Air Pollution Effects on Plant Growth

F. LEH and J. B. M U D D. Department of Biochemistry and Statewide Air Pollution Research Center,. University of California, Riverside, Calif. 92502. ...
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3 Reaction of Ozone with Lysozyme F.

LEH

and

J. B.

MUDD

Department of Biochemistry and Statewide Air Pollution Research Center,

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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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LEH AND MUDD

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 .

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

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AIR

POLLUTION EFFECTS ON PLANT GROWTH

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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

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

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LEH AND MUDD

Ozone with Lysozyme

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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.

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

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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)·

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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

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

3. LEH AND MUDD

Ozone with Lysozyme

27

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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

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

(ml)

-

AIR

28

POLLUTION EFFECTS ON PLANT GROWTH

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.

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=

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.

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 .

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30

AIR POLLUTION EFFECTS ON PLANT GROWTH

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.

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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.

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

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

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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.

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

AIR

32

POLLUTION EFFECTS ON PLANT GROWTH

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

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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

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

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

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

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

8

•s

Ι

3

ο 8

c! 8

> ζ

β

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AIR POLLUTION EFFECTS ON PLANT GROWTH

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.

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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

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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

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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.

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

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ON PLANT

GROWTH

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Ozone with Lysozyme

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