Reaction of Ozone with Lysozyme - ACS Symposium Series (ACS

3 Reaction of Ozone with Lysozyme F.

LEH

and

J. B.

MUDD

Department of Biochemistry and Statewide Air Pollution Research Center,

Downloaded by CORNELL UNIV on October 23, 2016 | http://pubs.acs.org Publication Date: June 1, 1974 | doi: 10.1021/bk-1974-0003.ch003

University of California, Riverside, Calif. 92502

Abstract Hens egg l y s o z y m e is inactivated b y o z o n e in 1.5-2.5 m o l a r ratios. The pH optimum for t h e native and o z o n i z e d l y s o z y m e is t h e same. O z o n i z e d l y s o z y m e b e h a v e s differently from native l y s o z y m e on ion e x c h a n g e c o l u m n s and in polyacrylamide gel electrophoresis but t h e product behaves as a single polypeptide chain in b o t h analyses. Amino acid a n a l y s e s showed that a t 95-100% inactivation t h e modified residues a r e tryptophan, tyrosine and m e t h i o n i n e . C h a n g i n g t h e pH o f t h e reaction mixture f r o m 4.6 t o 10.2 d e c r e a s e s t h e amount of t r y p t o p h a n reacting ( 1 . 8 t o 1 . 3 m o l e s / m o l e l y s o z y m e ) , increases t h e amount o f tyrosine reacting ( 0 . 1 3 - 0 . 7 3 m o l e s / m o l e l y s o z y m e ) , and d o e s n o t change t h e amount of m e t h i o n i n e reacting (0.8 m o l e s / m o l e lysozyme). Analysis of t h e fragments o b t a i n e d by cyanogen b r o m i d e c l e a v a g e shows that m e t h i o n i n e 105 is c o n v e r t e d t o m e t h i onine sulfoxide. R e v e r s i o n of this residue to m e t h i o n i n e d o e s not restore activity. D i g e s t i o n of t h e o z o n i z e d l y s o z y m e with trypsin and isolation of the tryptophan-containing fragments showed that t h e modified tyrosine is residue 23 a n d t h e modified t r y p t o p h a n residues a r e 108 and 1 1 1 . Circular dichroism spectra of o z o n i z e d l y s o z y m e c o n f i r m e d t h e modification of t h e amino acids and also showed considerable destruction of t h r e e dimensional structure. E x p e r i m e n t s with c a r b o x y m e t h y l chitin indicated that t h e binding site of t h e ozonized l y s o z y m e is unaffected. Introduction Ozone i s an i m p o r t a n t p o l l u t a n t o f u r b a n a t m o s p h e r e s , and i s g e n e r a t e d b y p h o t o c h e m i c a l r e a c t i o n s on p r i m a r y p o l l u t a n t s e m i t t e d from a u t o m o b i l e exhaust ( 1 ) . Ambient ozone c o n c e n t r a t i o n s c a u s e a c u t e damage t o v e g e t a t i o n . The b i o c h e m i c a l b a s i s f o r t h i s damage i s unknown a l t h o u g h s e v e r a l p o s s i b i l i t i e s have been s u g g e s t e d . T h i s paper p r e s e n t s r e s u l t s a p p l i c a b l e t o the s t u d y o f p l a n t damage e v e n t h o u g h t h e s u b j e c t o f s t u d y i s h e n s

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Dugger; Air Pollution Effects on Plant Growth ACS Symposium Series; American Chemical Society: Washington, DC, 1974.

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Ozone with Lysozyme

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egg

lysozyme. Exposure o f a n i m a l s t o ozone l o w e r s t h e i r r e s i s t a n c e t o b a c t e r i a l i n f e c t i o n o f t h e l u n g s ( 2 ) . Ozone e x p o s u r e a l s o c a u s e s c h a n g e s i n t h e p r o p e r t i e s o f m a t e r i a l t h a t can b e l a v a g e d f r o m the lungs: t h e r e i s i n a c t i v a t i o n o f t h e enzymes a c i d p h o s phatase, ^-glucuronidase and lysozyme, and t h e r e i s a tendency f o r t h e s e enzymes t o have b e e n r e l e a s e d f r o m t h e a l v e o l a r macrophage c e l l s ( 3_). I n v i v o i n a c t i v a t i o n o f l u n g l y s o z y m e h a s b e e n s t u d i e d (4)» a n d i t h a s a l s o b e e n r e p o r t e d t h a t t h e c o n t e n t o f l y s o z y m e i n human t e a r s i s l o w e r e d d u r i n g e x p o s u r e t o a m b i e n t a i r p o l l u t i o n (5_). These o b s e r v a t i o n s have s t i m u l a t e d o u r i n t e r e s t i n a s t u d y o f t h e mechanism o f l y s o z y m e i n a c t i v a t i o n b y o z o n e s i n c e i t may b e i n v o l v e d i n e l i c i t i n g t h e t o x i c r e s p o n s e . A p r e v i o u s s t u d y o f t h e r e a c t i o n o f ozone w i t h lysozyme d i s s o l v e d i n a n h y d r o u s f o r m i c a c i d gave r i s e t o t h e c o n c l u s i o n t h a t t h e o n l y amino a c i d r e s i d u e s a f f e c t e d i n t h e e a r l y s t a g e s o f t h e r e a c t i o n were t h e t r y p t o p h a n r e s i d u e s 108 a n d 1 1 1 (6)· C o n v e r s i o n o f t h e s e r e s i d u e s t o Ν -formyl-kynurenine d i d n o t c a u s e l o s s i n enzyme a c t i v i t y . Imoto et_ al* ( 7 ) have p o i n t e d o u t t h a t t h i s r e s u l t i s anomalous s i n c e m o d i f i c a t i o n s o f t r y p t o p h a n 108 ( e . g . w i t h i o d i n e ) n o r m a l l y c a u s e s i n a c t i v a t i o n . We hoped t h a t o u r s t u d y w o u l d r e s o l v e t h i s a n o m a l y . 1

M a t e r i a l s a n d Methods Ozonolysis. Ozone was g e n e r a t e d f r o m o x y g e n b y s i l e n t e l e c t r i c d i s c h a r g e , and bubbled through the r e a c t i o n mixtures f r o m a c a p i l l a r y t i p . The o z o n e c o n c e n t r a t i o n was m o n i t o r e d s p e c t r o p h o t o m e t r i c a l l y a t 350 nm b y t h e K I method a s p r e v i o u s l y d e s c r i b e d (8)* Material. 3 X c r y s t a l l i z e d , d i a l y z e d a n d l y o p h i l i z e d Grade 1 Hen E g g W h i t e Lysozyme was o b t a i n e d f r o m Sigma C h e m i c a l Company, a n d u s e d w i t h o u t f u r t h e r p u r i f i c a t i o n . P u r i t y o f the p r o t e i n was v e r i f i e d b y b o t h g e l e l e c t r o p h o r e s i s o n a c r y l a m i d e and c h r o m a t o g r a p h y o n a c o l u m n p a c k e d w i t h a n i o n e x c h a n g e r e s i n ( C l " f o r m ) Sephadex DEAE. The sample showed a s i n g l e peak i n these analyses. C h r o m a t o g r a p h i c A n a l y s i s . The s a m p l e s o f n a t i v e a n d o z o n i z e d lysozyme (lysozyme t r e a t e d w i t h ozone j u s t t o t h e p o i n t o f c o m p l e t e i n a c t i v a t i o n ) were a n a l y z e d b y c o l u m n c h r o m a t o g r a p h y . The column (0.8 X 56 cm.) c o n t a i n i n g DEAE-Sephadex A-50 ( C l " f o r m ) r e s i n , was e q u i l i b r a t e d w i t h 0.1 M T r i s C l b u f f e r , pH 8.3, a n d l o a d e d w i t h a b o u t 2-4 mg o f p r o t e i n . A l i q u o t s e l u t e d w i t h 0.1 M T r i s - C l pH 8.3 were c o l l e c t e d a n d a b s o r b a n c e a t 278 nm was m e a s u r e d . The n a t i v e l y s o z y m e e l u t e d e a r l i e r t h a n the ozonized products. T h i s d i f f e r e n c e may be a s s o i c a t e d w i t h both aggregation o f p r o t e i n and i o n i c behavior o f the r e s i d u e s .


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G e l E l e c t r o p h o r e s i s . T h i s method was u s e d i n t h e d e t e r m i n a t i o n o f t h e p u r i t y o f n a t i v e l y s o z y m e and i d e n t i f i c a t i o n o f o z o n ized products. D i f f e r e n t g e l c o n c e n t r a t i o n s ( 7 , 8 , 9 , 1 0 % ) and b u f f e r s o l u t i o n s (0.25M b o r a t e , pH 8.7; 0.025M p h o s p h a t e , pH 7.1) were t r i e d and t h e b e s t r e s u l t s were o b t a i n e d w i t h 7% g e l i n pH 8.7 b u f f e r . Enzymic A c t i v i t y . Lysozyme a c t i v i t y was d e t e r m i n e d b y f o l l o w i n g the r a t e of l y s i s o f d r i e d Micrococcus l y s o d e i k t i c u s c e l l s a c c o r d i n g t o t h e method o f Shugar (9)· A s s a y s were r u n a t r o o m t e m p e r a t u r e i n 0.1M p h o s p h a t e b u f f e r pH 7.0, w i t h an enzyme c o n c e n t r a t i o n o f a b o u t 0.05 mg/ml. A s o l u t i o n o f n a t i v e l y s o z y m e a t t h e same p r o t e i n c o n c e n t r a t i o n was a l w a y s a s s a y e d as s t a n d a r d , a l o n g w i t h o z o n i z e d lysozymes. Cyanogen B r o m i d e D i g e s t i o n . I n o r d e r t o c l e a v e t h e d i s u l f i d e b o n d s , t h e o z o n i z e d l y s o z y m e ( 0 . 6 pmole) was t r e a t e d w i t h d i t h i o t h r e i t o l ( 1 0 ) (8 mg. 110%) i n 9 M u r e a f o r 20 h r . The u r e a was t h e n removed by p a s s i n g t h e s o l u t i o n t h r o u g h a c o l u m n c o n t a i n i n g Sephadex G-25. A l i q u o t s were c o l l e c t e d , c h e c k e d f o r a b s o r b a n c e a t 278 nm, l y o p h i l i z e d and t h e n a l l o w e d t o r e a c t w i t h c y a n o g e n b r o m i d e ( 1 1 ) (50 m o l a r e x c e s s r e l a t i v e t o m e t h i o n i n e ) i n 70% f o r m i c a c i d T l ml.) f o r 24 h r . The m i x t u r e was l y o p h i l i z e d and d i s s o l v e d i n 0.2N HAc. T h r e e components s e p a r a t e d by p a s s a g e t h r o u g h a Sephadex G-25 ( 3 X 100 cm.) column were c o l l e c t e d f o r amino a c i d a n a l y s i s . T r y p t i c D i g e s t i o n . The o z o n i z e d l y s o z y m e s o l u t i o n a f t e r t r e a t m e n t w i t h d i t h i o t h r e i t o l i n 9 M u r e a a t pH 8.0 as d e s c r i b e d a b o v e , was c a r b o x y m e t h y l a t e d w i t h s o d i u m i o d o a c e t a t e (0.02 M) a t pH 4.6 and 25°C. f o r 24 h r , and p u r i f i e d by p a s s a g e t h r o u g h a Sephadex G-25 c o l u m n . H y d r o l y s i s o f l y s o z y m e by t r y p s i n ( 1 % ) , and s e p a r a t i o n o f t h e p r o d u c t s were p e r f o r m e d u n d e r t h e c o n d i t i o n s a s p r e v i o u s l y d e s c r i b e d by J o l i e s e t a l . ( 1 2 ) . C h a r a c t e r i z a t i o n o f E n z y m e - S u b s t r a t e Complex by use o f C M - C h i t i n ( C a r b o x y m e t h y l C h i t i n J T C M - c h i t i n was p r e p a r e d by c a r b o x y m e t h y l a t i o n o f c h i t i n a c c o r d i n g t o t h e method o f I m o t o , H a y a s h i and F u n a t s u ( 1 3 ) . The o z o n i z e d l y s o z y m e ( 1 . 3 mg) s o l u t i o n s a t d i f f e r e n t pHs were n e u t r a l i z e d w i t h NaOH o r HC1 t o pH 8.0 and t h e p o u r e d i n t o t h e column (1.5 X 4 cm.) c o n t a i n i n g w h i t e c o t t o n - l i k e C m - c h i t i n (-65 mg.), w h i c h was e q u i l i b r a t e d w i t h 0.1 M T r i s - C l b u f f e r pH 8.0. A l i q u o t s were e l u t e d f i r s t w i t h 0.1 M T r i s - C l pH 8.0 and t h e n w i t h 0.2 M HAc. The a b s o r b a n c e o f t h e f r a c t i o n s was m e a s u r e d s p e c t r o p h o t o m e t r i c a l l y a t 280 nm. R e d u c t i o n o f O z o n i z e d Lysozyme. The r e d u c t i o n o f m e t h i o n i n e s u l f o x i d e r e s i d u e s o f t h e p h o t o - o x i d i z e d l y s o z y m e was a c h i e v e d by a l l o w i n g t h e enzyme t o r e a c t w i t h 2 - m e r c a p t o e t h a n o l i n aqueous



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s o l u t i o n (14)· The same p r o c e d u r e was a p p l i e d t o r e d u c e t h e methionine s u l f o x i d e residue o f ozonized lysozyme. 10 mg o f o z o n i z e d l y s o z y m e i n 10 m l o f 0.1 M p h o s p h a t e b u f f e r a t pH 7.0 was a l l o w e d t o r e a c t w i t h 200 m l o f 5% a q u e o u s 2 - m e r e a p t o e t h a n o l a t room t e m p e r a t u r e u n d e r n i t r o g e n a t m o s p h e r e f o r 24 h r . A f t e r e x t e n s i v e d i a l y s i s a g a i n s t s e v e r a l c h a n g e s o f c o l d , d i s t i l l e d w a t e r , t h e pH o f t h e t h i o l - f r e e s o l u t i o n was a d j u s t e d t o 8.3 b y t h e a d d i t i o n o f 0.1 M T r i s - C l b u f f e r s o l u t i o n . The s o l u t i o n was k e p t a t 38°C f o r 12 h r i n t h e p r e s e n c e o f a t r a c e o f 2 - m e r c a p t o e t h a n o l ( 0 . 2 mg) a n d t h e n c o n c e n t r a t e d , c h r o m a t o g r a p h e d o n Sephadex G-25 c o l u m n ( 1 . 5 X 100 cm.) a n d e l u t e d w i t h 0.2 Ν a c e t i c a c i d . The f r a c t i o n w h i c h h a d a b s o r p t i o n a t 278 nm was c o l l e c t e d a n d l y o p h i l i z e d . The r e c o v e r e d p r o d u c t was d i s s o l v e d i n 0.1M p h o s p h a t e b u f f e r f o r t h e l y t i c a c t i v i t y t e s t . C i r c u l a r D i c h r o i s m M e a s u r e m e n t s . C i r c u l a r d i c h r o i s m mea s u r e m e n t s were c a r r i e d o u t b y u s i n g a C a r y m o d e l 6002 s p e c t r o p o l a r i m e t e r c a l i b r a t e d w i t h d-10 camphor s u l f o n i c a c i d . A l l m e a s u r e m e n t s were r u n a t room t e m p e r a t u r e i n t h e same 1 cm, q u a r t z c e l l o v e r t h e n e a r - u l t r a v i o l e t r e g i o n ( 2 5 0 - 3 3 0 nm). P r o t e i n c o n c e n t r a t i o n s were a p p r o x i m a t e l y 0.4%. Circular d i c h r o i s m d a t a were p r e s e n t e d a s mean r e s i d u e e l l i p t i c i t y [ Θ ] , i n degrees X cm X decimole"" . The same mean r e s i d u e w e i g h t 112.4 was e m p l o y e d f o r n a t i v e l y s o z y m e a n d i t s o z o n i z e d p r o d u c t s , s i n c e t h e d e v i a t i o n c a u s e d b y t h e o z o n o l y s i s o f f e w amino a c i d r e s i d u e s o f lysozyme i s f a r l e s s than the experimental e r r o r . 2

1

Amino A c i d A n a l y s e s . Samples o f n a t i v e a n d o z o n i z e d l y s o zyme s were h y d r o l y z e d i n e v a c u a t e d , s e a l e d t u b e s w i t h 6N h y d r o c h l o r i c a c i d a t 110 C f o r 24 h r . A f t e r b e i n g c o o l e d t o room t e m p e r a t u r e , t h e s o l u t i o n was a d j u s t e d t o pH 2 w i t h NaOH s o l u t i o n and b r o u g h t t o mark i n a v o l u m e t r i c f l a s k w i t h s o d i u m c i t r a t e b u f f e r pH 2.3. A p o r t i o n c o n t a i n i n g a s u i t a b l e amount o f t h e amino a c i d s was a p p l i e d t o a Beckman 120B amino a c i d a n a l y z e r . M e t h i o n i n e , m e t h i o n i n e s u l f o x i d e a n d t r y p t o p h a n were d e t e r m i n e d a f t e r a l k a l i n e h y d r o l y s i s , s i n c e t h e y a r e known t o d e g r a d e d u r i n g a c i d h y d r o l y s i s . F o r t h i s p r u p o s e , 3N NaOH was u s e d . The h y d r o l y s e s w e r e p e r f o r m e d i n s e a l e d e v a c u a t e d s i l i c o t u b e s a t 100°C f o r 17 h r . Then t h e s o l u t i o n s were c o o l e d , a c i d i f i e d t o a b o u t pH 2 w i t h c o n e . HC1 a n d a n a l y z e d f o r t h e amino a c i d com position. M o d i f i c a t i o n o f Tryptophan Residues. Tryptophan residue a l s o c o u l d be determined q u a n t i t a t i v e l y b y a m o d i f i c a t i o n w i t h a s u l f e n y l a t i n g agent such as 2 - n i t r o p h e n y l s u l f e n y l c h l o r i d e i n 30% a c e t i c a c i d ( 1 5 ) . S i n c e b o t h o x i d i z e d a n d s u l f e n y l a t e d t r y p t o p h a n gave c h a r a c t e r i s t i c a b s o r p t i o n a t 365 nm, t h e e x t e n t o f t r y p t o p h a n m o d i f i c a t i o n was c a l c u l a t e d f r o m t h e mean d i f f e r ence between t h e s u l f e n y l a t e d p r o t e i n and the k y n u r e n i n e . The a b s o r p t i o n c o n t r i b u t e d b y k y n u r e n i n e was c o m p a r a t i v e l y weak.


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26

S p e c t r o p h o t o m e t r y M e a s u r e m e n t s . The measurements o f u l t r a v i o l e t s p e c t r a were c a r r i e d o u t w i t h a C a r y 15 s p e c t r o p h o t o m e t e r . The c o n t e n t s o f t r y p t o p h a n and t y r o s i n e were c a l c u l a t e d d i r e c t l y f r o m two a b s o r b a n c e s a t 288 and 280 nm b y s o l v i n g two s i m u l t a n e o u s e q u a t i o n s a s r e p o r t e d b y E d e l h o c h (16)·


Results L o s s o f t h e A c t i v i t y and t h e Change i n P r i m a r y S t r u c t u r e . Ozone c a u s e s t h e l o s s o f e n z y m i c a c t i v i t y i n l y s o z y m e a s shown i n F i g u r e 1. The enzyme i s i n a c t i v a t e d l i n e a r l y u n t i l t h e a c t i v i t y i s r e d u c e d t o 5%. P r o l o n g i n g exposure o f lysozyme t o ozone causes f u r t h e r d e c r e a s e i n enzymic a c t i v i t y . C o n t r o l ex p e r i m e n t s , u s i n g g a s s t r e a m s w i t h o u t o z o n e , were c a r r i e d o u t i n t h e same c o n d i t i o n s and o v e r t h e same p e r i o d s . F i g u r e 2 shows t h a t t h e a c t i v i t y o f b o t h c o n t r o l and o z o n i z e d l y s o z y m e v a r i e s w i t h pH c h a n g e . A l t h o u g h t h e o z o n i z e d p r o d u c t shows more e x t e n s i v e dependence on pH t h a n t h e n a t i v e o n e , t h e optimum i s t h e same. In order t o analyse the p r o p e r t i e s o f the i n a c t i v a t e d l y s o zyme, t h e enzyme was s u b j e c t e d t o f u r t h e r a n a l y s i s b y e l e c t r o p h o r e s i s and c h r o m a t o g r a p h y . B o t h a n a l y s e s o f t h e samples a t v a r i o u s pHs, i n a c t i v a t e d t o t h e e x t e n t o v e r 9 5 % , i n d i c a t e d t h a t t h e p r o d u c t i s composed o f one p e a k . The o z o n i z e d l y s o z y m e moved s l o w e r t h a n t h e n a t i v e l y s o z y m e on DEAE-Sephadex and t h e p r o d u c t s a t d i f f e r e n t pHs were r e a d i l y d i s t i n g u i s h e d f r o m e a c h o t h e r ( F i g . 3 ) . H o w e v e r , a d i f f u s e b a n d was o b s e r v e d f o r o z o n i z e d lysozyme as d i s t i n c t from a sharp band f o r n a t i v e lysozyme i n p o l y a c r y l a m i d e g e l ( 1 7 ) . The p r e s e n c e o f o n l y one b a n d s u g g e s t s t h a t t h e o z o n o l y s i s does n o t cause t h e c l e a v a g e o f p e p t i d e bonds and t h e r e m a i n i n g a c t i v i t y i s n o t due t o t h e p r e s e n c e o f a s m a l l amount o f u n m o d i f i e d l y s o z y m e . P r e v i o u s s t u d y o f t h e r e a c t i o n o f ozone w i t h lysozyme i n anhydrous f o r m i c a c i d has demonstrated t h a t i n v e s t i g a t i o n o f t h e v a r i a t i o n s i n the u l t r a v i o l e t s p e c t r a o f lysozyme p r o v i d e d use f u l i n f o r m a t i o n about t h e a l t e r a t i o n s o f p r i m a r y s t r u c t u r e b r o u g h t a b o u t b y o z o n e ( 6 ) . The u l t r a v i o l e t s p e c t r u m o f l y s o z y m e r e s u l t s p r i n c i p a l l y f r o m f o u r c h r o m o p h o r i c amino a c i d r e s i d u e s : t r y p t o p h a n , t y r o s i n e , c y s t i n e , and p h e n y l a l a n i n e w i t h t h e m a j o r c o n t r i b u t i o n s from tryptophan (E2Q3 4815; E28O 5690) and tyrosine ( E ^ = 385; Ε ο 1280; r e s i d u e s . The c o n v e r s i o n o f t r y p t o p h a n i n t o Ν -formyl-kynurenine b y o z o n e c a n be f o l l o w e d b y o b s e r v i n g a d e c r e a s e o f t h e maximum a b s o r b a n c y a t 280 nm w h i l e two p e a k s a r i s e a t 260 and 320 nm, c o r r e s p o n d i n g t o t h e two maxima o f Ν -formyl-kynurenine. The more r a p i d d i s a p p e a r a n c e o f a b s o r b a n c e a t 280 nm a s t h e pH was c h a n g e d f r o m 4.6 t o 10.2 and t h e l e s s e r f o r m a t i o n o f Ν - f o r m y l - k y n u r e n i n e i n t h e more b a s i c s o l v e n t , suggested t h a t the o x i d a t i o n o f t y r o s i n e r e s i d u e s occurs and i s p H - d e p e n d e n t . Amino a c i d a n a l y s e s r e v e a l t h a t c y s t i n e and p h e n y l a l a n i n e d i d n o t r e a c t w i t h o z o n e u n d e r t h e s e r e a c t i o n =

=

=

8 8

2 8

1

1

1


3. LEH AND MUDD

Ozone with Lysozyme

27


Figure 1. Inactivation of lysozyme by ozone. Volumes of 4.8 ml of O.J M buffer containing 2.814 mg of lysozyme were exposed to a gas stream of ozone in oxygen (1.8 nmole/min ozone). Aliquots were removed for enzyme assay as described in Materials and Methods.

Figure 2. pH dependence of native and ozonized lysozyme activity. Vol umes of 1.0 ml of 0.1 M buffer contain ing 1.91 mg lysozyme were exposed to a gas stream of 0 /0 at a flow rate of 20 ml/min for 8 min. Ozone delivery was 1.5 nmoles/min. Samples were assayed at the different pHs as de scribed in Materials and Methods. 3

2

5.0

6.0

7.0 pH

τ

ι .·

Figure 3. Chromatography of native and ozonized lysozyme on DEAE Sephadex. Samples of lysozyme at the three different pHs were inactivated 95-100% by ozone. The samples were applied to a column of DEAE-Sephadex A-50 (0.8 X 56 cm) and eluted with 0.01M Tris-HCl pH 8.3 and a gradient of 0.01M NaCl. Fractions of 2 ml were collected. The results of four columns are plotted.

8.0

I

H

EFFLUENT

10.0

9.0

1

1

NATIVE LYSOZYME 0—0 OZONIZED pH 4.6 · — · « pH 7.0 Ο — o pH 10.2 Δ — -Δ

»

VOLUME


(ml)

-

AIR

28


c o n d i t i o n s ; t h i s f a c t and t h e o b s e r v a t i o n o f u.v. s p e c t r a t h a t k y n u r e n i n e makes o n l y a s m a l l c o n t r i b u t i o n ( E 2 8 O ) t o t a l a b s o r b a n c e a t 280 nm make i t r e a s o n a b l e t o c a l c u l a t e t h e e x t e n t o f t r y p t o p h a n and t y r o s i n e m o d i f i c a t i o n f r o m a b s o r b a n c e measurements a t 288 and 280 nm b y t h e method o f E d e l h o c h ( 1 6 ) . F u r t h e r c o n f i r m a t i o n c a n be o b t a i n e d by a d e t e r m i n a t i o n o f t r y p t o p h a n f r o m t h e a b s o r p t i o n b a n d a t 365 nm d e v e l o p e d b y sulfenylation with 2-nitrophenyl sulfenyl chloride (15). Both t h e s e methods g i v e r e s u l t s i n good agreement w i t h t h o s e o b t a i n e d f r o m amino a c i d a n a l y s e s ( T a b l e I ) . A t pH 10.2, t h e u l t r a v i o l e t s p e c t r u m o f l y s o z y m e ( F i g . 4) shows a s h a r p change o f 280 and 288 nm b a n d s a f t e r t r e a t m e n t w i t h e q u i m o l a r amounts o f o z o n e . A t t h i s p o i n t , t h e l y s o z y m e r e t a i n e d 40% o f i t s a c t i v i t y . This behavior contrasted w i t h the l i n e a r c h a n g e s o f 280 and 288 nm a b s o r b a n c e a t pH 4.6 and 7.0.


=

2 1 0

t o

t n e

Location o f the Modified Residues. In order t o determine t h e l o c a t i o n and number o f amino a c i d r e s i d u e s r e a c t e d w i t h o z o n e , t h e o z o n i z e d s a m p l e s were a l l o w e d t o u n d e r g o c y a n o g e n b r o m i d e c l e a v a g e and t r y p s i n d i g e s t i o n s . B o n a v i d a , M i l l e r and S e r c a r z ( 1 1 ) have d e m o n s t r a t e d t h a t t h e r e a c t i o n o f n a t i v e l y s o z y m e w i t h CNBr i s s o l v e n t - d e p e n d e n t . No r e a c t i o n o c c u r s i n e i t h e r 2N HC1 o r 10% f o r m i c a c i d , b u t t h e m e t h i o n i n e c a n be q u a n t i t a t i v e l y c o n v e r t e d i n t o h o m o s e r i n e l a c t o n e w i t h concomitant cleavage o f the methionyl peptide i n 70% f o r m i c a c i d . C l e a v a g e o f t h e m e t h i o n y l p e p t i d e bonds o f HEL s h o u l d r e s u l t i n t h e f o r m a t i o n o f two new f r e e amino g r o u p s a t r e s i d u e 13 ( l y s i n e ) and r e s i d u e 106 ( a s p a r a g i n e ) ; t h e r e a c t i o n o f CNBr w i t h o z o n i z e d HEL w o u l d be e x p e c t e d t o g i v e p a r t i a l c l e a v a g e o f the methionyl p e p t i d e bond, because the o x i d i z e d product o f methionine m e t h i o n i n e s u l f o x i d e w i l l not r e a c t w i t h cyanogen b r o m i d e ( 1 8 ) (Scheme 1 ) . F o r s e p a r a t i o n o f C N B r - t r e a t e d HEL p e p t i d e s , t h e o z o n i z e d HEL was p r e t r e a t e d w i t h d i t h i o t h r e i t o l (DTT) i n t h e p r e s e n c e o f 9M u r e a t o c l e a v e t h e d i s u l f i d e b o n d s . A f t e r r e m o v a l o f CNBr and f o r m i c a c i d u n d e r r e d u c e d p r e s s u r e , t h e p r o d u c t was d i s s o l v e d i n 0.2N HAc and chromâtographed on a Sephadex G-25 c o l u m n . T h r e e p e a k s were o b t a i n e d a s shown i n F i g u r e 5. Amino a c i d a n a l y s e s ( T a b l e I I ) i n d i c a t e d t h a t t h e f i r s t o f t h e s e p e a k s i s t h e sum o f t h e c o r e p o l y p e p t i d e LJJ_ ( r e s i d u e s 1 3 - 1 0 5 ) , p l u s Ljy ( r e s i d u e s 1 3 - 1 2 9 ) ; t h e s e c o n d i s t h e c a r b o x y l t e r m i n a l p e p t i d e LJJLI ( r e s i d u e s 1 0 6 - 1 2 9 ) ; and t h e t h i r d i s t h e a m i n o - t e r m i n a l d o d e c a p e p t i d e Lj_ ( r e s i d u e s 1 - 1 2 ) . LJJ_ l l l 1 V a b s o r b a t 280 nm, w h e r e a s LJL has no t r y p t o p h a n o r t y r o s i n e r e s i d u e s and shows no a b s o r p t i o n a t 280 nm, b u t c a n be detected with ninhydrin a f t e r a l k a l i n e hydrolyses. This r e s u l t r e v e a l s t h a t ozone causes t h e o x i d a t i o n o f the m e t h i o n i n e r e s i d u e 105 o f l y s o z y m e . In order to determine the p o s i t i o n s o f the modified t r y p t o p h a n r e s i d u e s , t h e o z o n i z e d l y s o z y m e a t pH 7.0 was r e d u c e d , c a r b o x y m e t h y l a t e d and d i g e s t e d w i t h t r y p s i n . The tryptophanS

L

L


Dugger; Air Pollution Effects on Plant Growth ACS Symposium Series; American Chemical Society: Washington, DC, 1974. 0.18

0.15 0.84

3.2

1.90

tyrosine

methionine

0.13

c* 1.84

0.77

0.45

A 1.63 0.47

Β 1.62 0.44

C 1.65

r e a c t e d w i t h ozone pH 7.0

0.72

0.71

A 1.34 0.73

Β 1.31

pH 10.2

—

0.72

C 1.36

A* = amino a c i d a n a l y s e s ; B* = E d e l h o c h ' s method ( 1 6 ) ; C* = S c o f f o n e ' s method ( 1 5 ) .

B* 1.81

A* 1.83

pH 4.6

6.0

Untreated (unit)

Amino a c i d c o n t e n t o f u n t r e a t e d a n d o z o n i z e d l y s o z y m e

tryptophan

Amino a c i d

Table I .


30


Figure 4. Changes in UV absorbance of ozonized lysozyme. Volumes of 3.0 ml of O.I M buffer containing 0.59-1.11 mg lysozyme (ini tial amounts of protein were different) were exposed to 0 /0 at 20 ml/min. Ozone de livery was 1.6 nmoles/min. Absorbance spec tra were recorded at intervah and changes at 280 and 288 nm were plotted.


3

J

I

100

200

I

I

I

300 400 0 , , nmoles

2

I

500

600

Figure 5. Separation of ozonized lyso zyme CNBr fragments. The ozonized lysozyme was treated with CNBr as described in the Materiah and Meth ods. The peptide mixture was applied to a column of Sephadex G-25 (3 χ 100 cm) and eluted with 0.2N HAc at a flow rate of 0.5 ml/min. Fractions were assayed by measuring UV absorbance on ninhydrin color after alkaline hy drolysis. 30

50

70

EFFLUENT VOLUME (ml)

I

I

I

I

f

*278 0.4

3 ïêmmKuuxaxxxxxy/

10 20 30 20 40 60 80 EFFLUENT VOLUME (ml)

Figure 6. Binding of ozonized lysozyme to CM-chitin. Samples of lysozyme (1.3 mg) ozonized at different pHs to > 9 5 % inactivation were applied to a column of CMchitin (1.5 χ 4 cm). The elution sequence was firstly Tris-Cl and secondly 0.2N HAc.


3.

LEH AND MUDD

Table

31

Ozone with Lysozyme

I I . Amino a c i d c o m p o s i t i o n o f o z o n i z e d a n d c y a n o g e n b r o m i d e t r e a t e d Hen E g g - W h i t e Lysozyme L (100%) 1

Amino a c i d

theory 1.0

Lysine

found 1.13

l l l (23%) theory found L

0.23

0.26

Histidine


Arginine Aspartic

1.0

0.88

acid

Threonine

acid

1.0

0.84

1 1

1 V

theory

found

4.77

4.72

1

0.99

0.92

1.17

9.08

9.04

0.69

0.58

20.31

19.85

0.23

0.24

6.77

6.93

10.00

9.86

3.77

3.82

2.00

1.93

Serine Glutamic

L (23%)+L (77%)

0.23

0.29

Proline Glycine

1.0

0.91

0.46

0.43

10.54

10.71

Alanine

3.0

2.95

0.69

0.74

8.31

8.24

Half-cystine

1.0

0.91

0.46

0.58

6.54

6.40

Valine

1.0

0.93

0.46

0.54

4.54

4.52

0.23

0.29

5.77

5.73

0.23

0.41

6.77

6.80

3.00

2.55

2.00

2.21

5.31

4.06

Methionine* Isoleucine Leucine

1.0

0.96

Tyrosine* Phenylalanine

1.0

Tryptophan*

0.86 0.69

0.31

^ozonized residues.


AIR

32


c o n t a i n i n g p e p t i d e s were s e p a r a t e d by t h e method d e s c r i b e d by J o l i e s e t al» ( 1 2 ) . The amino a c i d a n a l y s e s d a t a a r e l i s t e d i n T a b l e I I I . The l o s s o f t r y p t o p h a n w i t h t h e a p p e a r a n c e o f N'formyl-kynurenine i n peptide T ( r e s i d u e s 9 8 - 1 1 2 ) , and t h e complete r e s i s t a n c e o f tryptophan i n peptides T ( r e s i d u e s 117125), T ( r e s i d u e s 2 2 - 2 3 ) , and T 1 7 ( r e s i d u e s 6 2 - 6 8 ) , i n d i c a t e d t h a t o f t h e s i x t r y p t o p h a n r e s i d u e s o n l y 108 and 111 a r e o x i d i z e d . The change o f t h e c o n t e n t o f t y r o s i n e r e s i d u e i n p e p t i d e Tm shows t h a t o n l y t h e t y r o s i n e a t p o s i t i o n 23 i s m o d i f i e d . 1 6

1 0


llf

R e d u c t i o n o f O z o n i z e d Lysozyme. M e t h i o n i n e s u l f o x i d e can r e v e r t t o methionine w i t h the generation of the l y t i c a c t i v i t y f o r p h o t o - o x i d i z e d l y s o z y m e ( 1 4 ) . We t e s t e d w h e t h e r t h e o z o n i z e d l y s o z y m e c o u l d be r e a c t i v a t e d by c h e m i c a l r e d u c t i o n . The o z o n i z e d l y s o z y m e was t r e a t e d w i t h 2 - m e r c a p t o e t h a n o l , d i a l y s e d , p u r i f i e d by p a s s a g e t h r o u g h a column o f Sephadex G-25 and l y o p h ilized. The p r o d u c t showed no i n c r e a s e i n i t s l y t i c a c t i v i t y . T h i s i s not s u r p r i s i n g because r e s i d u e s o t h e r than methionine are o x i d i z e d , b u t i t may be c o n c l u d e d t h a t t h e o x i d a t i o n o f m e t h i o n i n e a l o n e c a n n o t a c c o u n t f o r enzyme i n a c t i v a t i o n . E s t i m a t i o n o f the B i n d i n g S i t e . T r y p t o p h a n - 1 0 8 shows a s p e c i f i c r e a c t i o n w i t h i o d i n e , d i s t i n g u i s h i n g i t from other tryptophan r e s i d u e s o f l y s o z y m e . When t r y - 1 0 8 i s s e l e c t i v e l y o x i d i z e d by i o d i n e , l y s o z y m e c o m p l e t e l y l o s e s i t s a c t i v i t y . Nevert h e l e s s , t h e l y s o z y m e s t i l l shows t h e a b i l i t y t o f o r m an enzymes u b s t r a t e complex w i t h C M - c h i t i n . This observation contributes t o t h e c o n c l u s i o n t h a t t r y - C 2 i s an e s s e n t i a l b i n d i n g s i t e f o r a complex f o r m a t i o n ( 1 3 ) . A l l o z o n i z e d lysozymes formed s t r o n g c o m p l e x e s w i t h C M - c h i t i n and c o u l d o n l y be e l u t e d b y 0.2N HA ( F i g . 6 ) . T h i s f u r t h e r c o n f i r m s t h a t two t r y p t o p h a n r e s i d u e s (108 and 111) a r e i n d i s p e n s i b l e f o r t h e h y d r o l y t i c a c t i o n o f l y s o z y m e , and t h a t i n a c t i v a t i o n by o z o n e c a n n o t be a t t r i b u t e d t o i n h i b i t i o n of substrate binding capability. C

C o n f o r m a t i o n . The n e a r u l t r a v i o l e t CD ( c i r c u l a r d i c h r o i s m ) s p e c t r a o f n a t i v e l y s o z y m e a t pH 4.6, 7.0 and 10.2 ( F i g . 7A) a r e c h a r a c t e r i z e d by t h r e e p o s i t i v e p e a k s a t 294, 288 and 282 nm and a l a r g e n e g a t i v e band a t 268-266 nm. These c i r c u l a r d i c h r o i s m p a t t e r n s a r e much l i k e t h o s e r e p o r t e d by o t h e r s ( 1 9 , 2 0 , 2 1 ) . I n c r e a s i n g pH b r i n g s a b o u t i n c r e a s i n g l y p o s i t i v e e l l i p t i c i t y v a l u e s a t 255 nm and a t 295 nm, a s w e l l as a l t e r n a t i o n i n t h e p o s i t i o n s o f t h e l a r g e n e g a t i v e b a n d a t 268 nm. The change i n e l l i p t i c i t y a t 255 nm p a r a l l e l s t h e i o n i z a t i o n o f t h e t y r o s i n e r e s i d u e s i n w h i c h two o f t h e t h r e e t y r o s i n e r e s i d u e s a r e r e v e r s i b l y i o n i z e d . T h i s i n t e r p r e t a t i o n has b e e n r e a c h e d by t h e t y r o s i n e t i t r a t i o n (20,22). F i g u r e 7B r e p r e s e n t s t h e n e a r - u l t r a v i o l e t CD s p e c t r a o f o z o n i z e d lysozyme i n t h r e e d i f f e r e n t b u f f e r s o l u t i o n s . Comparing t h e CD s p e c t r a o f n a t i v e and o z o n i z e d l y s o z y m e s , one o b s e r v e s



1 Arsinine «ozonized r e s i d u e s .

1.16

0..96 1

0.93

1

0.88

0.98

1

acid

Glutamic 1

2.01 2 3.08 3

1.16

1

1.24

1

acid

Aspartic

Lysine

1.93 2 0.37 2

1.15

1.14

1

1

0.55

1

1

2.20

0.97

1.02

1.11

2

0.95

1.03

1

1

1.16

1

1

1

L

Tryptophan*

Tyrosine*

Isoleucine

Leucine

0.93

1

Half-cystine

Valine

0.89

1 0.95

1.01

1

0.94

1

Serine

1

2.04

2

2.15

2

1.12

1

Alanine

Threonine

2.03

2

2.06

2

1.04

1

Glycine

χ

I I I . T r y p s i n d i g e s t i o n f r a c t i o n s o f o z o n i z e d Hen Egg W h i t e Lysozyme Nn. o f t r y p t i c p e p t i d e s a c c o r d i n g t o J o l i e s e t a l . ( 1 2 ) , m m Φ Amino a c i d 17 *16 14 t h e o r y found t h e o r y found t h e o r y found t h e o r y found

Table


8

•s

Ι

3

ο 8

c! 8

> ζ

β



250

270

290

310

250

270 290

310

330

λ ( nm)

Figure 7. CD spectra of native (A) and ozonized (B) lysozyme. Reproducibility of θ for each spec trum was within 5% indicated by vertical bar. Other details are given in Materials and Methods.

Scheme 1. Ozone treatment and CNBr fragmentation of lysozyme.



3.

LEH AND MUDD

Ozone with Lysozyme

35

several interesting features. I n general, the c i r c u l a r dichroism s p e c t r a l o s e t h e i r f i n e s t r u c t u r e ; t h e p o s i t i v e b a n d a t 295 nm and t h e n e g a t i v e b a n d a t 268 nm d i s a p p e a r . A t t h e l o w e r pH, a new b r o a d peak c e n t e r e d a t 300 nm i s o b s e r v e d w h i l e t h e i n t e n s i t i e s o f t h e b a n d s o f n a t i v e l y s o z y m e a t 282 nm a n d 288 nm a r e g r a d u a l l y r e d u c e d d e p e n d i n g o n t h e pH o f t h e s o l u t i o n . A pron o u n c e d e f f e c t i s o b s e r v e d f o r l y s o z y m e o z o n i z e d a t pH 10.2 a r i s i n g f r o m d r a m a t i c s t r u c t u r a l c h a n g e . A s i t i s known t h a t t h e n e a r - u l t r a v i o l e t CD o f l y s o z y m e i s e n t i r e l y d e p e n d e n t o n t h e n a t i v e t e r t i a r y s t r u c t u r e o f t h e p r o t e i n , t h e spectrum can be c o m p l e t e l y a b o l i s h e d due t o u n f o l d i n g t h e m o l e c u l e . This r e s u l t c l o s e l y resembles t h e e f f e c t o f guanidine h y d r o c h l o r i d e i n denat u r i n g lysozyme ( 2 3 ) . Discussion D i s t i n c t changes i n s e v e r a l p r o p e r t i e s o f lysozyme o c c u r a f t e r r e a c t i o n w i t h o z o n e . The l y t i c a c t i v i t y o f t h e o z o n i z e d l y s o z y m e shows t h e same t r e n d a t v a r i o u s pHs a s t h e n a t i v e enzyme ( F i g . 2 ) ; t h i s may s u g g e s t t h a t t h e pK v a l u e s o f t h e i o n i z a b l e g r o u p s i n v o l v e d i n c a t a l y s i s have n o t b e e n a l t e r e d b y o z o n p l y s i s . The amino a c i d c o m p o s i t i o n o f o z o n i z e d l y s o z y m e d i f f e r s f r o m t h a t o f t h e n a t i v e enzyme i n t h r e e r e s i d u e s — m e t h i o n i n e , tryptophan and t y r o s i n e . None o f t h e o t h e r amino a c i d s i s a f f e c t e d b y o z o n e . The e x t e n s i v e l o s s o f e n z y m i c a c t i v i t y must be a s c r i b e d t o t h e o x i d a t i v e m o d i f i c a t i o n o f t h e s e t h r e e amino a c i d r e s i d u e s i n t h e lysozyme. In an e a r l i e r experiment, J o r i e t a l . (14) r e p o r t e d t h a t methionyl residues are important i n maintaining the t e r t i a r y s t r u c t u r e o f l y s o z y m e . The i n t r o d u c t i o n o f a p o l a r c e n t e r i n t o the a l i p h a t i c s i d e c h a i n o f m e t h i o n i n e , a s a consequence o f t h e c o n v e r s i o n o f t h e t h i o e t h e r f u n c t i o n t o t h e s u l f o x i d e , may b r i n g a b o u t a s t r u c t u r a l change o f t h e l y s o z y m e m o l e c u l e w h i c h , i n t u r n , reduces the c a t a l y t i c e f f i c i e n c y . When o z o n i z e d l y s o z y m e was t r e a t e d w i t h 2 - m e r c a p t o e t h a n o l i n a n aqueous s o l u t i o n a c c o r d i n g t o t h e p r o c e d u r e o f J o r i et_ a l . ( 1 4 ) , t h e enzyme d i d n o t show any i n c r e a s e i n i t s a c t i v i t y . T h i s may be e x p l a i n e d i n t w o ways. I n o n e , s u c h r e a c t i o n s a r e c o m p l i c a t e d b y many s i d e r e a c t i o n s , e.g. s u l f h y d r y l - d i s u l f i d e i n t e r c h a n g e , a g g r e g a t i o n a n d p r e c i p i t a t i o n o f t h e m o d i f i e d enzyme ( 2 4 - 2 6 ) . In the other, the f a i l u r e t o r e c o v e r t h e a c t i v i t y o f t h e enzyme may b y a s s o c i a t e d w i t h t h e extensive oxidation o f other residues. T r y p t o p h a n 108 i s r e c o g n i z e d t o be a n a c t i v e s i t e i n p r o m o t i n g t h e h y d r o l y s i s o f 3 ( 1 , 4 ) - g l y c o s i d i c l i n k a g e s b e t w e e n amino s u g a r r e s i d u e s i n p o l y s a c c h a r i d e components o f t h e b a c t e r i a l c e l l walls. T h i s r e s i d u e i s shown t o o c c u p y t h e c l e f t a s w e l l a s t r y p t o p h a n 62 a n d 6 3 , a n d i s i n a h y d r o p h o b i c r e g i o n . Tryptophan r e s i d u e s 62 a n d 108 a r e i n d i s p e n s a b l e f o r t h e a c t i o n o f l y s o z y m e , and t r y p t o p h a n 62 i s known t o b e t h e o n l y b i n d i n g s i t e f o r t h e complex f o r m a t i o n ( 1 3 ) . O x i d a t i o n o f tryptophan-108 i s e x p e c t e d



36

AIR


to cause the lysozyme t o completely l o s e i t s c a t a l y t i c a c t i v i t y , b u t t o r e t a i n c o m p l e t e l y i t s a b i l i t y t o f o r m an e n z y m e - s u b s t r a t e complex w i t h C M - c h i t i n . T h i s p r e d i c t i o n i s i n agreement w i t h o u r o b s e r v a t i o n w i t h o z o n e and o t h e r s i n t h e r e a c t i o n o f t h e lysozyme w i t h i o d i n e (27,28). The h i g h s u s c e p t i b i l i t y o f t r y p t o phan 108 t o e l e c t r o p h i l e s l i k e o z o n e and i o d i n e may s u p p o r t t h e c o n c l u s i o n t h a t t h e same mechanism o p e r a t e s i n b o t h c a s e s . The i n d o l e Ν o f t r y - 1 0 8 i s hydrogen-bonded t o a main c h a i n c a r b o x y and t h e 2 - p o s i t i o n i s i n c o n t a c t w i t h t h e c a r b o x y o f g l u t a m i c 35 (27). Both i n t e r a c t i o n s would i n c r e a s e the e l e c t r o n d e n s i t y o f t h e r i n g and so a c t i v a t e i t w i t h r e s p e c t t o a t t a c k by t h e e l e c t r o phile. C i r c u l a r d i c h r o i s m was u s e d t o e l u c i d a t e t h e c o n f o r m a t i o n a l change o f l y s o z y m e upon o z o n o l y s i s . I n n a t i v e l y s o z y m e , t h e s h o r t e r w a v e l e n g t h n e g a t i v e band i s a s s i g n e d t o a d i s u l f i d e t r a n s i t i o n and t h e l o n g e r w a v e l e n g t h p o s i t i v e b a n d s a r e c o n t r i b u t e d t o by t y r o s i n e and t r y p t o p h a n ( 2 9 ) . By s y s t e m a t i c e x a m i n a t i o n o f t h e e f f e c t o f pH and c h e m i c a l m o d i f i c a t i o n , i t i s p o s s i b l e t o c o r r e l a t e the changes o f the observed e l l i p t i c i t y from d i f f e r e n t c h r o m o p h o r e s w i t h t h e r e a c t i o n o f o z o n e . The r e s u l t s i n F i g u r e 7A i n d i c a t e t h a t l i t t l e a l t e r n a t i o n i n s t r u c t u r e o c c u r s by v a r y i n g pH b e t w e e n 4.6 and 10.2. The o x i d a t i o n o f t r y p t o p h a n 108 and 1 1 1 , h o w e v e r , a b o l i s h e s t h e p o s i t i v e e l l i p t i c i t y a t 294 nm ( 2 1 ) , due t o t h e i n t e r r u p t i o n o f t h e c o u p l i n g o f t h e t r a n s i t i o n i n r e s i d u e s 108 and 111 w i t h i d e n t i c a l t r a n s i t i o n s i n o t h e r t r y p t o p h a n r e s i d u e s s u c h a s 2 8 , 6 2 , and 63 ( 3 0 ) . O t h e r e v i d e n c e s u p p o r t s t h i s c o n c l u s i o n f r o m t h e c i r c u l a r d i c h r o i s m s t u d i e s on t h e o x i d a t i o n o f t r y p t o p h a n 108 b y i o d i n e ( 2 1 ) and on t h e b i n d i n g o f o l i g o m e r s o f N - a c e t y l - D - g l u c o s a m i n e t o t r y - 1 0 8 ( 2 1 ) . Com p a r i s o n o f t h e d i f f e r e n t s p e c t r a a t t h r e e pHs shows t h a t a c c e s s i b l e t y r o s i n e r e s i d u e s c o n t r i b u t e a p o s i t i v e b a n d n e a r 282 nm (20) and a n e g a t i v e b a n d a t 255 nm. I o n i z a t i o n o f these t y r o s i n e r e s i d u e s l e a d s t o a d i m i n u t i o n o f b o t h p o s i t i v e and n e g a t i v e ellipticity. The change p a r a l l e l s t h e change o f pH. A t h i g h pH, t h e i o n i z e d t y r o s i n e r e s i d u e i s p r e f e r e n t i a l l y a t t a c k e d by t h e o z o n e . T h i s i n d e p e n d e n t s t u d y f u r t h e r i n d i c a t e s t h a t t h e pH e f f e c t s on t h e r e a c t i o n s o f o z o n e w i t h p r o t e i n a r e s i m i l a r t o t h o s e w i t h f r e e amino a c i d s ( 8 ) . P a r a l l e l i n g t h e a p p e a r a n c e o f t h e a b s o r p t i o n a t 320 nm i n u.v. s p e c t r a , t h e new b r o a d e r band a t 299-304 nm i n t h e CD s p e c t r u m i s t e n t a t i v e l y a s s i g n e d t o t h e c o n t r i b u t i o n from N-formyl-kynurenine. Straight-chain or l a r g e - r i n g d i s u l f i d e s c h a r a c t e r i s t i c a l l y g e n e r a t e a CD b a n d b e t w e e n 249 and 260 nm ( 2 9 , 3 1 ) . The o p t i c a l a c t i v i t y a r i s e s from i n h e r e n t dissymmetry, from asymmetric per t u r b a t i o n or from both ( 3 2 ) . I f the e x t e r n a l l y p e r t u r b i n g group i s u n a l t e r e d r e l a t i v e t o a f i x e d coordinate system, the r o t a t i o n a r o u n d t h e d i s u l f i d e bond ( c h a n g e i n s c r e w c o n f i g u r a t i o n ) w o u l d change t h e s i g n o f t h e o p t i c a l l y a c t i v e b a n d . B a r n e s , W a r r e n and Gordon ( 1 9 ) r e c e n t l y d e m o n s t r a t e d t h a t t h e 258 nm band i n l y s o z y m e a r i s e s p r i m a r i l y f r o m t h e i n t r a m o l e c u l a r d i s u l f i d e bands and



3.

LEH AND MUDD

Ozone with Lysozyme

37

a l t e r s w i t h t h e c o n f o r m a t i o n a l c h a n g e s . B a s e d o n t h e amino a c i d a n a l y s i s showing t h a t the d i s u l f i d e bonds a r e not r e a c t e d w i t h o z o n e , t h e i n t e n s i t y o f t h e CD b a n d n e a r 255-265 nm c o u l d b e u t i l i z e d f o r t h e d i a g n o s i s o f s t r u c t u r a l change. T h i s band i s completely a b o l i s h e d i n the s p e c t r a o f ozonized lysozyme ( F i g . 7 B ) i n d i c a t i n g t h e change o f s t r u c t u r e a c c o m p a n y i n g t h e m o d i f i c a t i o n o f amino a c i d r e s i d u e s b y o z o n e . H o l l a d a y a n d S o p h i a n o p o u l o s ( 2 j 0 c o n c l u d e d t h a t t h e b a n d s a t 288 a n d 294 nm a r e due " n e a r l y c o m p l e t e l y t o t h e t e r t i a r y a n d q u a t e r n a r y s t r u c t u r e " o f lysozyme and they are completely a b o l i s h e d d u r i n g denaturing. S i n c e t h e s e bands a r i s e from t h e chromophores t y r o s i n e and tryptophan which r e a c t w i t h ozone, the i n f o r m a t i o n f r o m t h e CD s p e c t r a c o n c e r n i n g t h r e e d i m e n s i o n a l s t r u c t u r e i s l e s s dependable.


38

AIR

POLLUTION

EFFECTS

ON PLANT

GROWTH

Literature cited L e i g h t o n , P. A. " P h o t o c h e m i s t r y of Air Pollution," p . 3 0 0 , A c a d e m i c Press, New York. ( 1 9 6 1 ) . 2. Goldstein, E . , Tyler, W. S., Hoeprich, P. D. a n d Eagle, C. Arch. Int. Med. ( 1 9 7 1 ) 127:1099-1102. 3. H u r s t , D.J.,G a r d n e r , D. E. a n d Coffin, D. L. Reticuloendothel. S o c . ( 1 9 7 0 ) 8:288-300. 4. H o l z m a n , R. S., G a r d n e r , D. E. a n d Coffin, D. L. J. Bact. ( 1 9 6 8 ) 96:1962-1966. 5. S a s p e , A. T., B o n a v i d a , B., S t o n e , W. a n d Sercarz, Ε. E . Amer. J. O p h t h a l . ( 1 9 6 8 ) 66:76-80. 6. Previero, Α., Coletti-Previero, M.-A. a n d Jolles, P.J.Mol. Biol. ( 1 9 6 7 ) 24:261-268. 7. I m o t o , T., J o h n s o n , L. N., North, A. C. T., Phillips, D. C. and R u p l e y , J. A. In "The EnzymesVII."665-808, e d . B o y e r , P. D. A c a d e m i c Press, New Y o r k . (1972). 8. Mudd, J. B., Leavitt, R., Ongun, A. a n d McManus, T. T. Atmos. Environ, ( 1 9 6 9 ) 3: 669-82. 9. S h u g a r , D. B i o c h i m . B i o p h y s . A c t a ( 1 9 5 2 ) 8:302. 1 0 . Cleland, W.W. B i o c h e m . ( 1 9 6 4 ) 3:480. 1 1 . B o n a v i d a , B., Miller, A. a n d Sercarz, Ε. E. B i o c h e m . ( 1 9 6 9 ) 8:968-79. 1 2 . Jolles, J., Jauregui-Adell, J., Berrier, J. a n d Jolles, P. B i o c h i m . B i o p h y s . A c t a ( 1 9 6 3 ) 78:668-89. 1 3 . I m o t o , T., H a y a s h i , K. a n d F u n a t s u , M. J. B i o c h e m . ( 1 9 6 8 ) 64:387-92. 14. Jori, G., Galiazzo, G., Marzotto, A. and Scoffone, E. J. Biol. Chem. (1968) 243:4272-78. 1 5 . S c o f f o n e , E . , F o n t a n a , A. a n d Rocchi, R. B i o c h e m . ( 1 9 6 8 ) 7:971-979. 1 6 . E d e l h o c h , H. B i o c h e m . ( 1 9 6 7 ) 6:1948-54. 17. Finlayson, A. J. C a n . J. B i o c h e m . ( 1 9 6 9 ) 47:31-7. 1 8 . G r o s s , E. a n d W i t k o p , B. J. Biol. Chem. ( 1 9 6 2 ) 237:1856. 1 9 . B a r n e s , K. P., W a r r e n , J. R. a n d G a r d o n , J. A. J. Biol. Chem. ( 1 9 7 2 ) 247:1708-12. 20. H a l p e r , J. P., Latovitzki, Ν., Bernstein, H. a n d B e y c h o k , S. P r o c . N a t . A c a d . Sci. USA ( 1 9 7 1 ) 68:517-22. 2 1 . T e i c h b e r g , V. J., K a y , C.M. a n d S h a r o n , N. E u r o p . J. B i o c h e m . ( 1 9 7 0 ) 16:55-59. 2 2 . I k e d a , K. a n d H a m a g u c h i , K. J. B i o c h e m . ( T o k y o ) ( 1 9 6 9 ) 6 6 : 513-520. 2 3 . H o l l a d a y , L. A. a n d S o p h i a n o p o u l o s , A. J., J. Biol. Chem. ( 1 9 7 2 ) 247:1976-79. 24. Epstein, C. J. a n d G o l d b e r g e r , R. F. J. Biol. Chem. ( 1 9 6 3 ) 238:1380-1383. 2 5 . K a n a r e k , L . , B r a d s h a w , R. A. a n d Hill, R. L. J. Biol. Chem. ( 1 9 6 7 ) 240:pc 2755-2757 ( 1 9 6 5 ) ; 242:3789-3798 ( 1 9 6 7 ) . 26. I m a i , K., Takagi, T. a n d I s e m u r a , T. J. B i o c h e m . ( T o k y o ) ( 1 9 6 3 ) 53:1-6. Downloaded by CORNELL UNIV on October 23, 2016 | http://pubs.acs.org Publication Date: June 1, 1974 | doi: 10.1021/bk-1974-0003.ch003

1.


3.

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27.

39

H a r t d e g e n , F. J. a n d Rupley, J. A. J. Amer. Chem. S o c . ( 1 9 6 7 ) 89:1743-1745. B l a k e , C. C. F. Proc. Roy. S o c . ( 1 9 6 7 ) 167 B:435-438. B e y c h o k , S. Proc. N a t . A c a d . Sci USA ( 1 9 6 5 ) 53:999-1005. Zuclich, J. J. Chem. P h y s . ( 1 9 7 0 ) 52:3586-3591. B e y c h o k , S. a n d Breslow, E . J. Biol. Chem ( 1 9 6 8 ) 2 4 3 : 151-54. L i n d e r b e r g , J. a n d Michl, J. J. Amer. Chem. S o c . ( 1 9 7 0 ) 92:2619-25.


28. 29. 30. 31. 32.

Ozone with Lysozyme


Reaction of Ozone with Lysozyme - ACS Symposium Series (ACS

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