IRESEARCH Carbonyls from Irradiated Proteins Chemists find t h e y can f o l l o w r a d i a t i o n d a m a g e in tissue b y s t a n d a r d c h e m i c a l tests CHEMICAL
RESEARCHERS continue
to
make progress in understanding what happens w h e n biological systems are irradiated. Latest development: Proof that radiation causes attack o n the peptide linkage in protein to produce carbonyl bonds. One result: a simple color test on irradiated tissue treated with 2,4-dinitrophenylhydrazine lets chemists follow carbonyl production in
irradiated biological systems. In fact, they can spot radiation effects at doses down to several h u n d r e d roentgens. Doses of this size lie in the range w h e r e d a m a g e stops short of death. Being able to see these effects b y a straight forward chemical m e a n s will hopefully be of much value i n studying t h e m . The proof a n d results have b e e n obtained by University of California
Irradiating Proteins Cleaves Peptide Chain o
H
o:
II I U !„ •C-NH-C-CrRi
Radiolysis
>-
O II R-C-NH2
o o
II II C-C-R, I
+
R2
H O I II H 2 N—C—C—R i
0
Peptide link
Hydrolysis
R—C—OH
•¥
R2 This reaction is presumed to go through an imino intermediate, although there are other ways that might give the same results:
O
H O
Hydroxy! radical
II 1 II R—C—NH—C—C-f^
o n
o II
chemists Warren M. Garrison, Michael E. Jayko, and Boyd M . W e e k s . Using t h e university's 60-inch cyclotron, these chemists have irradiated proteins (in a q u e o u s solutions containing oxygen) to form high molecular weight fragments containing carbonyl bonds. Using chemical techniques, they have isolated fragments a n d point of attack. W i t h carbonyl formation being a key effect of irradiation, Garrison h a s a standard organic chemistry r e a g e n t at h a n d to detect and follow its formation—2,4-dinitrophenyEhydrazine. This reagent gives h y d r a z o n e derivatives w h e n allowed to react with protein fragments. These can then he s e p a r a t e d and examined b y both chemical a n d spectrophotometric means. So far, the California scientists have studied pepsin and gelatin, two proteins about which a good deal is alr e a d y known. In b o t h cases, they find t h a t about one carbonyl is formed p e r 100 electron volts of absorbed energy. T h e y find, as well, t h a t u p o n hydrolysis of the "protein carbonyls" they get keto acids in rough proportion to t h e amino acids in the original proteins. • Radiolysis vs. Hydrolytic Cleavage. These data m e a n to Garrison, Jayko, and W e e k s t h a t radiationi n d u c e d reactions of aqueous p r o teins lead to cleavage of t h e peptide chain. But instead of a n acid and an amino group (hydrolysis p r o d u c t s ) , they get an amide a n d a carbonyl group. By postulating an imino intermediate, they get t h e simplest mechanism
o
H20
II R—C—NH 2
R—C-N=C—C-R, I
R2
O II
0 II
c—c- •R,
+
I R2
Imino intermediate Results with labeled compounds suggest that free radicals formed by irradiating solute may make the reaction go like this:
O II
H O I
II
Irradiation
R-C-NH-C-C-R, I H
3
O H O II I II R-C-NH-C—C-R, I
O H O II I II R-C-NH-C-C-R,
C14H2 I
O il
Irradiation
H—C 1 4 H,-C-OH
C=0 I OH
O II 14 C H,—C-OH O O II 1 4 II HO-C-C H2—CH-C-OH I NH-, Aspartic acid
50
C&EN
APRIL
7,
1958
Hydrolysis
t h a t e x p l a i n s t h e r e a c t i o n s of p r o t e i n in irradiated solutions. T h e y p o i n t o u t t h a t t h e a c t i o n of r a d i a t i o n o n s o l u t e s is i n d i r e c t a n d p r o c e e d s t h r o u g h f o r m a t i o n of f r e e r a d i c a l s f r o m w a t e r . Garrison a n d his coworkers h a v e o n e f u r t h e r p i e c e of i n f o r m a t i o n t o b a c k u p t h e i r i d e a s o n t h e r o u t e of f r e e r a d i cal attack. W h e n they irradiate p e p sin i n d i l u t e C 1 4 - l a b e l e d a c e t i c a c i d ( C 1 4 H 3 C O O H ) with no oxygen present a n d then hydrolyze, they are able to separate C14-labeled aspartic a c i d plus several other C14-labeled acids. The other C14-labeled acids are f o r m e d b y h o o k u p of C14H2COOH r a d i c a l s a t t h e a - c a r b o n of t h e a m i n o a c i d s in t h e p o l y p e p t i d e c h a i n . • In B i o l o g i c a l S y s t e m s . All t h i s is b y w a y of p r o v i n g t o t h e i r o w n s a t i s f a c t i o n t h a t c a r b o n y l f o r m a t i o n is o n e of t h e m a j o r effects w h e n p r o t e i n , a s i m p l i f i e d b i o l o g i c a l s y s t e m , is i r r a diated. A n d to prove that they could extend their m e t h o d to more representative biological systems, they irradia t e d s o m e r a t liver h o m o g e n a t e . T h e y found that they got a high molecular w e i g h t h y d r a z o n e fraction w h i c h t u r n e d an intense m a g e n t a color w h e n it w a s m i x e d w i t h m e t h a n o l a n d p o t a s s i u m h y d r o x i d e ( e v i d e n c e of t h e p r e s e n c e of t h e h y d r a z o n e c h r o m o phore). This means, Garrison points o u t , t h a t c h e m i s t s c a n follow c a r b o n y l production in irradiated biological systems—in fact, c a n spot r a d i a t i o n effects a t d o s a g e s d o w n t o s e v e r a l hundred roentgens. Below this level, color d e v e l o p m e n t in the control—unirradiated tissue—begins to interfere.
Prodigal Property A r e d i s c o v e r e d p r o p e r t y of s e m i c o n d u c t o r s is b e i n g u s e d t o m a k e l a b o r a t o r y - m o d e l , m i n i a t u r e amplifiers t h a t o p e r a t e a t 1 0 0 0 c . p . s . w i t h a g a i n of over 15 decibels. All-but-forgotten w o r k b y W . H. B r a t t a i n a n d J o h n B a r d e e n , d o n e a t Bell L a b s m o r e t h a n 10 years a g o , h a s b e e n p u t to u s e t h e r e in d e v e l o p i n g n e w i d e a s o n t r a n sistor a c t i o n in semiconductor/elect r o l y t e i n t e r f a c e d e v i c e s , J. F . D e w a l d told the American Physical Society, m e e t i n g in Chicago. I n the experimental device m a d e b y Bell r e s e a r c h e r s , a h e x a g o n a l , rodlike c r y s t a l of v e r y p u r e z i n c o x i d e is t h e s e m i c o n d u c t o r ; it is i m m e r s e d in a highly conducting electrolyte. A platinum electrode placed nearby s e r v e s as t h e g r i d e l e m e n t .
PAPI
(Polymethylene
Bell L a b s ' J. F . D e w a l d electrolyte/semiconductor
polyphenylisocyanate)
works with amplifier
Inherently, Z n O h a s a large energy gap. T h i s m e a n s t h a t it is r e l a t i v e l y difficult t o e x c i t e o r b i t a l e l e c t r o n s t o high e n e r g y levels. Therefore, the crystals c a n b e o p e r a t e d u n d e r conditions of h i g h e r e n r i c h m e n t — a t a h i g h e r p o t e n t i a l b e t w e e n t h e e n d s of t h e r o d . I m m e r s e d in a conductive solution, o n e e n d of t h e p u r e c r y s t a l is c a t h o d i c a l l y b i a s e d w i t h r e s p e c t to t h e solution, a n d t h e o t h e r e n d is a n o d i c a l l y biased. S o m e w h e r e in b e t w e e n is a n e u t r a l p o i n t w h i c h is u n b i a s e d . As this n e u t r a l p o i n t shifts b a c k a n d f o r t h u n d e r t h e i n f l u e n c e of v a r y i n g b i a s i n g g r i d v o l t a g e s , t h e r e s i s t a n c e of t h e crystal changes, passing a current which follows t h e d r i v i n g f r e q u e n c y very closely. A fairly e x t e n d e d r a n g e of l i n e a r r e s p o n s e is o b t a i n e d . T h e z i n c o x i d e c r y s t a l is s u p p o r t e d by copper leads, and a platinized platinum grid completes the assembly. A f t e r i n s u l a t i n g all w i r e s a n d c o n n e c tions e x c e p t t h e g r i d , t h e a s s e m b l y is i m m e r s e d in a 5 % s o d i u m t e t r a b o r a t e and boric acid solution a n d hermetically s e a l e d i n a s m a l l g l a s s t u b e t o avoid electrolyte evaporation. S m a l l size is r e q u i r e d t o g i v e h i g h frequency operation. The smallest u n i t s m a d e s o far u s e c r y s t a l s a b o u t 0.3 m m . l o n g a n d 0 . 1 5 m m . i n " d i a m e t e r . " U s i n g a flat p l a t e c r y s t a l i n s t e a d of t h e h e x a g o n a l r o d m i g h t r a i s e t h e present low output p o w e r levels a p preciably w i t h o u t a n y over-all increase in s i z e o r a n y c h a n g e in o t h e r o p e r a t i n g c h a r a c t e r i s t i c s , Bell s a y s . a
HOT STRENGTH
PAPI b a s e d u r e t h a n e f o a m s , coatings and elastomers retain their strength after p r o l o n g e d exposure to t e m p e r a t u r e s above the useful range of c o n v e n t i o n a l urethanes. EXCELLENT ADHESION PAPI provides excellent adhesion for r u b b e r s , v i n y l s a n d other s u b s t r a t e c o a t i n g s w i t h polyesters and p o l y a m i d e s . EASILY USED _ - _ - _ - « « — - — - — - .
PAPI is easily used i n formulations due t o its very low volatility.
Write for descriptive information.
ARWIN PAPl
is a Registered Trade APRIL
7,
1958
Name C&EN
51
