Methionine Sulfoxide and Other Combined Amino Acids in the

7 Methionine Sulfoxide and Other Combined Amino Acids in the German Cockroach S. MARK HENRY, RICHARD J. BLOCK, AND THOMAS W. COOK

Downloaded by FUDAN UNIV on December 8, 2016 | http://pubs.acs.org Publication Date: January 1, 1964 | doi: 10.1021/ba-1964-0044.ch007

Boyce Thompson Institute for Plant Research, Inc., 1086 North Broadway, Yonkers, Ν. Y.

A l k a l i n e h y d r o l y s i s of c o c k r o a c h r e s i d u e s subsequent to e x t r a c t i o n under n i t r o g e n w i t h 80% ethanol y i e l d e d methionine and m e t h i o nine sulfoxide in a r a t i o of 10 to 1. A d d i t i o n a l evidence for the p r e s e n c e of c o m b i n e d m e thionine sulfoxide w a s obtained by m e a s u r i n g the amount of methionine sulfoxide-S in acid and enzymatic hydrolyzates after assimila tion of Na S O . The data are believed to be indicative of naturally occurring peptide- or polysaccharide-bound methionine sulfoxide. Other combined amino acids were determined by ion exchange chromatography of the 5% trichloroacetic acid-insoluble cockroach res idues after hydrolysis with acid or alkali. βAlanine, normally present only in the soluble fraction of an organism, was found in the in soluble, proteinaceous residue. 35

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s u l f o x i d e , C H · SO · C H C H ( N H ) · C O O H , i s r e p o r t e d by Dent(2) to be of r a r e o c c u r r e n c e i n b i o l o g i c a l f l u i d s . H o w e v e r , Dent (2, 3) states that methionine, C H · S · C H C H ( N H ) · C O O H , i s e a s i l y o x i d i z e d to the s u l f o x i d e , p a r t i c u l a r l y on paper c h r o m a t o g r a m s d u r i n g d e velopment w i t h phenol. Indeed, i t a p p e a r s that whenever methionine sulfoxide i s detected, methionine should be suspected as the native form. R e p o r t s of the p r e s e n c e of methionine sulfoxide a r e l e s s frequent w i t h r e s p e c t to p r o t e i n h y d r o l y z a t e s than to free a m i n o a c i d e x t r a c t s . T h i s i s at l e a s t p a r t i a l l y due to the fact that r e d u c t i o n and other changes o c c u r upon h y d r o l y s i s w i t h h y d r o c h l o r i c a c i d (16). The extent of d e c o m p o s i t i o n depends on the c o n d i t i o n s of h y d r o l y s i s and m a y a p p r o a c h 100%. One o c c a s i o n a l l y finds a p u b l i s h e d r e p o r t of an a m i n o a c i d a n a l y s i s of p r o t e i n i n w h i c h data f o r methionine s u l f o x i d e but not m e t h i o 3

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Stekol; Amino Acids and Serum Proteins Advances in Chemistry; American Chemical Society: Washington, DC, 1964.

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nine a r e g i v e n . T h i s i s u s u a l l y due to inadvertent t r e a t m e n t of the h y d r o l y z a t e i n s u c h a way as to cause oxidation of the n o r m a l l y o c c u r r i n g f o r m of the a m i n o a c i d . O n l y a s m a l l number of p r o t e i n s and peptides have been a n a l y z e d by v a r i o u s i n v e s t i g a t o r s by methods designed s p e c i f i c a l l y to d e t e r m i n e whether methionine s u l f o x i d e i s one of the constituent a m i n o a c i d s . One of these, an analog of the α - m e l a n o c y t e - s t i m u l a t i n g h o r m o n e f r o m b o v i n e p i t u i t a r y glands (9), w a s found to contain the s u l f o x i d e . H o w e v e r , the authors e x p r e s s e d s o m e c o n c e r n o v e r the p o s s i b i l i t y of o x i d a t i o n d u r i n g i s o l a t i o n of the s a m p l e . The p r e s e n t i n v e s t i g a t i o n was undertaken to d e t e r m i n e i f the m e thionine sulfoxide found by s e v e r a l i n v e s t i g a t o r s i n i n s e c t t i s s u e i s a n o r m a l constituent of the a m i n o a c i d p o o l and of p r o t e i n . T h e a m i n o a c i d c o m p o s i t i o n of c o c k r o a c h p r o t e i n w a s s i m u l t a n e o u s l y d e t e r m i n e d both q u a l i t a t i v e l y and q u a n t i t a t i v e l y , s i n c e s u c h data a r e a v a i l a b l e f o r few s p e c i e s of i n s e c t s . Experimental L a b e l e d methionine and methionine sulfoxide w e r e detected r a d i o m e t r i c a l l y after a s s i m i l a t i o n of N a S 0 by c o c k r o a c h e s . T h e c o n v e r s i o n of i n o r g a n i c sulfur to methionine w a s shown p r e v i o u s l y (5) to be due to i n t r a c e l l u l a r s y m b i o t i c b a c t e r i a i n these i n s e c t s . In addition to this technique, unlabeled compounds w e r e separated by c o l u m n c h r o m a tography and m e a s u r e d c o l o r i m e t r i c a l l y . L a b e l i n g of Sulfur Compounds i n T i s s u e w i t h S . T h e G e r m a n c o c k r o a c h , B l a t t e l l a g e r m a n i c a ( L . ) , s e l e c t e d f o r t h i s study because of the l a r g e amounts of free methionine s u l f o x i d e o b s e r v e d i n p r e v i o u s i n v e s t i g a t i o n s (5), w a s r e a r e d on a f o r t i f i e d dog b i s c u i t diet (5) at 2 4 ° C . F u l l - g r o w n nymphs w e r e used without attempting to s e p a r a t e the s e x e s . A n aqueous s o l u t i o n of N a S 0 w a s injected i n t o the h e m o c e l e of 20 c o c k r o a c h e s by means of a m i c r o m e t e r - c o n t r o l l e d h y p o d e r m i c s y r inge fitted w i t h a 30-gage s t e e l needle. T h e i n s e c t s w e r e subsequently incubated at 2 5 ° C . f o r 2 days p r i o r to b e i n g s a c r i f i c e d . E x t r a c t i o n P r o c e d u r e s . E t h a n o l E x t r a c t i o n . T e n of the S - i n j e c t e d i n s e c t s w e r e homogenized i n about 5 m l . of hot 80% ( v . / v . ) ethanol w i t h a T e n B r o e c k t i s s u e g r i n d e r . The homogenate w a s centrifuged and, after decanting the supernatant f l u i d , the i n s o l u b l e r e s i d u e was washed three t i m e s w i t h 80% ethanol. T h e supernatant l i q u i d s w e r e c o m b i n e d and d r i e d i n v a c u o . T o p r e v e n t autoxidation of m e t h i o n i n e , as m u c h of the p r o c e d u r e as p o s s i b l e w a s conducted i n an i n e r t (nitrogen) a t m o s p h e r e using nitrogen-saturated solvents. T r i c h l o r o a c e t i c A c i d E x t r a c t i o n . T h e r e m a i n i n g 10 i n s e c t s injected w i t h N a S 0 w e r e homogenized i n 5% ( w . / v . ) t r i c h l o r o a c e t i c a c i d and the homogenate was kept at 1 0 0 ° C . i n a w a t e r bath f o r 30 m i n u t e s . T h e homogenate was centrifuged, and the r e s i d u e w a s washed once w i t h 5 m l . of 5% t r i c h l o r o a c e t i c a c i d and then w i t h d i s t i l l e d w a t e r . T h e s e f r a c t i o n s w e r e added to the o r i g i n a l supernatant s o l u t i o n and the c o m bined m i x t u r e w a s d r i e d i n vacuo. T h e p r o t e i n a c e o u s r e s i d u e was then washed t h r e e t i m e s w i t h a t o t a l of about 25 m l . of a c i d i f i e d ethanol (2 3 5

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d r o p s of 6 N H C l p e r 50 m l . of ethanol) and t h r e e t i m e s w i t h a s i m i l a r quantity of ether. A l l solvents w e r e saturated w i t h n i t r o g e n to m i n i m i z e oxidation. T r i c h l o r o a c e t i c a c i d e x t r a c t i o n was a l s o the method of c h o i c e i n p r e p a r i n g n o n r a d i o a c t i v e p r o t e i n f o r a n a l y s i s by c o l u m n c h r o m a t o g raphy. T y p i c a l l y , 20 m a l e and 20 f e m a l e adult G e r m a n c o c k r o a c h e s w e r e m a i n t a i n e d on a 3% ( w . / v . ) g l u c o s e diet for 3 to 5 d a y s . They w e r e then homogenized i n 20 m l . of 5% ( w . / v . ) t r i c h l o r o a c e t i c a c i d . The h o mogenate w a s p l a c e d i n a b o i l i n g w a t e r bath f o r 30 minutes, after w h i c h i t w a s centrifuged. The i n s o l u b l e m a t e r i a l was washed once w i t h each of the f o l l o w i n g s o l v e n t s : w a t e r , hot acetone, and ethanol-benzene (5 to 95, v . / v . ) , and t w i c e w i t h a c i d i f i e d ethanol followed by e t h y l ether. The r e s i d u e was then d r i e d either under a s t r e a m of n i t r o g e n o r i n a v a c uum d e s i c c a t o r . In both s e x e s the d r y r e s i d u e c o m p r i s e d about 17% of the l i v e weight of the i n s e c t . F o r quantitative d e t e r m i n a t i o n s of bound methionine s u l f o x i d e and tryptophan, 10 c o c k r o a c h e s of e a c h s e x w e r e homogenized and w e r e e x t r a c t e d w i t h the above s o l v e n t s saturated w i t h n i t r o g e n . P r e p a r a t i o n of E x t r a c t s f o r C h r o m a t o g r a p h y . The d r i e d ethanolic e x t r a c t w a s d i s s o l v e d i n 2 m l . of 10% ( v . / v . ) 2 - p r o p a n o l , shaken w i t h an equal v o l u m e of c h l o r o f o r m , and centrifuged. T h e c h l o r o f o r m l a y e r w a s d i s c a r d e d . The t r i c h l o r o a c e t i c a c i d e x t r a c t w a s taken up i n 2 m l . of w a t e r and e x t r a c t e d four t i m e s w i t h an equal v o l u m e of ether to r e m o v e t r i c h l o r o a c e t i c a c i d and l i p i d e s . The ether e x t r a c t w a s d i s c a r d e d . H y d r o l y s i s of P r o t e i n . T h e r a d i o a c t i v e , proteinaceous r e s i d u e s f r o m the s u l f a t e - i n j e c t e d i n s e c t s w e r e separated into e q u a l f r a c t i o n s , one of w h i c h w a s h y d r o l y z e d w i t h a c i d and the other w i t h p r o t e o l y t i c e n z y m e . A n a l y s e s of unlabeled p r o t e i n w e r e made after a c i d o r a l k a line h y d r o l y s i s . A c i d H y d r o l y s i s . T w e n t y - f i v e m i l l i l i t e r s of 6 N h y d r o c h l o r i c a c i d was a d d e d ' t o 250 m g . of d r y s a m p l e and the s u s p e n s i o n was r e f l u x e d for 16 h o u r s . T h e e x c e s s h y d r o c h l o r i c a c i d w a s r e m o v e d by e v a p o r a tion on a s t e a m bath o r on a r o t a r y v a c u u m e v a p o r a t o r . T h e a m i n o a c i d s w e r e then d i s s o l v e d i n 20 m l . of 10% 2 - p r o p a n o l . S a m p l e s so p r e p a r e d w e r e u s e d d i r e c t l y f o r a n a l y s i s by paper c h r o m a t o g r a p h y . T h o s e s a m p l e s to be a n a l y z e d by i o n exchange c h r o m a t o g r a p h y w e r e d e c o l o r i z e d w i t h a s m a l l amount of c h a r c o a l ( D a r c o G - 6 0 ) and f i l t e r e d through paper. A 2 - m l . aliquot of the f i l t r a t e was d r i e d and r e d i s s o l v e d i n s o d i u m c i t r a t e buffer at the p H and m o l a r i t y r e q u i r e d f o r a p p l i c a t i o n to the r e s i n . A l k a l i n e H y d r o l y s i s . P r o t e i n s a m p l e s to be a n a l y z e d f o r t r y p t o phan o r methionine sulfoxide w e r e h y d r o l y z e d w i t h b a r i u m h y d r o x i d e (16). F i f t y m i l l i g r a m s of p r o t e i n w a s the u s u a l quantity h y d r o l y z e d . A f t e r b a r i u m had been r e m o v e d f r o m the h y d r o l y z a t e (16), the p H of the s o l u t i o n was adjusted to 2.2 and the f i n a l v o l u m e to 22.5 m l . T h e e n t i r e p r o c e d u r e was conducted under n i t r o g e n to m i n i m i z e o x i d a t i o n . E n z y m a t i c H y d r o l y s i s . O n e - h a l f of the S - l a b e l e d proteinaceous r e s i d u e w a s suspended i n 95 m l . of d i s t i l l e d w a t e r and the p H was a d justed to about 7.5 w i t h 2 N a m m o n i u m h y d r o x i d e . F i v e m i l l i l i t e r s of ethanol and 10 m g . of S t r e p t o m y c e s g r i s e u s p r o t e a s e ( P r o n a s e , 6 P U 35

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p e r g r a m ) w e r e added and the d i g e s t i o n m i x t u r e w a s incubated at 4 0 ° C . (13). A f t e r 3 d a y s , the i n s o l u b l e m a t e r i a l w a s r e m o v e d by f i l t r a t i o n and the f i l t r a t e was d r i e d i n v a c u o and r e d i s s o l v e d i n 2 m l . of 10% 2 - p r o panol f o r c h r o m a t o g r a p h y . A s i n the above p r o c e d u r e s , n i t r o g e n - s a t u r a t e d s o l v e n t s and nitrogenous a t m o s p h e r e s w e r e used whenever p o s s i b l e i n p r e p a r i n g the h y d r o l y z a t e s . M e t h i o n i n e - S C o n t r o l E x p e r i m e n t . T o d e t e r m i n e what percentage of the methionine sulfoxide found i n h y d r o l y z a t e s and e x t r a c t s i s f o r m e d as a r e s u l t of d e g r a d a t i o n of methionine d u r i n g p r e p a r a t i o n and c h r o matography of the s a m p l e s , the f o l l o w i n g e x p e r i m e n t was conducted. T e n anesthetized c o c k r o a c h e s w e r e p l a c e d i n the t i s s u e g r i n d e r , to w h i c h w a s added 100 μ ΐ . of a s o l u t i o n of m e t h i o n i n e - S (1400 counts p e r minute p e r m i c r o l i t e r ) . T h e i n s e c t s w e r e homogenized w i t h hot 80% ethanol and e x t r a c t e d at the s a m e t i m e and i n the s a m e way a s the i n s e c t s injected w i t h sulfate. A n o t h e r 10 i n s e c t s w e r e e x t r a c t e d w i t h 5% t r i c h l o r o a c e t i c a c i d to w h i c h m e t h i o n i n e - S had been added. The p r o t e i n a c e o u s r e s i d u e s w e r e separated into equal a l i q u o t s and m e t h i o n i n e - S was added to each. One aliquot f r o m each type of e x t r a c t i o n w a s h y d r o l y z e d w i t h a c i d and the other w i t h p r o t e o l y t i c e n z y m e as d e s c r i b e d above. T h i s c o n t r o l e x p e r i m e n t w a s conducted s i m u l t a n e o u s l y w i t h the s u l f a t e - i n j e c t i o n e x p e r i m e n t and under the s a m e c o n d i tions. M e t h i o n i n e Sulfoxide A d s o r p t i o n C h e c k . S - l a b e l e d methionine sulfoxide was p r e p a r e d by o x i d i z i n g m e t h i o n i n e - S w i t h p e r o x i d e (6). F i v e m i c r o l i t e r s (ca. 20,000 counts p e r minute p e r m i c r o l i t e r ) of an aqueous s o l u t i o n of the s u l f o x i d e w a s injected i n t o each of 20 c o c k r o a c h e s . T h e f i r s t 10 w e r e i m m e d i a t e l y i m m e r s e d i n hot 80% ethanol, and the r e m a i n d e r i n hot 5% t r i c h l o r o a c e t i c a c i d to be homogenized and e x t r a c t e d . T h e e x t r a c t i o n p r o c e d u r e s w e r e s i m i l a r to those d e s c r i b e d above, except that p r e c a u t i o n s to p r e v e n t o x i d a t i o n w e r e not taken and the supernatant l i q u i d s , except f o r the a c i d i f i e d ethanol and ether w a s h e s , w e r e not c o m b i n e d but w e r e c o l l e c t e d s e p a r a t e l y i n 1 0 0 - m l . volumetric flasks. The protein residues were hydrolyzed i n 6N H C l and, l i k e the other f r a c t i o n s , w e r e then d i l u t e d to 100 m l . w i t h w a t e r for r a d i o m e t r i c a n a l y s i s . P a p e r C h r o m a t o g r a p h y and R a d i o m e t r y . F o r c h r o m a t o g r a p h y on p a p e r 46 x 57 c m . sheets of W h a t m a n N o . 3 f i l t e r p a p e r w e r e u s e d . T e n m i c r o l i t e r s of h y d r o l y z a t e o r 30 μ ΐ . of e x t r a c t w a s a p p l i e d and the c h r o m a t o g r a m s w e r e developed i n the f o l l o w i n g s y s t e m s . S y s t e m 1. T h e c h r o m a t o g r a m s w e r e developed i n the f i r s t d i r e c tion w i t h 60 p a r t s of ethanol, 20 p a r t s of t e r t - b u t y l a l c o h o l , 5 p a r t s of 58% ( w . / v . ) N H O H , and 15 p a r t s of H O , and i n the second d i r e c t i o n w i t h 14 p a r t s of t e r t - b u t y l a l c o h o l , 3 p a r t s of 88% ( w . / v . ) f o r m i c a c i d , and 3 p a r t s of H 0 (10). S y s t e m 2. T h e c h r o m a t o g r a m s w e r e developed i n the f i r s t d i r e c t i o n w i t h 450 p a r t s of η - b u t y l a l c o h o l , 50 p a r t s of g l a c i a l a c e t i c a c i d , and 125 p a r t s of H 0 , and i n the second d i r e c t i o n w i t h 2 p a r t s of n - b u t y l a l c o h o l , 2 p a r t s of m e t h y l e t h y l ketone, and 1 p a r t of H 0 . A s h a l l o w d i s h , c o n t a i n i n g 4 N N H O H to r e p l a c e the c y c l o h e x y l a m i n e u s e d by M i z e l l and S i m p s o n (11), w a s p l a c e d i n the c h r o m a t o g r a p h i c c h a m b e r 35


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f o r development i n the second d i r e c t i o n . In o r d e r to m a i n t a i n u n i f o r m c o n d i t i o n s , a l l h y d r o l y z a t e s o r e x t r a c t s w e r e c h r o m a t o g r a p h e d s i m u l t a n e o u s l y i n the s a m e c h a m b e r . A c h r o m a t o g r a m of a standard a m i n o a c i d m i x t u r e w a s used to locate the p o s i t i o n s of methionine, methionine s u l f o x i d e , and a second methionine sulfoxide spot f o r m e d as a r e s u l t of oxidation of methionine d u r i n g d r y i n g of the solvent used i n the f i r s t d i r e c t i o n . R a d i o a c t i v i t y w a s m e a s u r e d on the c h r o m a t o g r a m s w i t h an e n d window counting tube (4) f o l l o w i n g v i s u a l i z a t i o n of the a m i n o a c i d s w i t h n i n h y d r i n . The e x t r a c t s and h y d r o l y z a t e s f r o m the methionine s u l f o x i d e a d s o r p t i o n test w e r e d i l u t e d to 100 m l . w i t h w a t e r and 1 - m l . aliquots w e r e t r a n s f e r r e d to s a m p l e pans and d r i e d f o r the e s t i m a t i o n of r a d i o activity. Column Chromatography. A m i n o acids were determined i n hydroc h l o r i c a c i d h y d r o l y z a t e s w i t h a 1 5 0 - c m . c o l u m n of A m b e r l i t e I R - 1 2 0 (17) u s i n g the T e c h n i c o n A u t o A n a l y z e r . Quantitative m e a s u r e m e n t s w e r e made c o l o r i m e t r i c a l l y (17). A n a l t e r n a t e method of a n a l y s i s made use of a 1 3 3 - c m . c o l u m n of T e c h n i c o n c h r o m o b e a d r e s i n and a m o d i f i cation of the s u l f u r s y s t e m d e s c r i b e d by P i e z and M o r r i s (14). A l t h o u g h this method gave e x c e l l e n t s e p a r a t i o n of a l l c l a s s e s of a m i n o a c i d s , i t s value was l i m i t e d by the f a i l u r e to s e p a r a t e c y s t i n e f r o m g l u c o s a m i n e . C y s t i n e v a l u e s , t h e r e f o r e , w e r e d e t e r m i n e d f r o m the I R - 1 2 0 c h r o m a t o g r a m s alone. A l t h o u g h l y s i n e , h i s t i d i n e , and a r g i n i n e w e r e s e p a r a t e d on the c h r o m o b e a d r e s i n , these and tryptophan w e r e a n a l y z e d r o u t i n e l y on a 1 5 - c m . c o l u m n of A m b e r l i t e I R - 1 2 0 (17) s i n c e a complete r e c o r d of the b a s i c a m i n o a c i d s could be obtained i n l e s s than 3 h o u r s b y t h i s method.

Results Soluble M e t h i o n i n e Sulfoxide. T h e p e r c e n t a g e s of s o l u b l e m e t h i o nine s u l f o x i d e - S found on c h r o m a t o g r a m s of c o c k r o a c h e s injected 2 days p r e v i o u s l y w i t h N a S 0 a r e g i v e n i n T a b l e I. C o r r e s p o n d i n g p e r centages f r o m the c o n t r o l i n s e c t s e x t r a c t e d i n the p r e s e n c e of e x o g e nous m e t h i o n i n e - S a r e given i n the s a m e t a b l e . The amount of o x i d a t i o n (up to 50%) o c c u r r i n g i n the c o n t r o l i s somewhat s u r p r i s i n g . That t h i s i s not e n t i r e l y due to o x i d a t i o n d u r i n g c h r o m a t o g r a p h y i s i n d i c a t e d by the low l e v e l s of methionine s u l f o x i d e found on paper c h r o m a t o g r a m s of the m e t h i o n i n e - S s o l u t i o n used f o r the c o n t r o l e x p e r i m e n t . M o s t of the f r e e , l a b e l e d sulfoxide i n the s u l f a t e - i n j e c t e d i n s e c t s i s , t h e r e f o r e , due to spontaneous, n o n e n z y m a t i c o x i d a t i o n r e s u l t i n g f r o m i n t e r a c t i o n w i t h other components of the e x t r a c t , p r o b a b l y d u r i n g i s o l a t i o n o r c h r o matography. T h e m o s t l i k e l y t i m e of o c c u r r e n c e i s when the s a m p l e i s d r y i n g after b e i n g a p p l i e d to the p a p e r . H o w e v e r , the percentage i n the sulfate-injected i n s e c t s i s c o n s i d e r a b l y h i g h e r than i n the c o n t r o l i n s e c t s . W h i l e the amount of o x i d a t i o n may be influenced s l i g h t l y by the addition of a s m a l l amount of methionine (control) i t i s b e l i e v e d that the difference r e p r e s e n t s a c t u a l sulfoxide i n the 80% e t h a n o l - and 5% t r i c h l o r o a c e t i c a c i d - e x t r a c t a b l e f r a c t i o n of the c o c k r o a c h . P e p t i d e - B o u n d M e t h i o n i n e Sulfoxide. T h e p e r c e n t a g e s of m e t h i o 35

3 5

2

4

35

35


90

ADVANCES IN CHEMISTRY SERIES Si

Table I. Methionine Sulfoxide-S in Soluble Fraction of Cockroaches Extracted by Two Methods and Chromatographed in Two Solvent Systems 35

Chromatographic Extraction Solvents Method a


System 1

System 2

Methionine Sulfoxide-S as % of Total Reduced and Oxidized Methionine-S Immediate Source of on Chromatogramb S in Amino Acids 35

35

Ethanol 80%

61 43

Injected Na S 0 Methionine-S added to homogenate

Trichloro acetic acid 5%

66 50


Ethanol 80%

67 40



74


35

2

4

35

35

2

4

35

35

2

4

35

35

2

4

35

a

Solvent systems described in text. b Methionine sulfoxide-S in solution used for control was found to be 7-10% of total S measured on chromatograms of aliquot of solution. 35

35

nine s u l f o x i d e found on c h r o m a t o g r a m s of c o c k r o a c h h y d r o l y z a t e s ( T a b l e Π) i n these e x p e r i m e n t s a r e m u c h l o w e r than i n the free a m i n o a c i d f r a c t i o n s (Table I). A l t h o u g h these d i f f e r e n c e s a r e due p a r t i a l l y to v a r i a t i o n s i n the c h r o m a t o g r a p h i c p r o c e d u r e , the v e r y low p e r c e n t a g e s i n the a c i d h y d r o l y z a t e s i n d i c a t e the i n v o l v e m e n t of other f a c t o r s . F o r e x a m p l e , b e s i d e s r e d u c t i o n of methionine s u l f o x i d e d u r i n g r e f l u x i n g w i t h h y d r o c h l o r i c a c i d , spontaneous s u l f o x i d e f o r m a t i o n f r o m m e t h i o nine a p p e a r s to be i n h i b i t e d i n the p r e s e n c e of the s m a l l amounts of h y d r o c h l o r i c a c i d r e m a i n i n g i n the s a m p l e after e v a p o r a t i o n of the e x c e s s a c i d used f o r h y d r o l y s i s . It i s a l s o noted that a s i n g l e a l i q u o t of h y d r o l y z a t e w a s a p p l i e d to the p a p e r a s c o m p a r e d w i t h t h r e e a p p l i c a tions of the 80% ethanol o r 5% t r i c h l o r o a c e t i c a c i d e x t r a c t . T h e a d d i t i o n a l d r y i n g r e q u i r e d w i t h three a p p l i c a t i o n s i s undoubtedly r e s p o n s i ble for greater oxidation. M o r e i m p o r t a n t than t h e s e p o s s i b l e d i f f e r e n c e s i n the methionine sulfoxide content of the e x t r a c t a b l e f r a c t i o n and the p r o t e i n a r e the d i f f e r e n c e s i n bound sulfoxide between the c o n t r o l and the s u l f a t e - i n j e c t e d i n s e c t s . R e g a r d l e s s of the method of e x t r a c t i o n o r of h y d r o l y s i s , the amount of bound methionine s u l f o x i d e - S m the sulfate-injected i n s e c t s i s higher than that f o r m e d as a r e s u l t of o x i d a t i o n d u r i n g h y d r o l y s i s and c h r o m a t o g r a p h y of the p r o t e i n (control). A s i n the s o l u b l e f r a c t i o n s , these d i f f e r e n c e s m a y be p a r t i a l l y a r e f l e c t i o n of a r e d u c t i o n oxidation shift actuated by the p r e s e n c e of exogenous methionine i n the c o n t r o l . Inspection of the data s h o w s , i n a d d i t i o n , that the percentage 35


7.

91

HENRY ET AL Methionine Sulfoxide in the German Cockroach Table II. Methionine Sulfoxide-S" in Hydrolyzates of Proteinaceous Material Remaining after Extraction by Two Methods 35

Methionine Sulfoxide-S as % of Total Reduced and Immediate Source of Oxidized Methionine-S Chromatographic Extraction on Chromatogram S in Amino Acids Solvents Method 35

35

a


System 1

System 2

Acid hydrolyzate

Enzyme hydrolyzate

14 6

16 11



10 4

23 12


Ethanol 80%

11 3

26 12


23 17


Ethanol


35

2

4

35

35

2

4

35

35

2

4

35

35

2

4

35

a Solvent systems described in text. 35

of methionine s u l f o x i d e - S found on c h r o m a t o g r a m s of a c i d h y d r o l y zates i s a l w a y s l o w e r than on those of e n z y m e - d i g e s t e d p r o t e i n . T h i s i s due to r e d u c t i o n of the sulfoxide d u r i n g h y d r o l y s i s (16) w i t h a c i d . A l k a l i n e h y d r o l y s i s of p r o t e i n f r o m a m i x e d population of m a l e and f e m a l e c o c k r o a c h e s w a s found, by i o n exchange c h r o m a t o g r a p h y , to y i e l d a s a m p l e containing methionine and methionine sulfoxide i n a r a t i o of 10 to 1. T o d e t e r m i n e whether any of t h i s sulfoxide was f o r m e d as a r e s u l t of the a n a l y t i c a l p r o c e d u r e , another s a m p l e of the p r o t e i n was h y d r o l y z e d s i m u l t a n e o u s l y w i t h a s a m p l e of methionine alone and a s a m p l e of c o c k r o a c h p r o t e i n w i t h added methionine. A l l t h r e e s a m p l e s w e r e t r e a t e d i n an i d e n t i c a l m a n n e r , c a r e being taken to p r e v e n t o x i d a t i o n . The h y d r o l y z a t e s , after r e m o v a l of b a r i u m , w e r e c h r o m a t o g r a p h e d on the 1 5 0 - c m . c o l u m n . Table III. Methionine and Methionine Sulfoxide in Alkaline Hydrolyzates

Methionine Sulfoxide

Sample Hydrolyzed Cockroach protein Protein plus methionine Methionine

Methionine, G./16 g. N G./16 g. Ν

% of total

2.00 9.03

0.23 0.64

10.3 6.7

7.24

0.13

1.8



92


Quantitative data (Table ΙΠ) show that l i t t l e o x i d a t i o n o c c u r s when a p u r e s a m p l e of methionine i s t r e a t e d w i t h hot a l k a l i and c h r o m a t o graphed. A g r e a t e r amount o c c u r s when methionine i s subjected to the conditions of a l k a l i n e h y d r o l y s i s i n the p r e s e n c e of p r o t e i n . H o w e v e r , i f the p r o t e i n sulfoxide v a l u e i s deducted f r o m the t o t a l sulfoxide value of this s a m p l e , the percentage of methionine s u l f o x i d e i s found to be c o n s i d e r a b l y l o w e r than the 10% u s u a l l y found i n c o c k r o a c h p r o t e i n . In a r e p l i c a t e e x p e r i m e n t , c o c k r o a c h e s w e r e e x t r a c t e d w i t h 80% ethanol and a p o r t i o n of the p r o t e i n a c e o u s r e s i d u e w a s t r e a t e d w i t h e t h y l ether to d e t e r m i n e the effect, i f any, of ether w h i c h m a y contain p e r o x i d e s . T h i s and a n untreated p o r t i o n w e r e then h y d r o l y z e d i n a l k a l i and c h r o m a t o g r a p h e d . T h e a c t u a l quantities p e r unit of n i t r o g e n w e r e not d e t e r m i n e d but the r a t i o s of methionine to methionine sulfoxide w e r e i n the s a m e o r d e r a s those i n d i c a t e d i n T a b l e Ι Π . T h e e t h y l ether had no effect. T h e r e m a i n d e r of the above s a m p l e w a s h y d r o l y z e d w i t h added methionine i n a l k a l i , a n d , a s i n T a b l e Ι Π , a s m a l l e r p e r c e n t a g e of m e thionine sulfoxide w a s found than when c o c k r o a c h p r o t e i n w a s h y d r o l y z e d alone. T h e data a r e , t h e r e f o r e , i n a c c o r d w i t h the r e s u l t s o b tained u s i n g the isotope method. A d s o r p t i o n D a t a . C o n s i d e r i n g the p o s s i b i l i t y that the methionine sulfoxide found i n c o c k r o a c h t i s s u e h y d r o l y z a t e s m i g h t be due t o a d s o r p t i o n of s o l u b l e methionine s u l f o x i d e to a c t i v e s i t e s on p r o t e i n s , p o l y p e p t i d e s , e t c . , the a d s o r p t i o n check d e s c r i b e d i n a p r e v i o u s s e c t i o n w a s conducted. T h e data i n T a b l e I V show that, under the conditions u s e d , m o r e than 99% of exogenous methionine s u l f o x i d e i s r e m o v e d even before the f i n a l steps i n the e x t r a c t i o n p r o c e d u r e . A m a x i m u m of

Table IV. Efficiency of Two Extraction Procedures in Removal of Soluble Methionine Sulfoxide-S" from Tissues

Extraction Method Ethanol 80%

Fraction Hot 80% ethanol Cold 80% ethanol 1st wash 2nd wash 3rd wash Residue after acid hydrolysis

Total Trichloro acetic acid 5%

Total

Hot 5% trichloroacetic acid Cold 5% trichloroacetic acid Water Combined HCl-ethanol and ether fractions Residue after acid hydrolysis

C.P.M.

% of Total

9,830

90.58

874 100 16 32

8.05 0.92 0.14 0.29

10,852

99.98

8,518 306 49 9

95.78 3.44 0.55 0.10

11

0.12

8,893

99.99


7.

HENRY ET AL

Methionine Sulfoxide in the German Cockroach

93

0.3% i s a d s o r b e d . A n a l y s i s of the data f r o m the other e x p e r i m e n t s shows that the quantity of methionine sulfoxide i n the p r o t e i n f r a c t i o n of the G e r m a n c o c k r o a c h i s g r e a t l y i n e x c e s s of t h i s . O t h e r C o m b i n e d A m i n o A c i d s . The data f r o m two c h r o m a t o g r a p h i c a n a l y s e s on c h r o m o b e a d r e s i n and one on A m b e r l i t e I R - 1 2 0 w e r e a v e r aged to give the data shown i n T a b l e V . T h e tryptophan v a l u e i s f r o m a

Table V. Amino Acids of German Cockroach Protein


Amino acid

Grams Amino Acids/16 Grams Ν Male

Female

Arginine Histidine Lysine

7.3 4.3 9.2

7.0 4.6 9.5

Tyrosine Phenylalanine

8.2 5.0

7.4 5.4

Cystine Methionine

1.0 2.1

0.6 2.8

Serine Threonine

5.0 4.8

5.1 4.8

Leucine Isoleucine Valine

8.9 5.1 8.1

8.7 4.8 7.9

Glutamic acid Aspartic acid

13.9 10.1

13.0 10.1

Glycine Alanine

9.0 10.5

8.1 8.9

Proline

6.0

6.1

β -Alanine

0.2

0.2

Methionine sulfoxide Tryptophan

0.2 1.1

Hydrolyzing Medium

Acid

Alkali

s i n g l e d e t e r m i n a t i o n on the 1 5 - c m . c o l u m n of A m b e r l i t e I R - 1 2 0 . E x cept f o r the s u l f u r a m i n o a c i d s , there appear to be no s e x - l i n k e d d i f f e r e n c e s . M o r e o v e r , s i n c e c y s t i n e and m e t h i o n i n e a r e both subject to c o n s i d e r a b l e d e s t r u c t i o n w i t h b o i l i n g h y d r o c h l o r i c a c i d , the v a l u e s given f o r these a m i n o a c i d s s h o u l d be substantiated by other methods. The p r e s e n c e of β - a l a n i n e i s u n u s u a l i n that t h i s a m i n o a c i d i s g e n e r a l l y thought to be nonexistent i n p r o t e i n s . It i s n o r m a l l y found e i t h e r uncombined o r a s a constituent of s m a l l peptides s u c h a s a n s e r i n e and c a r n o s i n e .



94


Conclusions The r e s u l t s of both p a p e r and i o n exchange c h r o m a t o g r a p h y i n d i cate that methionine sulfoxide contributes to a s m a l l extent to the s u l fur a m i n o a c i d content of p r o t e i n s a n d / o r i n s o l u b l e peptides of the G e r m a n c o c k r o a c h . N e u m a n n , M o o r e , and Stein (12) r e p o r t that t r a c e s of methionine sulfoxide a r e found i n a l k a l i n e h y d r o l y z a t e s of r i b o n u c l e a s e , l y s o z y m e , t r y p s i n o g e n , p e p s i n , c a t a l a s e , c h y m o t r y p s i n o g e n , bovine s e r u m a l b u m i n , and some other p r o t e i n s . The r e s u l t s of a n a l y s i s of a l k a l i n e h y d r o l y z a t e s of p e p s i n and bovine s e r u m a l b u m i n by the a u t h o r s i n d i c a t e that methionine sulfoxide m a y amount to as m u c h as 6.5% of the t o t a l weight of r e d u c e d and o x i d i z e d m e t h i o n i n e . T h i s amount m a y be expected as a r e s u l t of o x i d a t i o n d u r i n g s t o r a g e , h y d r o l y s i s , and c h r o m a t o g r a p h y (see T a b l e Ι Π ) . M a n y of the p r o t e i n s l i s t e d w e r e obtained f r o m c o m m e r c i a l s o u r c e s and w e r e p r o b a b l y i s o l a t e d without the p r e c a u t i o n s taken i n the p r e s e n t e x p e r i m e n t s to p r e v e n t o x i d a t i o n . T h e function, i f any, of methionine sulfoxide r e s i d u e s i n peptides o r p r o t e i n s i s a m a t t e r of conjecture. T h e r e i s no evidence that they a r e of any s t r u c t u r a l s i g n i f i c a n c e . Indeed, s i n c e v a r i o u s e n z y m e s s u c h as r i b o n u c l e a s e and c h y m o t r y p s i n a r e e i t h e r p a r t i a l l y o r c o m p l e t e l y i n a c t i v a t e d by oxidation of the methionine r e s i d u e s (15, 16), one h e s i tates to suggest any functional r o l e f o r the s u l f o x i d e . H o w e v e r , a r o l e i n the maintenance of o x i d a t i o n - r e d u c t i o n potential of a b i o l o g i c a l s y s t e m , as suggested by Dent (3), i s c o n c e i v a b l e . Kennaugh (8) r e p o r t s changes i n the quantity of β-alanine i n the c u t i c l e of the A m e r i c a n c o c k r o a c h , P e r i p l a n e t a a m e r i c a n a ( L . ) , d u r i n g hardening and tanning, but i t i s not c l e a r whether t h i s r e p r e s e n t s free o r c o m b i n e d β - a l a n i n e . D a t a p r e s e n t e d h e r e i n and a d d i t i o n a l unpub l i s h e d m a t e r i a l i n d i c a t e that the β-alanine i s , indeed, a constituent of the c u t i c u l a r p r o t e i n . T h e m o l a r r a t i o s of m o s t of the a m i n o a c i d s i n the p r o t e i n of the G e r m a n c o c k r o a c h a r e g e n e r a l l y s i m i l a r to those of v e r t e b r a t e s and other i n v e r t e b r a t e s w i t h r e s p e c t to whole a n i m a l p r o t e i n h y d r o l y z a t e s (1). H o w e v e r , h i s t i d i n e , l y s i n e , t y r o s i n e , l e u c i n e , i s o l e u c i n e , v a l i n e , and alanine a r e somewhat m o r e abundant i n c o c k r o a c h p r o t e i n and t h e r e i s l e s s c y s t i n e . The data v a r y s i g n i f i c a n t l y f r o m data p r e v i o u s l y r e p o r t e d on the a m i n o a c i d c o m p o s i t i o n of the G e r m a n c o c k r o a c h (7). The e a r l i e r a n a l y s i s , h o w e v e r , was conducted on i n s e c t s w i t h the e n t i r e head and d i g e s t i v e t r a c t r e m o v e d and the r e m a i n i n g p o r t i o n s of the body e x t r a c t e d w i t h l i p i d e s o l v e n t s o n l y . Acknow ledgm en t T h e a u t h o r s e x p r e s s t h e i r a p p r e c i a t i o n to P i e r r e F r o m a g e o t , S a c l a y , F r a n c e , f o r h i s v a l u a b l e suggestions and to W . J . R a y , J r . , R o c k e f e l l e r Institute, N e w Y o r k , and D . E . K o s h l a n d , J r . , B r o o k h a v e n N a t i o n a l L a b o r a t o r y , Upton, Ν . Y . , f o r t h e i r a d v i c e on a l k a l i n e h y d r o l y s i s . Literature

Cited

(1) Block, R. J., Weiss, K. W., "Amino Acid Handbook," p. 344, Charles C. Thomas, Springfield, Ill., 1956.



7.


95

(2) Dent, C. E., Biochem. J. 43, 169 (1948). (3) Dent, C. E., Science 105, 335 (1947). (4) Fuller, R. C., Ibid., 124, 1253 (1956). (5) Henry, S. M . , Block, R. J., Contribs. Boyce Thompson Inst. 20, 317 (1960). (6) Ibid., 21, 129 (1961). (7) Hilchey, J . D., Block, R. J., Ibid., 17, 380 (1954). (8) Kennaugh, J . H., J. Insect Physiol. 2, 97 (1958). (9) Lo, T., Dixon, J. S., L i , C. H., Biochim. Biophys. Acta 53, 584 (1961). (10) Margolis, D., Mandl, R. H., Contribs. Boyce Thompson Inst. 19, 509 (1958). (11) Mizell, M., Simpson, S. B., J r . , J. Chromatog. 5, 157 (1961). (12) Neumann, N. P., Moore, S., Stein, W. H., Biochemistry 1, 68 (1962). (13) Nomoto, M., Narahashi, Y., Murakami, M., J. Biochem. (Tokyo) 48, 593 (1960). (14) Piez, Κ. Α., Morris, L . , Anal. Biochem. 1, 187 (1961). (15) Ray, W. J., Jr., Koshland, D. E . , Jr., Brookhaven Symp. Biol. No. 13, 135 (1960). (16) Ray, W. J., Jr., Koshland, D. E . , Jr., J. Biol. Chem. 237, 2493 (1962). (17) Spackman, D. H., Stein, W. H., Moore, S., Anal. Chem. 30, 1190 (1958).

Received November 30, 1962. Research aided by tutes of Health, U. S. Public Health Service, Bethesda, Md.,

Grant

E-2608,

National


Insti

Methionine Sulfoxide and Other Combined Amino Acids in the

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