f o l d as compared to u n t r e a t e d egg w h i t e . Stepwise E l u t i o n . The r e s u l t s o f the o p t i m i z a t i o n experiments i n d i c a t e d t h a t m a n i p u l a t i o n o f the pH and the shape of the s imple 50 mL s a l t g r a d i e n t was i n s u f f i c i e n t f o r s a t i s f a c t o r y r e s o l u t i o n of lysozyme and a v i d i n . However, o p t i m i z a t i o n d i d a l l o w s e l e c t i o n o f the optimum pH f o r t h i s b u f f e r system as a p p r o x i m a t e l y pH 7 .9 . In f a c t 7.9 i s c l o s e to the upper b u f f e r i n g l i m i t f o r NaP so i t was not p o s s i b l e to p r o p e r l y e v a l u a t e h i g h e r pH's w i t h t h i s b u f f e r . S ince NaP was d e s i r a b l e f o r o ther reasons ( i . e . economy and low t o x i c i t y ) , 45 Table 2.2. Factor levels for simplex optimization and resulting avidin recovery and purity for lysozyme-avidin separation from egg white by Duolite C-464 chromatography. Factor levels Results pH [NaCl] of Power Avidin Avidin Response l i m i t buffer value purity* 3 y i e l d index 0 (M) ( P ) a (%) 7.30 0.610 1.550 8.43 0.643 1.774 7.58 0.742 1.774 7.58 0.643 2.446 7.77 0.665 1.699 7.96 0.687 1.550 7.87 0.676 1.550 .044 62.7 2.76 .044 71.8 3.16 .042 67.2 2.82 .024 54.5 1.31 .046 61.2 2.82 .045 57.2 2.57 .070 54.4 3.80 aPower value of gradient (P) such that Y = X p, where Y i s the NaCl concentration of the l i m i t buffer and X i s the corresponding elution volume fraction. ^Avidin purity in mg avidin/mg protein. cResponse index = avidin purity x avidin y i e l d . F i g u r e 2 . 3 . E l u t i o n p r o f i l e o f a d s o r b e d egg w h i t e p r o t e i n s f rom D u o l i t e C-464 (7 mL r e s i n ) u s i n g a 5 s t e p sodium c h l o r i d e e l u t i o n g r a d i e n t . 47 pH 7.9 was chosen f o r subsequent experiments. Stepwise e l u t i o n becomes p r a c t i c a l when pH ceases to be a v a r i a b l e since i t becomes possib l e to accurately determine the s a l t concentration that w i l l elute lysozyme and a v i d i n . This information was determined from l i n e a r gradient experiments and used to s e l e c t e l u t i o n buffers which would f i r s t elute a l l proteins adsorbed less strongly than a v i d i n , followed by an a v i d i n e l u t i o n buffer. Table 2.3 shows the e l u t i o n conditions and r e s u l t s of two experiments on the 7 mL column. The r e s u l t s of the t h i r d 2-step e l u t i o n experiment are i l l u s t r a t e d i n Figure 2.4. A v i d i n a c t i v i t y was completely excluded from the f i r s t peak and the p u r i t y of the pooled a v i d i n f r a c t i o n s was higher than previously. However, y i e l d was poor at 44%. None the less the 2-step approach was further investigated on a larger column. A 20 f o l d increase i n column volume, with corresponding increase i n flow rates and egg white a p p l i c a t i o n volume were undertaken. Lysozyme and a v i d i n e l u t i o n conditions were a l t e r e d s l i g h t l y to improve separation and decrease t a i l i n g (Table 2.3). The response value was s l i g h t l y improved. " E l u t i o n Looping" Although the response value of the 2-step e l u t i o n experiments were the best to date, further improvement was desired. The f r a c t i o n designated the "a v i d i n peak" i n fact contained only 8.8% a v i d i n while the "lysozyme peak" contained 72% a c t i v e lysozyme. This suggests that because of the s i m i l a r i t y i n charge properties of a v i d i n and Table 2.3. Results of 2-step elution for resolution of lysozyme and avidin fractions from egg white by Duolite C-46 4 chromatography on a 7 mL (1, 2) or a 170 mL (3) column. Elution conditions 1 3 Avidin purity (mg avidin/ mg protein) Avidin y i e l d (%) Response index a l ( i ) NaP, pH 8.45, 0.26M NaCl ( i i ) NaP, pH 8.45, 0.50M NaCl. . 117 44. 0 5.15 2(i) NaP, pH 8.43, 0.26M NaCl, ( i i ) NaP, pH 8.43, 0.43M NaCl. . 147 26.5 3. 85 3(i) NaP, pH 7.90, 0.13M NaCl, ( i i ) NaP, pH 7.90, 0.75M NaCl. .088 59 . 3 5 . 22 ^Response index = avidin purity x avidin y i e l d . b F i r s t step (i) for lysozyme elution and second step ( i i ) for avidin elution. Figure 2.4. Elution p r o f i l e of adsorbed egg white proteins from Duolite C-464 (170 mL resin) using a two step elution process. 50 lysozyme, together with the much lower abundance of a v i d i n , complete r e s o l u t i o n of the pro t e i n peaks would be d i f f i c u l t by conventional e l u t i o n conditions of ion exchange chromatography. The large t a i l i n g peak composed mainly of lysozyme i n e v i t a b l y contaminated the small a v i d i n containing peak. A novel e l u t i o n process was necessary for better r e s o l u t i o n and improved p u r i t y of recovered a v i d i n . Apparently an a v i d i n peak of high p u r i t y was d i f f i c u l t to achieve as long as the r a t i o of a v i d i n to other proteins (mainly lysozyme) on the column remained low. In an attempt to increase t h i s r a t i o , a scheme was investigated i n which a v i d i n was allowed to accumulate on the r e s i n through several cycles of egg white a p p l i c a t i o n and lysozyme e l u t i o n with low i o n i c strength buffer. The term " e l u t i o n looping" was coined to describe t h i s process. Two such multi-cycle experiments were performed on the 170 mL Duolite C-464 column and the r e s u l t s are summarized i n Table 2.4. Buffers and e l u t i o n conditions for lysozyme and a v i d i n were extrapolated from the gradient e l u t i o n and two step e l u t i o n experiments. The A 2 3 0 a v i d i n a c t i v i t y p r o f i l e s from a 5 cycl e experiment are presented i n Figure 2.5. Both experiments r e s u l t e d i n improved r e s o l u t i o n of a v i d i n from other proteins. The 5 cyc l e t r i a l , with a response value of 10.37, was two f o l d better than the previous best r e s u l t s . Both a v i d i n p u r i t y and recovery were improved. 51 Figure 2.5. P r o f i l e of adsorbed egg white proteins eluted from a Duolite C-464 column (170 mL) by the elution looping process. Five cycles of egg white application and lysozyme elution were followed by a single elution of avidin. 52 Table 2.4. Results of lysozyme-avidin separation from egg white by Duolite C-46 4 chromatography with m u l t i - c y c l e e l u t i o n looping. E l u t i o n conditions Avidin p u r i t y (mg a v i d i n / mg protein) Avidin y i e l d (%) Response index a 3 cycles on 170 mL column .075 77.0 5.78 5 cycles on 170 mL column . 129 80.4 10. 37 8 cycles on 470 mL column .295 79. 3 23. 34 16 cycles on 25 mL column . 409 74.0 30. 27 aResponse index = a v i d i n p u r i t y (mg a v i d i n / mg protein) x a v i d i n recovery (%). 53 Small P i l o t Scale T r i a l . The question of process scale-up i s an important aspect of any procedure which has p o t e n t i a l for i n d u s t r i a l a p p l i c a t i o n . Although space and equipment r e s t r i c t i o n s precluded t r u l y large scale experiments i n t h i s study, an experiment was completed using a 470 mL (5.0 cm x 24 cm) r e s i n column. A t o t a l of 14.2 L of egg white was applied to the column i n 8 c y c l e s , over a s i x day period. Although the egg white was kept at 4°C except when a c t u a l l y on the column, the column and apparatus were at room temperature. The r e s u l t s are summarized i n Table 2.4. SDS-PAGE p r o f i l e s of representative f r a c t i o n s are presented i n Figure 2.6. Capacity of Duolite C-464. The success of the 5 and 8 cycles experiments r a i s e d a question as to the l i m i t s of e l u t i o n looping; that i s how many cycles could be accommodated before a v i d i n or lysozyme response would diminish. The lysozyme capacity of Duolite C-464 has been examined under s i m i l a r conditions (Li-Chan et a l . , 1986). Approximately 19.3 mg of lysozyme was applied per mL of Duolite C-464 before rete n t i o n dropped below 90%. In the present study, a v i d i n loading of 24.8 mg/mL of r e s i n was consistent with greater than 90% re t e n t i o n on the column. With the assumption that lysozyme and a v i d i n compete for the same s i t e s on the r e s i n , these fi g u r e s allow an estimate of the maximum number of cycles before lysozyme binding drops below 90%. I f the egg white applied per cyc l e per mL r e s i n i s 4 mL and lysozyme 54 Figure 2.6. SDS-PAGE gel of lysozyme and avidin fractions recovered from egg white with 8 cycles of egg white application and lysozyme elution followed by a single avidin elution: A/ untreated egg white; B/ 1st cycle eluted egg white; C/ 4th cycle eluted egg white; D/ 8th cycle eluted egg white; E/ 1st cycle lysozyme; F/ 3rd cycle lysozyme; G/ 6th cycle lysozyme; H/ 8th cycle lysozyme; 1/ avidin fraction. 55 content i s 3.5 mg/mL egg white, then approximately 35% of the resin ion exchange groups would be available for avidin. This i s s u f f i c i e n t to adsorb 8.7 mg of avidin / mL of resin or 40 to 50 cycles of egg white application, depending upon the exact concentration of avidin in the egg white. Although a 40 cycle t r i a l was not undertaken, a 16 cycle experiment was performed on a small 25 mL column (Table 2.4 and Figure 2.7) with substantial further improvement in avidin purity. In p r a c t i c a l terms, i t i s doubtful whether extension of the technique to the apparent l i m i t of 40-50 cycles would result i n proportional improvement in response. Purity of recovered avidin was plotted against the t o t a l amount of avidin loaded onto Duolite C-464 (Figure 2.8). The avidin purity appeared to be approaching a maximum well before the estimated maximum resin loading of 8.7 mg/ml. Protein - Duolite C-464 Interaction. The net charge of a protein at a given pH has commonly been used to predict i t s behavior on ion exchange resins (Kopaciewicz et a l . , 1983). The net charge of a protein i s zero at i t s i s o e l e c t r i c point ( p i ) , and increasingly positive as the pH f a l l s or increasingly negative as the pH rises from the p i . According to the net charge approach, lysozyme (pl=10.7) should bind more strongly than avidin (pl=10.0) to a cation exchange resin at pH 7.9. In fact, we have shown th i s to be true when the cation exchanger i s CM-cellulose (Figure 3.1). However, the 56 F i g u r e 2 .7 . SDS-PAGE g e l of lysozyme and a v i d i n f r a c t i o n s recovered from egg white w i th 16 c y c l e s of egg white a p p l i c a t i o n and lysozyme e l u t i o n f o l l o w e d by a s i n g l e a v i d i n e l u t i o n : A / un t rea ted egg w h i t e ; B / 1st c y c l e e l u t e d egg w h i t e ; C / 8th c y c l e e l u t e d egg w h i t e ; D/ 16th c y c l e e l u t e d egg w h i t e ; E / 1st c y c l e lysozyme; F / 8th c y c l e lysozyme; G / 16th c y c l e lysozyme; H/ a v i d i n f r a c t i o n . 5 7 Figure 2.8. Relationship between the purity of the recovered avidin fractions and the concentration of accumulated avidin on the Duolite C-464 column. 58 reverse situation was observed on Duolite C-464. Either the net charge concept i s inadequate to explain the ele c t r o s t a t i c binding of these proteins to Duolite C-464 or some other binding mechanism contributes to the retention on the column. Duolite C-464, a co-polymer of methacrylic acid and d i v i n y l benzene, has s i g n i f i c a n t potential for hydrophobic interaction with proteins due to i t s aromatic ring content. This raises the question of whether hydrophobic interaction might favour the retention of avidin on the column, and account for the unusual elution pattern. A recently proposed model of protein interaction on ion exchangers allows estimation of el e c t r o s t a t i c and hydrophobic binding parameters by examining the effect of neutral s a l t concentration in the eluant on protein retention (El Rassi and Horvath, 1986; Horvath et a l . , 1985). Although t h i s model was developed for use with high performance ion exchange chromatography data (HPIEC), where considerably greater precision was possible, i t appeared applicable to open column data as well, a l b e i t with greater experimental error. Capacity constants (k), were determined for avidin and lysozyme at seven dif f e r e n t ammonium sulphate concentrations in 25mM sodium phosphate buffer, pH 7.3. Although pH 7.9 would have been more in keeping with the experimental conditions employed previously, 25mM sodium phosphate had insufficent buffering capacity at the higher pH. A higher phosphate concentration might have contributed 59 unduely to the t o t a l s a l t concentration and obscured the a f f e c t of ammonium sulphate concentration. The data were f i t t e d to the following equation: log k = A + B log m + Cm (4) where k i s the capacity f a c t o r , m i s the m o l a l i t y of ammonium sulphate, and A, B, and C are parameters for a given solute, solvent, r e s i n , pH and s a l t combination (Figure 2.9). The values of these constants together with R , the c o e f f i c i e n t of determination are presented i n Table 2.5. Parameters B and C are of p a r t i c u l a r i n t e r e s t . B, the l i m i t i n g slope at s u f f i c e n t l y low s a l t concentration i s a measure of s o l u t e - r e s i n i n t e r a c t i o n i n conditions which maximize 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 minimize hydrophobic i n t e r a c t i o n . C i s the corresponding parameter i n charge quenching, high s a l t conditions, when 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 are i n s i g n i f i c a n t and hydrophobicity governs p r o t e i n - r e s i n i n t e r a c t i o n s . In the case of a v i d i n and lysozyme, the absolute values of both B and C were greater for a v i d i n , i n d i c a t i n g a greater p o t e n t i a l for both e l e c t r o s t a t i c and hydrophobic i n t e r a c t i o n with Duolite C-464 at appropriate s a l t concentration. The manner i n which these data may be applied to the adsorption of lysozyme and a v i d i n from egg white was not immediately apparent. The di f f e r e n c e i n pH (7.9 vs 7.3), may a l t e r the i n t e r a c t i o n s l i g h t l y but experiments have 6 0 Figure 2.9. Plot of logarithmic capacity factors of lysozyme (*) and avidin (•) against the logarithm of ammonium sulfate molality. Elution was is o c r a t i c with 25 mM sodium phosphate buffer, pH 7.3, at different ammonium sulfate concentrations. 61 Table 2.5. E l e c t r o s t a t i c (B) and hydrophobic (C) interaction parameters for retention of avidin and lysozyme on Duolite C-464. Intercept (A) and R of multiple regression are also included. A B C R 2 Avidin -3.16 -4.53 1. 93 . 9653 lysozyme -2. 95 -3.90 1. 63 . 9026 62 shown the general elution pattern with phosphate buffers and sodium chloride gradients was similar between pH 7 and pH 9. In retrospect, the determination of B and C i n sodium chloride solutions rather than ammonium sulfate would have sim p l i f i e d interpretation. However, preliminary experiments showed that the elution order of lysozyme and avidin was the same in gradients of these two s a l t s (Table 2.6). The fact that avidin was adsorbed more strongly at high ammonium sulphate molality seemed to suggest that hydrophobic forces might help to explain the elution order. On the other hand, contradictory evidence also exists. When avidin and lysozyme were chromatographed on a l i p h a t i c HIC or Phenyl-Sepharose columns, lysozyme was found to bind more strongly than avidin (Table 3.2). This does not necessarily c o n f l i c t with the results on Duolite C-464 since the a c c e s s i b i l i t y and density of hydrophobic regions in both the resin and the protein are known to affect the capacity and s e l e c t i v i t y of HIC (Hofstee and O t i l l i o , 1978). However, i t does seem that there i s not a clear cut relationship between protein HIC binding potential and retention on Duolite C-46 4. Also, although retention of avidin and lysozyme at high ammonium sulphate molality i s c l e a r l y demonstrated, under the conditions used for i s o l a t i o n from egg white, both proteins were bound at low s a l t concentrations and ultimately eluted with increasing s a l t . Since increasing s a l t concentration would tend to strengthen hydrophobic bonds and only weaken e l e c t r o s t a t i c bonds, e l e c t r o s t a t i c 63 Table 2.6. Retention volumes of lysozyme and avidin adsorbed from egg white onto Duolite C-464 and eluted with linear gradients of 4 different s a l t s . Columns were equilibrated with 0.1M NaP, pH 7.5 and a l l gradients were in the same buffer. Molal surface tension increments (<r) of eluting s a l t s are also l i s t e d . Eluting s a l t <J Avidin eluant s a l t (M) volume at elution Lysozyme eluant volume s a l t (M) at elution NaCl 1.64 100 0. 40 60 0. 24 NH4C1 1. 39 71 0. 28 44 0.17 (NH 4) 2S0 4 2.16 35 0. 32 25 0.22 Na 2HP0 4 2.02 10 0.04 15 0.06 64 interactions must play a dominant role. The interaction parameters as determined here confirm that the e l e c t r o s t a t i c interaction of avidin with Duolite C-464 is also greater than that of lysozyme. A f i n a l indication of the dominant role of e l e c t r o s t a t i c forces in this separation was obtained from the effect of different, neutral s a l t s on the elution pattern. In preliminary experiments, gradients of d i f f e r e n t s a l t s (ammonium sulphate, sodium phosphate, ammonium chloride and sodium chloride) were used to elute lysozyme and avidin from Duolite C-464. Elution volumes with each s a l t are presented i n Table 2.6. According to the solvophobic theory of hydrophobic interaction chromatography, the strength of s a l t mediated hydrophobic bonds i s d i r e c t l y proportional to the molal surface tension increment (<r) of the s a l t (Melander and Horvath, 1977). In th i s case, a comparable trend was not observed. If on the other hand, one assumes the r e l a t i v e retention of avidin and lysozyme on Duolite C-464 i s primarily an e l e c t r o s t a t i c event, but one which i s not completely defined by the net charge of the proteins, the observed phenomena are consistant with previously reported results. Kopaciewicz et a l . , (1983) examined the influence of various s a l t s on protein retention by strong, sulphonic acid, cation exchangers. They found that the nature of both the cation and the anion in the s a l t gradient affected 65 protein retention times and that d i f f e r e n t proteins responded d i f f e r e n t l y to different s a l t s . In the case of lysozyme the retention time was greater i n sodium chloride gradients than in the sodium phosphate as observed here. However, i n the previous study, retention was greater in ammonium chloride than sodium chloride, while the reverse was observed i n thi s study. The observation that the net charge does not adequately predict the ion exchange behavior of proteins even in the absence of s i g n i f i c a n t hydrophobic binding has been reported as well (Kopaciewicz et a l . , 1983). The phenomena have been ascribed to charge asymmetry on the protein molecule. Even at i t s p i where net charge i s zero, proteins have many charged groups on the i r surfaces. If the charge d i s t r i b u t i o n i s not uniform, th i s w i l l lead to an uneven e l e c t r o s t a t i c potential depending on the orientation of the molecule with respect to the charged groups on the ion exchange resin. Thus proteins may be oriented on the ion exchanger i n a non-random fashion, leading to deviation from the ideal relationship between net charge and protein binding. The implications of an uneven d i s t r i b u t i o n of potential adsorption sites on protein surfaces has been examined with a generalized computer model of protein-adsorbant chromatography (Gorbanov et a l . , 1986). These authors demonstrated that even when the protein i s assumed to be spherical, d i f f e r e n t distributions of binding sites on 66 the protein surfaces lead to substantial differences in d i s t r i b u t i o n c o e f f i c i e n t s between mobile and stationary phases. CONCLUSIONS A single column cation exchange method was developed which allowed simultaneous recovery of lysozyme and avidin as separate peaks from undiluted egg white. Lysozyme was recovered at higher yields than reported for the i s o e l e c t r i c p r e c i p i t a t i o n methods often used in the industry (86% vs 60 - 80%) and in high purity. Avidin recovery was also as good or better than for previously reported ion exchange methods (74 - 80% vs 17 - 80%). The purity of the avidin f r a c t i o n (up to 40.9%) was higher than previously reported primary preparations although lower than i s desirable for some applications. Avidin was shown to have a greater potential for both e l e c t r o s t a t i c and hydrophobic interactions with Duolite C-464 than lysozyme. Under the chromatographic conditions of t h i s separation method however, e l e c t r o s t a t i c interactions were dominant. F i n a l l y , the elution looping method used in t h i s study may prove valuable for other protein p u r i f i c a t i o n problems, where a trace protein must be separated from a more abundant protein of similar charge. 67 I I I . Secondary P u r i f i c a t i o n of Avidin. INTRODUCTION. Mu l t i p l e p u r i f i c a t i o n steps have frequently been required f o r the p u r i f i c a t i o n of a v i d i n . The e a r l i e s t reported method for large scale a v i d i n i s o l a t i o n was that of Dhyse (1954). That method ou t l i n e d s i x p u r i f i c a t i o n steps: acetone p r e c i p i t a t i o n , washing, extract with sodium c h l o r i d e s o l u t i o n s , d i a l y s i s against water to p r e c i p i t a t e the pro t e i n s , r e s o l u b i l i z a t i o n i n s a l t s o l u t i o n s , and a second solvent p r e c i p i t a t i o n with ethanol. The resu l t a n t p r o t e i n product was about 50% active a v i d i n (Green, 1975). Melamed and Green (1963) used a preliminary batch adsorption on carboxymethylcellulose (CMC) followed by 3 column p u r i f i c a t i o n steps on CMC and a fourth ion exchange on Amberlite CG-50 to achieve a v i d i n of approximately 93% p u r i t y ( i . e . 13.5 units/mg p r o t e i n ) . G a t t i et a l . (1984) used the method of Melamed and Green (1963) with an a d d i t i o n a l g el f i l t r a t i o n step to obtain very high p u r i t y a v i d i n for X-ray c r y s t a l l o g r a p h i c a n a l y s i s . The method for simultaneous i s o l a t i o n of lysozyme and a v i d i n developed i n t h i s laboratory r e s u l t e d i n an a v i d i n containing f r a c t i o n with a maximum mean p u r i t y of 40.9%. Since some biochemical methods which use av i d i n recommend 80 - 92% p u r i t y (Heitzman and Richards, 1974), a d d i t i o n a l methods were examined for secondary p u r i f i c a t i o n 68 of avidin. CMC ion exchange, gel f i l t r a t i o n , metal chelate interaction chromatography (MCIC) and hydrophobic interaction chromatography (HIC) on both a l i p h a t i c and aromatic hydrophobic media were compared i n terms of their effectiveness for t h i s purpose. MATERIALS AND METHODS. Reagents. Carboxymethylcellulose (Whatman CM-52), Sepharose 6B, Sephadex G-75, iminodiacetic acid (IDA), and standard avidin and lysozyme proteins were obtained from Sigma Chemical Co. (St. Louis, MO). Butanediol d i g l y c i d y l ether (BGE) was obtained from Eastman Kodak Co. (Rochester N.Y.). P a r t i a l l y p u r i f i e d avidin preparations were recovered from egg white as outlined above or donated by Brookside Laboratories Inc. (Abbotsford, B.C.). CMC Ion Exchange. CMC was equilibrated with starting buffer (0.02M Tris-HCl, pH 8.2 - 9.0; 0.05M ammonium acetate, pH 9.0; or 0.012M sodium tetraborate, pH 9.0 - 10.2) and packed into a 1.5 cm x 25 cm column. Proteins were applied in starting buffer and washed with the same buffer or water, then eluted with increasing ionic strength solutions (sodium chloride or ammonium acetate) either as a linear gradient or as stepwise increases. Flow rates were 1.0 - 1.5 mL/min for sample 69 a p p l i c a t i o n and e l u t i o n but up to 5.0 mL/min for washing. Samples contained up to 5.3 mg a v i d i n /mL CMC. Gel F i l t r a t i o n . A 2.5 cm x 79 cm column of Sephadex G-75 was e q u i l i b r a t e d with 0.02M T r i s - H C l , pH 8.2, containing 0.1M sodium c h l o r i d e . Samples (lOmL) containing up to 50 mg av i d i n and up to 250 mg of pro t e i n were applied and eluted at a flow rate of 0.5 mL/min or less and c o l l e c t e d i n 7mL f r a c t i o n s . Metal Chelate In t e r a c t i o n Chromatography. Sepharose 6B was ac t i v a t e d and c r o s s l i n k e d with BGE as described by Sunberg and Porath (1974). IDA was bound to the ac t i v a t e d Sepharose by the method of Porath and 01in (1983). IDA-Sepharpse 6B was packed into a column (3.5 cm x 8.0 cm) and the upper one-third to one-half was saturated with cupric ions as indicated by the blue colour (about 15 mL of 50mM CuCl 2 ) - The column was washed with d i s t i l l e d water, then s t a r t i n g buffer (0.02M sodium phosphate, pH 7.7 with 0.5M sodium c h l o r i d e ) . Up to 200 mL of sample containing up to 15mg of a v i d i n and ll2mg of p r o t e i n were applied at a flow rate of 15 mL/h. Immediately a f t e r a p p l i c a t i o n , adsorbed proteins were eluted with a pH gradient generated using a two chamber device containing 400 mL of s t a r t i n g buffer i n the mixing chamber and 400 mL of 0.1M a c e t i c a c i d i n the resevoir chamber. Eluted material was c o l l e c t e d as 4.5 mL f r a c t i o n s . 70 Hydrophobic Interaction Chromatography. Hydrophobic interaction chromatography (HIC) of the avidin containing f r a c t i o n from the Duolite C-464 primary separation was performed on 6 separate 1.0 mL columns of a l k y l agarose with 0, 2, 4, 6, 8, or 10 carbon atoms per a l k y l chain. (Thus column #4 contained agafose-C H 2 - C H 2 - C H 2 - C H 3 . ) Pre-packed columns were obtained from Miles S c i e n t i f i c (Naperville, IL) . Each column was equilibrated with 0.05M Tris-HCl, pH 7.9, loaded with approximately 10 ug of protein and eluted i s o c r a t i c a l l y with star t i n g buffer. Primary avidin fractions were also chromatographed on a 1.5 cm x 20 cm column of Phenyl-Sepharose CL-4B (Pharmacia Inc.: Dorval, Quebec), equilibrated with a start i n g buffer of 0.1M sodium phosphate, pH 7.9, containing 1.0M sodium chloride. Samples (8 mL, 4.9 mg protein/mL, 0.5 mg avidin/mL) were applied in starting buffer and proteins were eluted f i r s t with starting buffer (130 mL), followed by 150 mL of 0.02M sodium phosphate, pH 7.9, with 0. 2M sodium chloride, or with d i s t i l l e d water at a flow rate of 0.5 mL/min. Eluant was collected as 11 mL fractions. Comparison of Zeta Potential. An estimate of the r e l a t i v e charge of 2 lysozyme samples in 0.01M borate buffer, pH 9.0 - 10.5, was obtained as follows. Protein solutions (2 mL of 0.1 % in buffer) were homogenized with 0.06 mL of 3,3'-dimethyl biphenyl (Aldrich Chemical Co. : Milwaukee, WIS) with the aid of a Tekmar 71 Sonic Disrupter (20 s at 20% of maximum output, with the microtip) . Emulsions were diluted 50 - 100 f o l d with the appropriate buffer and d i s t i l l e d water so as to have a conductance of 8 x 10~ 6 mhos. Zeta potential of the protein coated o i l droplets was determined with a Model 501 Lazer Zee meter (Pen Kern Inc., Bedford H i l l s , N. Y. ) . A l i n e comparison procedure was used to test for homogeneity of slopes and intercepts of the zeta potential vs pH lines of d i f f e r e n t protein samples (Ostle, 1950) RESULTS CMC Ion Exchange. CMC i s an accepted medium for the p u r i f i c a t i o n of avidin, due to i t s use by Melamed and Green (1963), Green and Toms (1970), and others. In the studies reported here i t was also found to be an ef f e c t i v e method. Avidin purity was increased substantially by t h i s treatment and yields were excellent (Figure 3.1; Table 3.1). Avidin concentrations of up to 38 mg avidin/mL CMC bed volume were possible without impairing avidin resolution or yields. A pH of 9.0 was chosen as the most appropriate for reasons which may not be apparent from an examination of Table 3.1. Any pH below 7.3 was unsuitable because i t resulted i n p r e c i p i t a t i o n of some egg proteins on the column. Buffers of pH 9.0 gave somewhat better resolution of avidin ( i . e . a greater ionic strength difference between the point i n the gradient at which avidin eluted and lysozyme 72 E l u a n t v o l u m e ( m L ) Figure 3.1. Chromatography of av i d i n and lysozyme r i c h egg white f r a c t i o n s on CMC i n 0.02M T r i s - H C l , pH 9.0: A 2 8 0 ( — ) ; ammonium carbonate molarity x 100 ( ); a v i d i n a c t i v i t y (--- ) ; and lysozyme a c t i v i t y (...). Table 3.1. Secondary chromatography of egg white p r o t e i n f r a c t i o n s containing a v i d i n and lysozyme on CMC. Avi d i n y i e l d and p u r i t y are reported as percentages. buffer a v i d i n p u r i t y a v i d i n y i e l d i n i t i a l f i n a l 0.05M ammonium 26.1 83.4 97.0 acetate, pH 9.0 0.01M t r i s - H C l , 26.1 82.9 98.1 pH 9.0 0.01M t r i s - H C l , 26.1 86.6 98.3 pH 8.2 0.02M T r i s - H C l , 13.8* 84.3* 94.8* pH 8.6 13.8* 88.7* 98.0* duplicate experiments. eluted) than pH 8.2 or pH 8.5 and equivalent r e s o l u t i o n to pH 9.88. Furthermore, pH 9.0 was compatible with the less t o x i c T r i s - H C l b u f f e r rather than the borate buffer used for higher pH's. Preliminary experiments, i n which adsorbed proteins were eluted with a l i n e a r sodium c h l o r i d e gradient i n d i c a t e d that 0.20M NaCl i n 0.01M Tr i s - H C l eluted a v i d i n but not lysozyme. Approximately 0.36M NaCl i n the same buffer was the minimum necessary for rap i d lysozyme e l u t i o n . An example of secondary chromatography of a v i d i n containing f r a c t i o n s from a primary Duolite C-464 separation of egg white at pH 9.0 with a stepwise gradient i s presented i n Figure 3.2. The e l u t i o n patterns of a v i d i n and lysozyme chromatographed on Duolite C-464 and CMC were s t r i k i n g l y d i f f e r e n t , i n s p i t e of the f a c t that both exchangers employ the same carboxylic a c i d ion exchange groups. A v i d i n i s released from CMC at a lower i o n i c strength than lysozyme at pH's from 7.9 to 9.0. However, when adsorbed to Duolite C-464 at s i m i l a r pH, lysozyme was eluted at a lower i o n i c strength than a v i d i n (Figure 2.3). This phenomenon w i l l be discussed further with the r e s u l t s of hydrophobic i n t e r a c t i o n chromatography and electrophoresis of a v i d i n and lysozyme containing f r a c t i o n s . Chromatography of 2 mg avidin/mL CMC was used to estimate the proportion of a v i d i n adsorbed to CMC at pH values from 9.00 to 10.20. Up to pH 9.88, v i r t u a l l y 100% of the a v i d i n was absorbed, while at pH 10.20, no a v i d i n was Figure 3.2. CMC chromatography of an egg white protein fract i o n r i c h in avidin and lysozyme. Figure 3.3. Adsorption of avidin to CMC at different pH's. 77 retained on the column (Figure 3.3). This i s consistant with the reported a v i d i n p i of 10 (Woolley and Longsworth, 1942) and provides i n d i r e c t evidence that the adsorption of a v i d i n to CMC i s predominantely i o n i c i n nature. Gel F i l t r a t i o n . SDS-PAGE of the a v i d i n containing f r a c t i o n s from the primary i s o l a t i o n on Duolite C-464 indicated that lysozyme was a major contaminant of a v i d i n . The molecular s i e v i n g a c t i o n of gel f i l t r a t i o n was expected to allow e f f i c i e n t separation of lysozyme and a v i d i n because of the four f o l d molecular weight d i f f e r e n c e between the two proteins ( i . e . 14,700 daltons vs 68,000 daltons) In f a c t , ge l f i l t r a t i o n of a crude egg white containing f r a c t i o n on Sephadex G-75 re s u l t e d i n a su b s t a n t i a l increase i n av i d i n p u r i t y (Figure 3.4). Pur i t y of the a v i d i n f r a c t i o n was 49.0% as opposed to 18.6% of the applied proteins. A v i d i n was t y p i c a l l y eluted at K a v = 0.23 while K a v (lysozyme) = 0.72. Sephadex G-75 was also e f f e c t i v e i n sequence with CMC secondary p u r i f i c a t i o n of av i d i n . When Sephadex G-75 was used as a t e r t i a r y p u r i f i c a t i o n technique, a v i d i n p u r i t y was increased from 77% to 97% (Figure 3.5). Examination of Figure 3.4 reveals the presence of some A 2 8 o absorbing material which eluted before and perhaps concurrent with a v i d i n . This material no doubt contributed to the UV absorbance of the a v i d i n peak. Although t h i s material was not i d e n t i f i e d , examination of the ele c t r o p h o r e t i c p r o f i l e of the f r a c t i o n applied to the gel 78 I S . O l — O S 1 3 6 1 8 4 2 3 2 2 8 0 3 2 8 3 7 6 E l u a n t v o l u m e ( m L ) Figure 3 . 4 . Gel f i l t r a t i o n of egg white fr a c t i o n s r i c h in av i d i n and lysozyme on Sephadex G-75: A 2gg ( — ) ; avidin ( ) . 79 Eluant Volume (mL) Figure 3.5. T e r t i a r y p u r i f i c a t i o n of a v i d i n on Sephadex G-75. Avidin f r a c t i o n s from a primary separation of egg white on Duolite C-464 were chromatographed next on CMC before a p p l i c a t i o n to the gel f i l t r a t i o n column.: A28O (—r-) » avidin, 80 f i l t r a t i o n column revealed a band corresponding in mobility to ovotransferrin (Figure 3.6). Gel f i l t r a t i o n was effective and convenient for laboratory scale separations but i s not ideal for larger scale separations because of the r e l a t i v e l y low capacity of gel f i l t r a t i o n columns and the fact that they are not easily automated. Thus a maximum of only about 2 mg of avidin may be applied per mL of column bed volume, assuming a sample volume of 0.1 column bed volumes, sample protein concentration of 10% and i n i t i a l avidin purity of 10%. Metal Chelate Interaction Chromatography (MCIC). Figure 3.7 shows the elution p r o f i l e of proteins on Sepharose 6B-BGE-IDA-Cu++. Almost a l l of the proteins of the applied protein f r a c t i o n (avidin containing f r a c t i o n from Duolite c-464 separation of egg white proteins) were bound to the column, as indicated by the low A 2 3 0 of the eluant following sample application. Upon elution with a decreasing pH gradient, 2 peaks were indicated by the A 2 3Q p r o f i l e . The f i r s t peak was r i c h in avidin a c t i v i t y . SDS-PAGE p r o f i l e s of applied and eluted protein fractions are presented in Figure 3.8. Lysozyme appears to be the major protein applied, with the avidin band being only f a i n t l y v i s i b l e . Avidin content of the applied f r a c t i o n was calculated to be 12.9%. The avidin r i c h f r a c t i o n eluted from the column on the other hand appeared to be mainly avidin (Figure 3.8), and in fact avidin purity was calculated to be approximately 75%. The second MCIC peak Figure 3.6. SDS-PAGE of a v i d i n f r a c t i o n before gel f i l t r a t i o n (A), duplicate samples at d i f f e r e n t concentrations of o v o t r a n s f e r r i n standard (B,C), and the a v i d i n f r a c t i o n from the gel f i l t r a t i o n separation (D). 82 Figure 3.7. MCIC separation of egg white fractions containing lysozyme and avidin: &28O ^'—^ ' p H (--); avidin ( . . ) . 83 F i g u r e 3 .8 . SDS-PAGE p r o f i l e s of f r a c t i o n s separa ted from egg white by D u o l i t e C-464 chromatography and MCIC chromatography: A , B / un t rea ted egg w h i t e ; C , H / a v i d i n f r a c t i o n from D u o l i t e C-464 ; D, E / peak 2 from MCIC; F , G / peak 1 from MCIC. 84 appeared to contain mainly lysozyme. No a v i d i n a c t i v i t y was detected i n t h i s f r a c t i o n . The y i e l d of a v i d i n from the column was 92% of that applied. The Sepharose-BGE-IDA-Cu + + column was shown to have high capacity to bind lysozyme. The h a l f point s a t u r a t i o n curve of such a column with pure lysozyme was determined. Approximately 95 mg lysozyme was bound per mL column bed volume before any detectable lysozyme was washed through the column and the 166 mg lysozyme/mL bed volume was applied to the column before the lysozyme concentration of the eluant was h a l f the lysozyme concentration applied to the column. Hydrophobic I n t e r a c t i o n Chromatography (HIC). P r o t e i n samples containing mainly lysozyme and a v i d i n were chromatographed on hydrophobic i n t e r a c t i o n agaroses containing a l k y l chains of varying length. The r e s u l t s are presented i n Table 3.2. Under the e l u t i o n conditions u t i l i z e d ( i s o c r a t i c e l u t i o n with 10 column volumes of s t a r t buffer) neither a v i d i n nor lysozyme was retained by i n t e r a c t i o n with a l i p h a t i c chains up to 6 carbons long. Eight and ten carbon a l k y l groups r e s u l t e d i n greater i n t e r a c t i o n and greater r e t e n t i o n of lysozyme than a v i d i n . However, r e s o l u t i o n was not s u f f i c i e n t to recommend the method for routine separations. This method i s believed to separate proteins based on a combination of the r e l a t i v e hydrophobicity and the a c c e s s a b i l i t y of hydrophobic pockets 85 Table 3.2. Retention of p r o t e i n , a v i d i n and lysozyme on hydrophobic chromatography columns with 0, 2, 4, 6, 8 or 10 carbon a l i p h a t i c sidechains. Retention (% of t o t a l ) lenath Protein Avidin Lysozyme 0 0 0 0 2 0 0 0 4 3 0 0 6 7 0 0 8 10 0 20 10 47 13 55 86 i n the three dimensional structure ( S h a l t i e l , 1974). Longer hydrophobic 'arms' are able to i n t e r a c t with less accessible hydrophobic pockets and thus can r e t a i n a larger v a r i e t y of proteins than shorter hydrophobic arms. Phenyl Sepharose I n t e r a c t i o n Chromotography Simi l a r p r o t e i n samples were separated on a Phenyl- Sepharose column (Figure 3.9). PSIC, although often r e f e r r e d to as aromatic hydrophobic i n t e r a c t i o n chromotography,is i n f a c t based on a somewhat d i f f e r e n t mechanism than a l i p h a t i c HIC. The aromatic e f f e c t per se i s presumably a d i r e c t i n t e r a c t i o n between ir electrons of aromatic r i n g s , although i t may p o s s i b l y be r e i n f o r c e d by hydrophobic i n t e r a c t i o n s between aromatic structures (Nakai and Li-Chan, 1987). Avidin was not retained by Phenyl-Sepharose i n the presence of 1.0M sodium c h l o r i d e but lysozyme was. P u r i t y of the a v i d i n f r a c t i o n was improved to ~62 %, with y i e l d s of approximately 84 %. Lysozyme was subsequently eluted with buffer containing 0.2 M NaCl, or with d i s t i l l e d water. Lysozyme-Avidin Interaction. The f a c t that a v i d i n was d i f f i c u l t to separate from lysozyme by any of the methods examined (Table 3.3) prompted the suggestion of some form of i n t e r a c t i o n between lysozyme and a v i d i n . In the case of i s o l a t i o n of lysozyme and a v i d i n from egg white with the c a t i o n exchange r e s i n Duolite C-464 (Figure 2.5) the 'lysozyme' and 'avidin' peaks Figure 3.9. Phenyl-Sepharose separation of a v i d i n and lysozyme: A 2 8 0 ( ); s a l t ( ); a v i d i n ( ); lysozyme 88 Table 3.3. Comparison of f i v e chromatographic methods for the secondary p u r i f i c a t i o n of crude a v i d i n . Chromatography method Y i e l d (%) F i n a l (mg a v i d i n p u r i t y / mg protein) gel f i l t r a t i o n (Sephadex G75) 98 0. 49 ion exchange (CM c e l l u l o s e ) 97 0. 87 MCIC (IDA-BDE) 92 0.75 HIC (C-10 a l i p h a t i c ) - 87 0. 50 HIC (Phenyl Sepharose) 84 0 .62 89 appeared to be well separated in that the A280 of intervening fractions dropped to approximately 1% of that of the avidin peak. None the less the avidin f r a c t i o n contained only 12.9% avidin. The remainder appeared, from SDS-PAGE of a similar f r a c t i o n , to be mainly lysozyme (Figure 2.6). The elution pattern of lysozyme alone on Duolite C-464 was examined. Three times c r y s t a l l i z e d lysozyme was loaded on a column and eluted i n a manner similar to that used to elute lysozyme and avidin when chromatographing egg white (Figure 3.10). Unexpectedly, lysozyme was eluted as two peaks. The bulk was eluted in the f i r s t peak with 0. 2M NaCl i n buffer. Isocratic elution of a second peak was possible but elution was accelerated in 0.5M NaCl. The net charge of lysozyme samples from each peak were compared as zeta potential at 5 points between pH 9.0 and pH 10.5 (Figure 3.11). No s i g n i f i c a n t difference was found between the slopes or intercepts of the zeta potential vs pH regression lines of the two samples at the 5% l e v e l . Lysozyme from the two peaks was also indistinguishable on native protein PAGE. A clear explanation for thi s phenomenon was not possible from th i s study, although multiple lysozyme peaks on cation exchange resins have been reported previously (Tellan and Stein, 1951; 1953). Lysozyme has been shown to form dimers above pH 5 (Sophianopoulos and Holde, 1964) and lysozyme dimers could conceivably have an altered charge d i s t r i b u t i o n on the surface of the complex so as to interact 1 11 21 31 F r a c t i o n Figure 3.10. Duolite C-464 chromatography of p u r i f i e d lysozyme i n sodium phosphate buffer. pH 7.9: A 2 8 0 ( — ) ; sodium chloride (...). Figure 3.11. A comparison of the r e l a t i v e charge of o i l droplets coated with protein from lysozme peak I (•) and lysozyme peak II (•) at different pH's. 92 d i f f e r e n t l y with the ion exchanger. Although dimers were not v i s i b l e i n native PAGE, the p o s s i b i l i t y can not be ruled out, since dimers may be broken with migration through the PAGE g e l . According to Sophianopoulous and Holde, (1964), 25-40% of the lysozyme would be present as a dimer at pH 7, but no dimer band was apparent on the PAGE. A secondary band was v i s i b l e at lower lysozyme concentrations but, assuming net charge i s the same as that of the monomer the p r o t e i n of the secondary band i s only s l i g h t l y larger i n molecular weight than that of the main band. Apparently therefore, lysozyme dimers do not survive electrophoresis. Whatever the source of the the second lysozyme peak on Duolite C-464 cat i o n exchange, t h i s band c e r t a i n l y contributes to the lysozyme contamination of the a v i d i n peak and may explain the poor separation on that medium. None the l e s s , other evidence e x i s t s for a lysozyme-avidin i n t e r a c t i o n . When pro t e i n f r a c t i o n s r i c h i n lysozyme and a v i d i n were separated on a CMC column, the a v i d i n peak was again well separated from the lysozyme peak as seen i n the A280 P r o f i l e « Even so, the SDS-PAGE p r o f i l e s of the a v i d i n f r a c t i o n revealed a d i s t i n c t p r o t e i n band i n the p o s i t i o n t y p i c a l of lysozyme (Figure 3.12). When the same sample was analysed with native PAGE, even at high concentrations, no band t y p i c a l of lysozyme was detected. Furthermore, when egg white with added a v i d i n was examined with native protein PAGE, the a v i d i n band showed a s l i g h t l y decreased mob i l i t y , as compared to pure av i d i n . This may be due to a 93 A B C D E F i g u r e 3.12. E l e c t r o p h o r e s i s of a v i d i n and lysozyme: A / a v i d i n f r a c t i o n from CMC chromatography on n a t i v e p r o t e i n PAGE; B / lysozyme on n a t i v e PAGE; C / h i g h l y p u r i f i e d a v i d i n on n a t i v e PAGE; D/ egg white wi th added a v i d i n on n a t i v e PAGE and E / sample A on SDS-PAGE. 94 s i m i l a r i n t e r a c t i o n of a v i d i n with lysozyme i n egg white. Unfortunately a p p l i c a t i o n of egg white alone to the native PAGE gel at concentrations high enough to v i s u a l i z e a v i d i n r e s u l t e d i n streaking which obscured the a v i d i n p o s i t i o n . Apparently the avidin-lysozyme i n t e r a c t i o n withstands the shear forces inherent i n electrophoresis while the previously reported lysozyme-lysozyme dimers do not. Both lysozyme and a v i d i n demonstrated p o t e n t i a l for hydrophobic i n t e r a c t i o n s on HIC and t h i s type of i n t e r a c t i o n may provide a basis f o r pr o t e i n - p r o t e i n i n t e r a c t i o n . 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 are u n l i k e l y given the charge s i m i l a r i t i e s of a v i d i n and lysozyme. CONCLUSIONS CMC ion exchange, g e l f i l t r a t i o n , MCIC, HIC and PSIC each allowed considerable p u r i f i c a t i o n of crude a v i d i n f r a c t i o n s (Table 3.3). In terms of r e s i n capacity, y i e l d s , and a v i d i n p u r i t y however, CMC ion exchange was superior. Where a v i d i n of greater than -87% p u r i t y i s required, e i t h e r g e l f i l t r a t i o n , MCIC, or PSIC would be e f f e c t i v e as an a d d i t i o n a l p u r i f i c a t i o n step. A comparison of SDS-PAGE and native p r o t e i n electrophoresis p r o f i l e s gave c l e a r evidence of i n t e r a c t i o n between a v i d i n and contaminating lysozyme i n p a r t i a l l y p u r i f i e d a v i d i n preparations. This i n t e r a c t i o n may also occur between the native proteins i n egg white, but has not been demonstrated with c e r t a i n t y . 95 IV. Stoichiometry of the Biotin-Avidin Interaction INTRODUCTION Removal of b i o t i n from the bi o t i n - a v i d i n complex has not been achieved without complete denaturation of the avidin (Green, 1975). This is hardly suprising given the high disassociation constant of the complex (K D = 1 0 - 1 5 ). Although the denaturation of avidin i s largely reversible, there i s no p r a c t i c a l procedure whereby avidin which has b i o t i n bound to i t may be restored to complete b i o t i n binding capacity. A recent report has stated that a f f i n i t y p u r i f i e d avidin normally has only about 75% of the theoretical b i o t i n binding capacity of 4 moles biotin/mole avidin (Mock et a l . , 1985) These authors suggested that this may be due to the fact that, since only one binding site/molecule i s necessary for interaction with the a f f i n i t y ligand, the a f f i n i t y methods cannot distinguish f u l l y active avidin from p a r t i a l l y active avidin. Alternatively, the b i o t i n bound to the avidin may have been leached from the a f f i n i t y columns. Whatever the explanation, decreased a c t i v i t y makes a f f i n i t y p u r i f i e d avidin less useful than avidin p u r i f i e d by other means and may explain the fact that a f f i n i t y p u r i f i e d avidin has a lower d o l l a r value in spite of high purity. Although many methods to measure the stoichiometry of b i o t i n - a v i d i n interaction have been described, two 96 pa r t i c u l a r methods stand out as simple, p r a c t i c a l procedures. Green, (1965) examined the interaction by t i t r a t i o n of a hydroxy azo benzoic acid (HABA) - avidin complex with b i o t i n . B i o t i n displaces HABA from the avidin and results i n the loss of a strong absorbance band at 500nm. A A 5 0 0 i s thus a measure of b i o t i n binding. When combined with protein content, determined from the absorbance at 280nm, an estimate of the ratio of available b i o t i n binding s i t e s to avidin molecules can be made. Mock et a l . (1985) used a fluorometric variation of Green's method i n which a fluorescent probe, 2-anilinonaphthalene-6-sulfonic acid was displaced from avidin by the binding of b i o t i n . In both methods, avidin samples must be assumed to be 100% pure because the absolute amount of avidin in the sample (as opposed to i t s b i o t i n binding capacity) was determined from the absorbance at 280nm and the reported molar extinction coefficent of avidin. This can lead to inaccuracies even with highly p u r i f i e d avidin. Also, neither method can be applied to avidin contaminated with a s i g n i f i c a n t amount of other protein. In this study, two alternate approaches to this problem were considered. In the f i r s t , phenylalanine content rather than t o t a l absorbance at 280nm was used to estimate t o t a l avidin concentration. In the second method, avidin was allowed to interact with b i o t i n immobilized on a column and the remaining number of b i o t i n binding s i t e s was determined. In t h i s way b i o t i n binding capacity of avidin could be determined even i n the presence of a high concentration of other proteins. MATERIAL AND METHODS Reagents. Avidin in an impure form ( ~0.25mg avidin/mg protein) was kindly donated by Brookside Laboratories, (Abbotsford, B.C.). When high purity was required, avidin was p u r i f i e d by carboxymethyl ce l l u l o s e followed by gel f i l t r a t i o n on Sephadex G-75. F i n a l avidin purity was approximately 0.98 mg avidin/mg protein. Avidin was freeze dried and stored, desiccated, at -20°C. 1-Anilinonaphthalene-8-sulfonic acid (ANS) was obtained from BDH Chemicals (Poole, England). N-acetyl-L-phenylalanine (NAPhe) was purchased from Sigma Chemical Co. (St.Louis, MO. ) . Guanidine-HCl (ultrapure grade) was purchased from Ald r i c h Chemicals (Milwaukee, WIS). Protein Assays Protein was estimated from absorbance at 280nm. When applicable avidin content was established using E ° = 15.4 (Green, 1975). Avidin Assays. Avidin a c t i v i t y was measured by two methods, f i r s t by the method of Green (1965) and secondly by a modification of the method of Mock et a l . (1985). The method of Green was described above. The second method was a fluorescent assay 98 for b i o t i n binding using the fluorescent probe 1,8-ANS. Two mL of buffer (0.2M sodium phosphate, pH 7.3) containing 1 to 10 jitM a v i d i n was mixed with 1 mL of 100 fiM 1,8-ANS i n buff e r . This mixture was then t i t r a t e d with 10 or 20 /JLL a l i q u o t s of 120 /iM b i o t i n and the r e l a t i v e i n t e n s i t y of fluoresence ( e x c i t a t i o n 350nm; emission 490nm) recorded. Fluoresence was measured with a Shimadsu RF-5 40 Spectrofluorophotometer (Kyoto, Japan). Relative Intensity (R.I.) was corrected for d i l u t i o n and p l o t t e d against the b i o t i n / a v i d i n r a t i o i n the cuvette. B i o t i n Assay. B i o t i n was assayed by a v a r i a t i o n of Green's (1965) method of a v i d i n assay. B r i e f l y , 0.5mL of a s o l u t i o n of 2mg/mL a v i d i n and 40 jxg/mL HABA i n 0. 2M sodium phosphate buffer was d i l u t e d to 2.5mL with buffer and A 5 0 0 determined. Up to 0.5mL of unknown b i o t i n s o l u t i o n was added and A A 5 Q 0 determined. A f t e r c o r r e c t i o n for d i l u t i o n , b i o t i n concentration was c a l c u l a t e d as follows. B i o t i n (M) = (v o l . assay x A A 5 0 0 )-f (vol. sample x 34000) (1) where 34000 i s the molar e x t i n c t i o n c o - e f f i c e n t at 500nm of the avidin-HABA complex. B i o t i n hydrazide was assayed by the same procedure. 99 Biotin-Avidin Interaction on an Immobilized B i o t i n Column Bi o t i n was bound to a column of sodium periodate oxidized Sephadex G-50 as follows: Sephadex G-50 was oxidized as described by Wilson and Nakane (1976). B r i e f l y , Sephadex G-50 was washed extensively with d i s t i l l e d water, then dried with suction on a Buchner funnel. Next 32g was weighed into 6 4mL of 0.05M sodium periodate, and incubated with reciprocal shaking (~150 rpm at room temperature) for t h i r t y minutes. The reaction was stopped by the addition of 22mL of 3M ethylene glycol and shaken an additional 30 minutes. The oxidized Sephadex (DAS-50) was washed extensively with d i s t i l l e d water and stored at 4°C. A 8.5mL (0.95cm x 12cm) column of DAS-50 was loaded with 8mL of lOmM b i o t i n hydrazide. The column was then washed with 20mL of citrate-phosphate buffer at pH 5.6. A protein sample containing avidin of known t o t a l b i o t i n binding a c t i v i t y was applied to the column, and washed with 20mL of buffer. The b i o t i n binding a c t i v i t y of the wash water was assayed and the t o t a l b i o t i n binding a c t i v i t y of avidin applied to the column (B-^ was calculated. Next 3mL of solution of known b i o t i n content was applied to the column and washed through with 25mL of buffer. The b i o t i n eluant was collected and assayed for b i o t i n . B i o t i n bound to previously adsorbed avidin was calculated (B 2). Assuming that each avidin molecule i s bound to a single immobilized b i o t i n ligand, the true avidin content adsorbed to the column (AT) may be calculated as 100 A.T.( moles) = B-^  ( moles) - B 2( moles). (2) Phenylalanine assay. A quantitive phenylalanine method by second derivative spectroscopy as described by B a l e s t r i e r i et a l . (1978) was employed. B r i e f l y , highly p u r i f i e d avidin was dissolved in 0.05M sodium phosphate buffer containing 6M Guanidine-HCl ( A 2 8 0 = 0.223), and heated to 80°C for 5 minutes to promote complete denaturation of the protein. Second derivative spectra in the 320 - 240nm range were determined on a Cary 210 spectrophotometer equipped with a Derivative/Log A accessory. Each spectrum was recorded fiv e times and the results averaged. The v e r t i c a l distance between the maximum at 25 6nm and the minimum at 25 9nm was determined in arbitary units from the recorded graph. Spectra were also recorded for similar samples with 0.10, 0.20, 0.30, and 0.40 umoles of added NAPhe. When the 256nm-25 9nm interpeak distance was plotted against the concentration of added NAPhe, a straight l i n e was obtained. The i n i t i a l concentration of phenylalanine in the sample was calculated from the intercept and slope of thi s l i n e with the following equation. C Q = i T a (1) where C Q i s the i n i t i a l concentration of phenylalanine, a is the slope of the line and i i s the y intercept. 101 RESULTS. Estimation of the molar r a t i o s of b i o t i n - a v i d i n i n t e r a c t i o n by the methods of Green (1965), or Mock et al.(1985), requires an estimate of a v a i l a b l e b i o t i n binding capacity and an independent estimate of a v i d i n concentration. In both of the previous studies, a v i d i n concentration was estimated from the absorbance at 280nm, u t i l i z i n g the molar e x t i n c t i o n c o e f f i c e n t of a v i d i n as reported by Green (1965). Green used displacement of HABA as a measure of b i o t i n binding, while Mock et a l . (1985) used displacement of the fluorescent probe 2,6 ANS. In t h i s study, HABA and 1,8 ANS were employed to monitor b i o t i n binding. A v i d i n concentration was estimated by two methods, absorbance at 280nm and phenylalanine content as determined from the second d e r i v a t i v e scan of UV absorbance (320-240nm) of the denatured a v i d i n sample. SDS-PAGE of the a v i d i n sample revealed the presence of a small band corresponding i n ele c t r o p h o r e t i c m o b i l i t y to lysozyme, but no other v i s i b l e contaminants. The Phe method of av i d i n estimation was expected to minimize the error due to lysozyme content because lysozyme contains only one Phe. residue/molecule while native a v i d i n contains 7 such residues. ANS Displacement by B i o t i n Mock et a l . (1985) found that 2-anilino naphthalene-6-suIfonate (2,6 ANS), exhibited a large increase i n fluorescence ( e x c i t a t i o n 328nm; emission 408nm), 102 when bound to a v i d i n and that t h i s band decreased dramatically when b i o t i n was bound to the a v i d i n , presumably because 2,6 ANS was replaced by b i o t i n . In t h i s study, 1-anilinonaphthalene-8-sulphate (1,8 ANS) behaved i n a s i m i l a r but not i d e n t i c a l manner. A large increase i n fluorescence was noted i n the presence of a v i d i n , although the band of i n t e r e s t was red s h i f t e d somewhat ( e x c i t a t i o n 350/emission 490). T i t r a t i o n with b i o t i n r e s u l t e d i n a l i n e a r decrease i n fluorescence (Figure 4.1). In Figure 4.1, r e l a t i v e fluorescence i s p l o t t e d against the molar r a t i o of b i o t i n to a v i d i n with a v i d i n estimated from A 23 0- The i n f l e c t i o n point of each curve was estimated from the intercept of the two l i n e a r portions of the curves, thus g i v i n g three estimates of the molar r a t i o of b i o t i n - a v i d i n i n t e r a c t i o n , with a mean value of 3.58 and a standard deviaton of 0.009. Estimation of b i o t i n binding capacity of the same sample by the HABA method, and using the same estimate of a v i d i n content, gave a mean b i o t i n / a v i d i n r a t i o of 3.88 with a standard d e v i a t i o n of 0.005. Phenylalanine Content The dependence of 256-25 9 nm interpeak distance on added concentration of NAPhe to the denatured a v i d i n sample i s shown i n Figure 4.2. The i n i t i a l phenylalanine concentration may be c a l c u l a t e d from the intercept and slope of the l i n e described i n the methods section. This value was then 103 Figure 4.1. Decrease in fluorescence of 1,8-ANS-avidin complex when t i t r a t e d with b i o t i n : 1 mM avidin ( o ) ; 5 mM avidin (*••); 10 mM avidin ( • ) . Figure 4.2. Dependence of the 256-259nm interpeak distance of the second derivative spectrum of avidin on the concentration of added NAPhe. 105 Table 4.1 Stoichiometry of b i o t i n - avidin interaction as determined by three methods. Sample Method Avidin (nmoles) B i o t i n (nmoles) A/B R a t i o a Avidin HABA/A 2 8 0 10. 20 39.58 3. 88 +. 005 ANS/A 2 8 0 10 . 20 36 .52 3.58±. 009 HABA/Phe 10. 5 39.58 3.8 ±. 19 ANS/Phe 10 . 5 36 .52 3.5 i . 18 Impure Avidin B i o t i n column 63.0 252.0 3. 9 4 ± . 06 aAverage of 3 determinations + standard deviation. 106 used to c a l c u l a t e a v i d i n concentration, based on the known phenylalanine content of a v i d i n of 7 residues per a v i d i n subunit (Green, 1975). Subsequently, a v i d i n concentration was used to determine the b i o t i n / a v i d i n r a t i o as reported for the HABA/Phe and 1,8 ANS/Phe methods i n Table 4.1. Column Method The molar r a t i o of b i o t i n and a v i d i n i n t e r a c t i o n was also estimated with use of a b i o t i n y l a t e d Sephadex G-50 column. When a v i d i n was allowed to bind to the b i o t i n y l a t e d column, other b i o t i n binding s i t e s of a v i d i n remained capable of binding b i o t i n . Quantitation of the r e s i d u a l b i o t i n binding of bound a v i d i n was accomplished by assaying the decrease i n b i o t i n concentration of a b i o t i n s o l u t i o n passed through the a v i d i n loaded column. The mean molar r a t i o of three such determinations together with the standard d e v i a t i o n i s presented i n Table 4.1. DISCUSSION. At l e a s t f i v e methods have been described for measuring b i o t i n binding by a v i d i n , including a method using enzyme l a b e l l e d b i o t i n (Gebauer and Rechnitz, 1980), one using 3H l a b e l l e d b i o t i n (Rettenmaier, 1980), one employing an enzyme cofactor l a b e l l e d b i o t i n (Carrico et a l . , 1976) and two methods which employ chromophores which are displaced from a v i d i n by the binding of b i o t i n (Mock et a l . ,1985 and Green, 1965). Of these, the method of Green 107 the displacement of HABA from avidin may be considered the method of choice because of i t s s i m p l i c i t y and sharply defined endpoint. Mock et a l . (1985) found th e i r 2,6 ANS method s l i g h t l y more sensitive than the method of Green (1965), but more time consuming. In t h i s study, 1,8 ANS was found to behave generally i n a similar fashion to 2,6 ANS. However, the 1,8 ANS method consistently underestimated b i o t i n binding capacity of avidin when compared to the HABA method. Phenylalanine content as determined from the second derivation u l t r a v i o l e t spectrum was useful as confirmation of the avidin content of pure, or almost pure samples. The results of the assay were consistant with the estimates based on the molar extinction c o e f f i c i e n t at 280nm. but with decreased precision. For example, t r i p l i c a t e determinations of one sample gave a mean of 0.690 ± 0.009 mg avidin/mL by the A 2 8 0 method and 0.71 + 0.03 mg avidin/mL by the phenylalanine method. Both methods may be combined with the two dye binding methods to y i e l d estimates of b i o t i n -avidin stoichiometry. The biotin-Sephadex method introduced here gave comparable results to the HABA-A23o procedure, a l b e i t with a s l i g h t l y greater standard deviation (Table 4.1). The new method did however have the very s i g n i f i c a n t advantage of not requiring extensive preliminary p u r i f i c a t i o n of avidin. This has obvious advantages of convenience but, perhaps more 108 s i g n i f i c a n t l y , i t decreases the opportunity for error due to contamination with extraneous b i o t i n during a protracted laboratory processing of the avidin samples. CONCLUSIONS. The molar r a t i o of avidin to available b i o t i n binding s i t e s was estimated by 5 methods. When dealing with highly p u r i f i e d avidin fractions the HABA-A2 g onm meth'od proposed by Green, (1965) was superior. A new method based on avidin a c t i v i t y as determined by the HABA method and avidin molarity based on phenylalanine content gave comparable results but with reduced precision. A second new method which u t i l i z e d a column of immobilized b i o t i n and which did not require extensive p u r i f i c a t i o n of avidin was also found to give similar results. This column method may be p a r t i c u l a r l y a t t r a c t i v e for routine determinations of b i o t i n binding capacity of commercial avidin preparations. 109 V. Quantitation of Proteins Immobilized on N i t r o c e l l u l o s e  Membranes using A v i d i n Mediated Peroxidase L a b e l l i n g . INTRODUCTION SDS-PAGE has become a standard and a l l but ind i s p e n s i b l e procedure for p r o t e i n a n a l y s i s . Detection of pr o t e i n bands on SDS-PAGE with the s e n s i t i v e Coomassie Blue R-250 s t a i n (Laemmli, 1970), or the u l t r a s e n s i t i v e s i l v e r s t a i n ( M e r r i l et a l . , 1980) are likewise common procedures. Quantitation of pr o t e i n bands by these procedures however i s often u n s a t i s f a c t o r y . The pr o t e i n concentration of Coomassie Blue stained bands may be estimated by densitometer tracings or by spectrophotometry estimation of eluted dye (Wong et a l . , 1985), but p r e c i s i o n i s often poor. Also, s t a i n i n g i n t e n s i t y has been shown to be an i n t r i n s i c function of s p e c i f i c proteins (Fishbein, 1972). In the case of s i l v e r s t a i n i n g the duration of the reaction with s i l v e r n i t r a t e and developer concentration e f f e c t the depth of color development through the gel cro s s - s e c t i o n (Pochling and Newholf, 1981), while some proteins are not stained at a l l (eg. pepsin, a v i d i n ) . In addi t i o n , proteins at the same concentration can y i e l d bands of d i f f e r e n t widths, further complicating the problems of uneven s t a i n penetration into the gels (Quitschke and Schechter, 1982). The t r a n s f e r of proteins from polyacrylamide electrophoresis gels to the surface of n i t r o c e l l u l o s e sheets 110 has gained popularity largely because the transferred protein, exposed on the n i t r o c e l l u l o s e membrane, becomes available for a variety of reactions and a n a l y t i c a l procedures which are impractical within the gel matrix (Towbin et a l . , 1979). Although electrophoretic transfer of proteins (Western Blotting) i s the most common method of immobilizing proteins on n i t r o c e l l u l o s e membranes, i t i s not the only approach. When electrophoretic transfer is unavailable or inappropriate, transfer from polyacrylamide gels by d i f f u s i o n i s possible. Protein solutions may also be spotted d i r e c t l y onto n i t r o c e l l u l o s e and simply allowed to dry. Staining and detection may be accomplished by a variety of methods, including amido black 10B, Coomassie blue, india ink, or c o l l o i d a l gold (Hancock and Tsang, 1983; Rohringer and Holden, 1985) . A highly sensitive protein detection system intended for v i s u a l i z a t i o n of membrane bound proteins u t i l i z i n g avidin i s commercially available. This procedure, which labels proteins with horseradish peroxidase v i a a bio t i n - a v i d i n linkage, was reported to routinely detect 30ng quantities of membrane bound protein (Anon., 1986b). The dark coloured bands may be subjectively ranked as to re l a t i v e intensity or estimated by reflectance spectrophotometry. This assay system i s marketed by BioRad (Richmond, CA. ) under the name B i o t i n Blot Protein Detection. I l l A major drawback to electrophoretic protein analysis i s the length of time necessary to run, stain, and destain samples before results can be evaluated. An apparatus for accelerated electrophoresis has become available recently, through the Pharmacia Phastsystem (Pharmacia Canada Inc., Dorval, Montreal) which allows i ' electrophoresis, staining, and destaining of protein samples on miniaturized 4cm x 4cm SDS-PAGE or native protein PAGE. The goal of the study reported here was to develop a method whereby proteins immobilized on n i t r o c e l l u l o s e sheets could be assayed quantitatively, using a modification of the Bio-Rad B i o t i n Blot protein detection method. This method was employed to assay protein applied d i r e c t l y to n i t r o c e l l u l o s e , electrophoretically transferred from 12cm x 12cm SDS-PAGE gels or d i f f u s i o n transferred from 4cm x 4cm SDS-PHAST gels. MATERIALS AND METHODS. Reagents. Acrylamide reagents, sodium dodecyl sulphate (SDS) and protein standards were purchased from Sigma Chemical Co. (St.Louis, MO). Nitrocellulose (30 cm x 30 cm membrane f i l t e r s , 0.45 fim pore size) were purchased from MSI (Honeoye F a l l s , N.Y.). PHAST gels (SDS-PAGE, 10-15% gradient gels) were purchased from Pharmacia (Pharmacia Canada ., Dorval, Que.). Biotin-Blot reagents were obtained from Bio-Rad (Richmond, CA.). 112 Prote i n Assays Concentrations of pr o t e i n i n s o l u t i o n were estimated by determining absorbance at 280nm using the appropriate e x t i n c t i o n c o e f f i c e n t ( i e . bovine immunoglobulin G (IgG), E 1 % @280 nm = 13.5; bovine B - l a c t o g l o b u l i n (B-Lg), E 1 % <§ 280 nm = 9.5). "Microdotting". Protein solutions (0.25-1.50 mg/mL) were i n 0.1M sodium phosphate buf f e r , -pH 6.5, with or without 2.5% SDS and 5% mercaptoethanol. When SDS and mercaptoethanol were used, p r o t e i n samples were heated with the reagents for 5 minutes i n a b o i l i n g water bath. N i t r o c e l l u l o s e membranes were b r i e f l y immersed i n phosphate buf f e r , then allowed to dry for 5 minutes on f i l t e r paper. Protein s o l u t i o n samples (lfiL) were applied with a Hamilton microsyringe and allowed to dry before proceeding. D i f f u s i o n B l o t t i n g of PHAST gels. PHAST SDS 10-15% polyacrylamide gradient gels were used according to the manufacturers d i r e c t i o n s . Protein samples were loaded onto the PHAST gels with the sample ap p l i c a t o r s which were supplied with the system (comb shaped p l a s t i c a p p l i c a t o r s ) . Each t i n e of the comb contained a c a p i l l a r y groove of s u f f i c e n t s i z e to pick up lfiL of sample when brought into contact with the sample s o l u t i o n . PHAST gels could not be e l e c t r o p h o r e t i c a l l y b l o t t e d due to the p l a s t i c backing of the pre-made gels. 113 G L A S S P L A T E P H A S T G E L N I T R O C E L L U L O S E MEMBRANE DRY F I L T E R P A P E R G L A S S P L A T E Figure 5.1. Diffusion blotting of protein bands from PHAST electrophoresis gels onto n i t r o c e l l u l o s e membranes. 114 Therefore, proteins were t r a n s f e r r e d to n i t r o c e l l u l o s e by passive d i f f u s i o n (Figure 5.1) for two hours. Following t h i s period the membrane was b i o t i n y l a t e d as described below. The depleted PHAST gels were s i l v e r stained to reveal the r e s i d u a l p r o t e i n bands, and used as a template for excising p a r t i c u l a r p r o t e i n bands from the membranes for p r o t e i n -bound peroxidase a c t i v i t y assays as o u t l i n e d below. Western B l o t t i n g . P r o t e i n samples were subjected to electrophoresis on SDS-PAGE e s s e n t i a l l y according to Laemmli (1970), on 12cm x 12cm polyacrylamide gels. Separation was at 100 v o l t s , constant voltage, i n 0.01M T r i s - g l y c i n e b u f f e r , pH 8.3, for 4 h at room temperature. When electrophoresis was complete, the gels were immediately "blotted" onto n i t r o c e l l u l o s e as described by Szewczyk and Kozloff (1985). B r i e f l y , the electrophoresis gel was placed on a wet Scotch-Brite pad (3M Canada Inc. London, Ont.), followed by a n i t r o c e l l u l o s e sheet wetted i n t r a n s f e r buffer (25mM Tris/192mM g l y c i n e , pH 8.3, containing 20% methanol), being c a r e f u l to avoid a i r bubbles under the membrane. The f i l t e r was then covered with two sheets of wet Whatman 3MM f i l t e r p a p e r and a second Scotch-Brite pad. The assembly was then placed i n a Pharmacia e l e c t r o p h o r e t i c destainer apparatus with the n i t r o c e l l u l o s e membrane toward the anode, and the destainer was f i l l e d with t r a n s f e r buffer. Protein t r a n s f e r s were c a r r i e d out for 90 minutes at 36 v o l t s . Following t r a n s f e r , r e s i d u a l p r o t e i n bands i n the gel were v i s u a l i z e d with 115 visualized, with s i l v e r stain (Merril et a l . , 1980) or with Coomassie B r i l l i a n t Blue R-250 (Laemmli, 1970). Nitr o c e l l u l o s e blots were biotinylated and labelled with peroxidase as outlined below. Particular protein bands were excised, using the residual protein bands of the PAGE as a template, and assayed for peroxidase labelled protein. Peroxidase Labelling of Immobilized Proteins. Protein bands were biotinylated, then allowed to interact with avidin-peroxidase conjugate as described in the Bio-Rad Biotin-Blot (Bio-Rad, Richmond, CA) protein detection k i t manual. B r i e f l y , the blot membrane was washed in two changes of 0.05M sodium borate, 0.2% Tween-20, pH 9.3 for 2 x 10 minutes with agitation. Next, i t was submerged in fresh borate-Tween-20 to which was added 200 pL of 75mM N-hydroxy succinimide biotinate (NHSB) i n dimethylformamide. After 15 minutes of agitation, the membrane was again washed in borate-Tween-20, pH 7.5. Next a 1:1000 d i l u t i o n of Bio-Rad Avidin-Horseradish Peroxidase conjugate reagent was prepared and the membrane was incubated i n t h i s solution for one hour with agitation. The labelled membrane was then washed i n four changes of Tris-NaCl buffer before proceeding to the peroxidase a c t i v i t y assay. Peroxidase A c t i v i t y of Immobilized, Labelled Proteins. Pyrogallol or guiacol were used as peroxidase electron donors. Assay conditions for peroxidase using pyrogallol were as follows: 0.32mL of assay buffer, 0. 1M sodium phosphate, pH 6.0; 0.16mL of 0.147M hydrogen peroxide; 0.32mL of 5% pyrogallol in H 20; 2.10 mL H20; temperature approximetely 22°C. The oxidation of pyrogallol was monitored as the increase i n absorbance at 420nm. Segments of n i t r o c e l l u l o s e , bearing protein bands, were cut to f i t in the bottom of a lcm x lcm x 5cm cuvette and topped with a magnetic s t i r bar. A l l reagents except hydrogen peroxide were added to the cuvette and used to zero the spectrophotometer (Cary 210). The hydrogen peroxide was then added and a stop watch started while s t i r r i n g vigorously from 30 to 180 seconds. At the end of that period, the absorbance was recorded. A similar procedure was employed with guiacol as the electron donor except that the buffer pH was 7.0, the electron donor solution consisted of 0.22mL of guiacol in d i s t i l l e d water, and the reaction was monitored at 470nm. (Chance and Maehly, 1954). RESULTS AND DISCUSSION. Choice of Electron Donor. Simple staining methods for peroxidase labels employ electron donors such as 4-chloro-l-naphthol or 3,3' diaminobenzidine tetrahydrochloride which y i e l d insoluble products when oxidized by the enzyme (Pearse, 1980). However, i n t h i s instance, where spectrophotometry assaying of products was intended, a more soluble product was 117 desirable. Two well known peroxidase electron donors were examined; guiacol and pyrogallol. In a preliminary experiment, pyrogallol was found to give greater s e n s i t i v i t y when microdots of bovine igG (20 jxg) on ni t r o c e l l u l o s e were labelled with peroxidase. Incubation periods of 30 seconds gave a mean AA420 of 0.17 with pyrogallol but gave a mean AA470 of only 0.005 with guiacol. Pyrogallol was used thereafter. Microdot Protein Assay. Bovine IgG was denatured with SDS and mereaptoethano1, then dotted onto n i t r o c e l l u l o s e sheets and labelled with peroxidase. The relationship between protein applied to the n i t r o c e l l u l o s e and peroxidase a c t i v i t y i s presented i n Figure 5.2. The relationship appears to be s u f f i c e n t l y linear within the range tested to make peroxidase a c t i v i t y a useful indicator of protein. The method was also sucessful without denaturation of the protein sample with SDS and mercaptoethanol. Native /3-lactoglobulin (B-Lg) microdots gave a roughly linear response (Figure 5.3), although the slope was less than that for denatured IgG. Apparently the B-Lg bound less peroxidase than the denatured IgG, but whether t h i s was due to decreased exposure of the alpha and epsilon amino groups to which NHSB binds in the undenatured protein, or due to an i n t r i n s i c difference between IgG and B-Lg was not determined. Figure 5.2. Assay of enzyme labeled IgG "microdots" on n i t r o c e l l u l o s e by peroxidase a c t i v i t y : means ± standard d e v i a t i o n of t r i p l i c a t e determinations. 119 .0100 .0094 • .0088 • .0082 • .0076 • o ^ .0070 1 .0064 • .0058 -.0052 • .0046 -.0040 0.50 0.75 1.00 B - L g (micrograms) 1.25 Figure 5.3. Assay of enzyme labeled B-Lg on n i t r o c e l l u l o s e by peroxidase a c t i v i t y : means ± standard deviation of t r i p l i c a t e determinations. 120 Certainly, simpler protein assays are available, although t h i s procedure was very sensitive and might be useful when only very small quantities of protein are available. As l i t t l e as 0.12/ig could be assayed in t h i s manner. By comparison approximately 50jU,g of protein i s required for the Lowry protein assay (Peterson, 1977) or a minimum of approximately lOjug protein for the Coomassie blue dye binding method (Bradford, 1976). Assay of Western Blot Protein Bands. Mixtures of bovine IgG and B-Lg were separated by electrophoresis (SDS-PAGE), Western blotted and biotinylated. Excised segments of n i t r o c e l l u l o s e corresponding to individual protein bands were then assayed for protein by the peroxidase l a b e l l i n g method. A plot of the quantity of B-Lg applied to the gel vs. peroxidase a c t i v i t y on the n i t r o c e l l u l o s e membrane did not show a linear trend (Figure 5.4) The peroxidase a c t i v i t y response could be somewhat increased by using longer incubation times in the peroxidase assay. However, when the r a t i o of B-Lg peroxidase response to IgG heavy chain peroxidase response was plotted against the o r i g i n a l r a t i o of B-Lg to IgG in the applied samples, a linear relationship was demonstrated (Figure 5.5). The r a t i o plot was p a r t i c u l a r l y interesting because i t may avoid several sources of error inherent in assaying absolute amounts of protein by t h i s method. To use t h i s method for absolute determination requires an assumption of 100% efficency of transfer from PAGE to 121 .0.32 0 1 1 1 H -0 7.5 15 30 B - L g (micrograms) Figure 5.4. Peroxidase a c t i v i t y of enzyme labeled. Western blots of B-Lg electrophoresis bands. A A 4 2 0 w a s determined over 30s, 60s or 90s periods of incubation. 1 2 2 0 J i 1 1 1 1— 0 0.5 1.0 1.5 2.0 B-Lg / IgG Ratio Figure 5.5. Assay of r e l a t i v e concentration of B-Lg and IgG bands on Western blots (12 cm x 12 cm gels) by a c t i v i t y of peroxidase labels. 123 n i t r o c e l l u l o s e , an assumption which i s c e r t a i n l y not j u s t i f i e d , given that r e s i d u a l p r o t e i n traces were r o u t i n e l y detected i n gels following b l o t t i n g . I f the r a t i o of proteins was determined i t was only necessary to accept error due to heterogeneous t r a n s f e r of d i f f e r e n t proteins i n the same lane. Errors i n sample a p p l i c a t i o n were likewise avoided. B l o t t i n g of PHAST Gels. The PHAST electrophoresis system of Pharmacia Ltd.-was a very convenient and rapid procedure for SDS and native p r o t e i n electrophoresis. Electrophoresis, s t a i n i n g , and destaining of proteins was accomplished i n 2 to 3 hours. The system used miniature 4cm X 4cm polyacrylamide gels cast d i r e c t l y on a t h i n p l a s t i c sheet s i m i l a r to Gelbond. Adaptation of the p r o t e i n assay to t h i s electrophoresis system presented three s p e c i a l problems. F i r s t , the p l a s t i c backing of the gels precluded e l e c t r o p h o r e t i c t r a n s f e r of proteins to n i t r o c e l l u l o s e and d i f f u s i o n b l o t t i n g was necessary. D i f f u s i o n b l o t t i n g was expected to be slower and less e f f i c i e n t than "Western" b l o t t i n g . Secondly,• the very small s i z e of the gels made the task of accurately excising i n d i v i d u a l bands from the n i t r o c e l l u l o s e b l o t more d i f f i c u l t . F i n a l l y , the sample a p p l i c a t i o n method used i n the PHAST system, although rapid and simple, does not lend i t s e l f to accurate c o n t r o l of sample volumes. Assay r e s u l t s r e f l e c t e d these increased sources of experimental error. 124 Table 5.1 Peroxidase a c t i v i t y of enzyme labeled n i t r o c e l l u l o s e b l o t s of pr o t e i n samples containing IgG heavy chain, separated on PHAST SDS electrophoresis gels. Only the the t o t a l p r o t e i n concentration was va r i e d between samples; the r e l a t i v e composition of the samples remained constant. Protein (ug) Peroxidase To t a l A c t i v i t y IgG H.C. ( A A 4 2 Q ) Ratio IgG H.C./Total 0. 38 . 0019 .0005 0 . 26 0. 50 .0047 .0010 0.21 0. 75 .0114 .0026 0. 23 0. 75 . 0099 .0022 0. 22 125 The concentration of the IgG heavy chain band r e l a t i v e to the rest of a protein sample was estimated in terms of peroxidase a c t i v i t y of PHAST blots (Table 5.1). The proportion of peroxidase a c t i v i t y associated with the IgG heavy chain remained f a i r l y constant even though the protein concentration was changed. In a similar experiment, peroxidase a c t i v i t y of labelled B-Lg bands and IgG heavy chain bands were plotted against the ra t i o of these two proteins i n the protein samples (Figure 5.6). Although a general indication of cor r e l a t i o n between the two ratios may be suggested (correlation c o e f f i c i e n t r = 0.69) the v a r i a b i l i t y of the results was too high for an accurate estimate of protein r a t i o s . This may be p a r t i a l l y due to the small amount of protein present i n each protein band, on the order of 0.2 fig, but the protein microdotting results indicated that the detection method was s u f f i c i e n t l y sensitive. Greater error probably arose from inaccurate cutting of the appropriate bands from the ni t r o c e l l o s e blots of the 4cm x 4cm PHAST gels. CONCLUSIONS Nitrocellulose membranes are the most commonly used membrane for protein b l o t t i n g of electrophoresis gels (Szewczyk and Kozloff, 1985) and a quantitative assay of protein blots could be useful i n many a n a l y t i c a l situations. For example, SDS-PAGE may be used to detect proteins for 1 2 6 3.0-2.5 + 2.0-X X o CN < <3 1.3 + CP m < 1.0 + •5 + 1.0 2 . 0 B - L g / IgG R a t i o 3.0-Figure 5 . 6 . Assay of relative concentrations of B-Lg and IgG bands on PHAST diffusion blots (4 cm x 4 cm gels) by a c t i v i t y of peroxidase labels. 127 which s p e c i f i c a c t i v i t y assays are unavailable or cumbersome such as B-Lg and IgG, or to estimate r e l a t i v e concentrations of individual proteins i n non-homogeneous solutions. As mentioned above, estimating protein concentrations i n PAGE from staining densities i s prone to error. Transfer b l o t t i n g followed by a spectrophotometry protein assay provides an i a t t r a c t i v e alternative. The spectrophotometry assay of peroxidase bound to protein immobilized on n i t r o c e l l u l o s e was found to be a highly sensitive assay of protein, with sufficent precision for many applications. The method may be applied to SDS denatured proteins or native proteins. Although standard curves would be required for absolute protein determinations, r e l a t i v e proportions of proteins i n samples of similar composition may be obtained d i r e c t l y . Although not investigated i n t h i s study, a p o s s i b i l i t y exists for even greater s e n s i t i v i t y of the protein assays with the selection of a more suitable detection system. This could consist of a better peroxidase electron donor, or of an e n t i r e l y d i f f e r e n t , more active enzyme label . 128 REFERENCES Ahvenainen, R., M. Heikonen, M. Kreula and P. Linko. 1980. Separation of lysozyme from egg white. Food Process Engr. 2:301. Akashi, A. 1970. 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