The effect of sulphur compounds on free radical reactions and formation in irradiated dry proteins

The effect of sulphur compounds on free radical reactions and formation in irradiated dry proteins

204 B I O C H I M I C A E T B I O P H Y S I C A ACTA BBA 45203 T H E E F F E C T OF S U L P H U R COMPOUNDS ON F R E E RADICAL REACTIONS AND FORMATI...

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204

B I O C H I M I C A E T B I O P H Y S I C A ACTA

BBA 45203 T H E E F F E C T OF S U L P H U R COMPOUNDS ON F R E E RADICAL REACTIONS AND FORMATION IN I R R A D I A T E D DRY P R O T E I N S B. B. SINGH AND ~/[. O. ORMEROD on secondment from: Biology Division, Atomic Energy Establishment, Trombay, Bombay (India), and Physics Department, Royal Military College of Science, Shrivenham, Swindon, Wilts (Great Britain)

(Received February 5th, 1965)

SUMMARY The free radical reactions occurring ill gamma-irradiated proteins: salmine, gelatin and zein, freeze-dried with mercaptoethylamine (cysteamine), cysteine or cystamine have been studied using electron spin resonance spectroscopy. It was found that at 77°K the energy was not absorbed randomly over the whole macromolecule and particularly an unpaired electron (probably an ion) was stabilised on a sulphur atom in the additive molecule. At room temperature the protein radicals migrated to the additive to give the radical, CHy-S'. This radical then reacted with a glycine residue according to the reaction: -CH~-S" +-CO-CH2-NH--~-CH2-SH +-CO-CH-NHSO that tile major effect of the added sulphur compounds was to accelerate the rate of migration of the initial protein radicals to a glycine residue. Also they accelerated the decay of total radicals in proteins.

INTRODUCTION

Irradiation of a dry protein at 77 ° K traps the primary free radicals, and in some cases, ions, formed by high energy radiation. They can then be studied using ESR. It has been shown that the sulphur-containing amino acids play an important part in the primary radical formation 1. On warming a low temperature irradiated sample, some of the radicals decay and the others migrate to the glycine or cysteine and cystine residues to give two types of free radicals both with a characteristic spectrum 2,~. The radical on the glycine residue, - C O - C H - N H - , has a doublet spectrum*, the other, - C H y - S " , a broad low field spectrum 5. If a freeze-dried mixture of a protein or nucleoprotein in the mercaptoethylamine is studied, an increased yield of the radical -CH~-S" is observed 6-s, due to the migration of protein radicals to the mercaptoethylamine through a hydrogen transfer reaction. It is postulated that this reaction accounts for the protection by mercaptoethylamine observed in nucleic acids and viruses. In this work we have investigated the effect of the compounds mercaptoethylamine, cystamine and cysteine on primary radical formation in some proteins which Abbreviation: ESR, electron spin resonance. Biochim. Biophys. dcta, lO9 (1965) 2o4-213

RADICAL REACTIONS IN IRRADIATED PROTEINS

205

contain little or no sulphur. We have also studied in more detail the subsequent reactions of the primary radicals with the added compounds. EXPERIMENTAL

The proteins were obtained from British Drug House, England. They were zein, gelatin, salmine sulphate and bovine serum albumin. The additives mercaptoethylamine (cysteamine), mercaptoethylamine-HC1, cystamine.HC1 and cysteine-HC1 were also commercial samples. Artificial molecular complexes of proteins and a sulphur compound were prepared ill two different ways. All the proteins except zein were dissolved in water--zein in 70 % ethanol-water mixture. In one series of experiments the proteins were titrated with mercaptoethylamine (free base). For 5 % by weight addition of mercaptoethylamine the changes in pH of the protein solutions were:

Zein Bovine serum albumin Gelatin Salmine sulphate

Initial pH

Final p H

6.4 4-9 5.0 2.6

9.2 9.8 8.6 8.75

The solutions were then freeze-dried. A second set of samples were prepared using mercaptoethylamine-HC1, which is neutral and did not change the pH. Similar complexes of zein were made with cystamine and cysteine. Since these compounds were only available in the form of their hydrochlorides the pH adjustments were made with NaOH. The freeze-dried samples were sealed in glass tubes in vacuo (lO-5 Torr). They were irradiated with 7 kC 6°Co rays at a dose rate of 3 MR h -1 to a dose of 5 MR at 77 ° K. The ESR observations were made at temperatures of 77 ° K, I 9 5 ° K and 300 ° K. The ESR spectra were recorded as the first derivative of the absorption curve and are shown as such in the figures. An X-band spectrometer with a transmission cavity was used with magnetic field modulation at 400 kcycles/sec for the low temperature spectra and lO5 kcycles/sec for the room temperature spectra. The power level was 0.5 mW at the sample. The radical concentrations were determined by numerical integration of the derivative curve and comparison with standard coal or molybdenum disulphide samples which had been calibrated against 2,2-diphenyl-I-picrylhydrazyl. Free radical yields are quoted as G-values i.e. the number of radicals per IOO eV of absorbed energy. They were measured from yield-dose curves, the lowest dose on which was 0.5 MR. It was found that the yield-dose curves were only linear up to about 2 MR. RESULTS

A t 77 ° K

On irradiation and observation at 77 ° K, bovine serum albumin gave an E S R spectrum which consisted of a broad asymmetrical line typical of the spectra obtained Biochim. Biophys. dcta, lO9 (1965) 2 o 4 - 2 i 3

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B. B. SINGH, M. G. ORMEROD

f r o m all t h e p r o t e i n s w i t h c y s t i n e o r c y s t e i n e r e s i d u e s . T h e p r o t e i n w i t h f e w or no c y s t i n e / c y s t e i n e r e s i d u e s g a v e s y m m e t r i c a l s p e c t r a zein a single line, g e l a t i n s e v e n b a d l y r e s o l v e d lines a n d s a l m i n e five lines. I t w a s f o u n d t h a t t h e a d d i t i o n of a s u l p h u r c o m p o u n d t o a s u l p h u r - c o n t a i n i n g p r o t e i n - - s u c h as b o v i n e s e r u m a l b u m i n h a d n o e f f e c t o n t h e E S R s p e c t r u m . I n t h e p r o t e i n s w h i c h c o n t a i n e d l i t t l e o r n o s u l p h u r , i n s o m e c a s e s t h e a d d i t i o n of a s u l p h u r c o m p o u n d h a d n o effect as h a s p r e v i o u s l y b e e n r e p o r t e d for s a l m i n e L I n o t h e r cases, a n a d d i t i o n a l l o w field line w a s o b s e r v e d w i t h a g - v a l u e of a b o u t 2.01. T h e n e w l o w field line g a v e t h e s p e c t r a a m a r k e d a s y m m e t r y m a k i n g t h e s y m m e t r i c a l s p e c t r a of z e i n a n d g e l a t i n s i m i l a r t o t h o s e of t h e s u l p h u r - c o n t a i n i n g p r o t e i n s (see F i g . I). I t h a s b e e n s u g g e s t e d t h a t t h i s low field line is d u e t o a n u n p a i r e d e l e c t r o n o n a s u l p h u r a t o m . I n t h i s p a p e r w e will r e f e r t o it as t h e p r i m a r y s u l p h u r r a d i c a l .

Zein + . . . . .

V

MercaptoethylomiIne'

Gelatin+

Mercaptoethylamir~ __

Mer'capioethylQrnirleV "J

J\

//~

~

2 0 ~ ~G

20G

Fig. I. Effect of mercaptoethylamine on the ESR spectra of proteins irradiated and observed at 77 ° K. Dose ~ 5 MR. TABLE I Protein

Additive (5 % by weight)

G radicals at 77 ° Ix" ( ~ 30 %)

Primary sulphur radical observed

Zein Zein Zein Zein Zein Zein Zein Gelatin Gelatin Gelatin Gelatin Salmine Salmine Salmine Salmine Salmine Bovine serum albumin Bovine serum albumin Bovine serum albumin

none mercaptoethylamine • HC1 mercaptoethylamine cysteine • HC1 cysteine cystamine- HCI cystamine none mereaptoethylamine. HC1 mercaptoethylainine cystamine. HC1 none mercaptoethylamine- HC1 mercaptoethylamine cysteine. HC1 cystamine - HCI none mercaptoethylamine. HC1 mercaptoethylamine

i-4 i .6 1.8 2. I 2.2 2.5 2.5 2.0 2.0 2.6 2.5 1.9 I. 8 2. i 2.6 i .9 2.8 2.8 2.8

no no yes yes yes yes yes no no yes yes no no yes yes yes yes yes yes

Bioehim. Biophys. Acta, lO9 (1965) 204 213

RADICAL REACTIONS

IN IRRADIATED

207

PROTEINS

The cases in which it was observed are shown in Table I. From these results two generalisations m a y be made. The primary sulphur radical is formed more readily in the presence of mercaptoethylamine as compared to mereaptoethylamine. HC1 and more efficiently on cystamine as compared to the sulphydryl compounds. Careful measurements of radiation yields (G-values) showed that when the primary sulphur radical was observed there was an increase in the G-value for radicals at 77 ° K. The only exception to this was salmine plus 5 % cystamine. HC1 but it must be emphasised that the expected increase was within the experimental error.

°oS

J

0.50] '3454

e 0351

~

"~0.30

°1°1 A/X o.o5t/ O~r

0

.

1

.

2

.

.

3

.

.

4. Dose

5

6

(mead)

F i g . 2. P r o d u c t i o n o f f r e e r a d i c a l s i n z e i n s a m p l e s i r r a d i a t e d a t 77 ° K . F q - - [ B , zein + mercaptoethylamine; x--x, z e i n + m e r c a p t o e t h y l a m i n e . HC1.

zein;

@--@3,

The radical-dose curves showed saturation at higher doses (see Fig. 2 in the case of zein). In particular the radical concentration in the presence of 5 % mercaptoethylamine. HC1 reached a lower saturation level and at a lower dose than in the pure protein or in other mixtures. This phenomenon would account for the lowering in radical yield by mercaptoethylamine-HC1 in bovine serum albumin reported by LIBBY et al. 9 who measured the yield at a dose of 5 MR whereas HENRIKSEN et al. s, who worked at lower doses, found no change in the radical yield. A t higher temperatures On warming to room temperature a low temperature irradiated sample of zein, gelatin or salmine, the E S R spectrum changed into a doublet similar to that observed in silk 4. There was little radical decay. The added sulphur compounds accelerated the protein radical decay. A careful comparison of proteins with added mercaptoethylamine, mercaptoethylamine. HC1, cystamine and cystamine. HC1 showed that a free base mercaptoethylamine and cystamine produced faster radical decays than the corresponding hydrochlorides and the disulphide almost doubled the effect of mercaptoethylamine. This is shown for zein in Fig. 3In gelatin and salmine there was also a growth of a low field structure in the Biochim. Biophys. Acta, lO9 (1965) 2 o 4 - 2 1 3

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B . B . SINGE, M. G. ORMEROD

E S R spectrum. This was the spectrum of the radical -CH2-S" which ORMEROD AND ALEXANDER 6,7 and HENRIKSEN et al. s have observed in proteins and nucleoproteins with added mercaptoethylamine. HC1. The final concentration of this radical was greater in the case of mercaptoethylamine as compared to mercaptoethylamine. HC1 or cystamine. HC1 although in all cases the concentration was less than that observed in a nucleoprotein-mercaptoethylamine. HC1 mixtureL In zein, the E S R spectrum of -CH2-S" never appeared. In zein the transformation from the initial radical to the "doublet" radical was very rapid. In gelatin the transformation could be followed at 253 ° K. In salmine it was even slower and could be followed at room temperature. In gelatin and salmine, in which the transformation could be easily studied, it was found that the added sulphur compounds accelerated the rate of transformation of the ESR spectrum to a 1.6! 15q I 1.4 4 13 2

~A.

A

12 ~ 8

A

0.94 084 @ 0.7~ 0.6~

0.44 027

!

O'1L 0 0

i 10

~9

A

100

1000

10000

Time (min)

Fig. 3- D e c a y of free radicals in zein at r o o m t e m p e r a t u r e . A - - A , zein; @ - - @ , zein + m e r c a p t o ethylamine-HC1; • @, zein + m e r c a p t o e t h y l a m i n e ; [3---[3, zein + c y s t a m i n e . HC1, I 1 - - 1 , zein + cystamine.

Gelot•

Gelotin + Mercoptoethylclmine

Gelotin + MercoptoethylQmine.HCl

I

H ~

i

20G

Fig. 4. Effect of m e r c a p t o e t h y l a m i n e and m e r c a p t o e t h y l a m i n e . HCI on E S R spectra of gelatin. D o s e = 5 M R . A, at 7 7 ° K ; 13, i o nlin at - - 2 o ° C ; C, IOO rain at - - 2 o ° C ; D, i d a y at r o o m temperature.

Biochim. Biophys. Acta, lO9 (1965) 2o4-213

/

f

J

/Y

H~

/ f

Solmine + M e r c a p t o e t h y l a m i n e

i

vr/-

f

Salmine + Mercaptoethylamine • NCl

Fig. 5. Effect of mercaptoethylamine and m e r c a p t o e t h y l a m i n e ' H C 1 on E S R spectra of salmine. Dose = 5 MR. A, at 77 ° K; B, immediately at room temperature; C, ioo rain at room t e m p e r a t u r e ; D, i day at r o o m temperature.

D

B-

Sol mine

to O

~Z ~q

0

*q

PV

(3

t~

t~

v

210

B . B . SINGH,

M. G. ORMEROD

doublet. This is shown q u a l i t a t i v e l y in Figs. 4 a n d 5 a n d q u a n t i t a t i v e l y for gelatin in Fig. 6. The a d d e d m e r c a p t o e t h y l a m i n e h a d less effect t h a n either m e r c a p t o e t h y l a m i n e . HC1 or c y s t a m i n e . HC1. o

70, !/ / 4o4

o 3° i u

~

~

// 30

0~0

g

16

I'5 2'0 2'5 3'0 30

40

45

50

55

60

65

70

75

80

85

80

T i m e rain

Fig. 6. D e v e l o p m e n t of d o u b l e t s t r u c t u r e i n E S R s i g n a l s of g e l a t i n s a m p l e s s t o r e d a t @ @, g e l a t i n + 5 o/ /o m e r c a p t o e t h y l a m i n e ; Q - - O , g e l a t i n + 5 O//o m e r c a p t o e t h y l a m i n e .

2o ° C. HC1.

DISCUSSION

Identification of the primary radicals I n t h e p r o t e i n s c o n t a i n i n g no sulphur, the p r i m a r y sulphur radical was only o b s e r v e d in the presence of s u l p h u r c o n t a i n i n g additives. Similar E S R spectral lines were also o b s e r v e d in s u l p h u r - c o n t a i n i n g proteins. W e therefore p o s t u l a t e t h a t these lines are due to u n p a i r e d electrons l o c a t e d on a s u l p h u r atom. BOX AND FREUND 1° a n d AKASAKA et al. u concluded t h a t the E S R s p e c t r u m o b s e r v e d in single c r y s t a l s of cystine d i h y d r o c h l o r i d e i r r a d i a t e d at 77 ° K arose from e i t h e r a hole or an electron l o c a t e d on the disulphide bond. TRUBY 12 s t u d i e d a m y l disulphide i r r a d i a t e d at 77 ° K a n d c o n c l u d e d t h a t the o b s e r v e d species were ionic. H e p r o p o s e d t h a t b o t h holes a n d electrons were f o r m e d a n d l o c a t e d on s u l p h u r a t o m s . I n light of these results SINGH AND ORMEROD1 h a v e suggested t h a t the p r i m a r y s u l p h u r r a d i c a l is an ionic species associated w i t h a s u l p h u r atom. A t present t h e r e is no evidence to i n d i c a t e w h e t h e r this is a positive or negative ion. Salmine c o n t a i n s 8o % arginine residues. The l o w - t e m p e r a t u r e s p e c t r u m of p u r e salmine was similar to t h a t o b t a i n e d from polyarginine. The five-line E S R s p e c t r u m of salmine was p r o b a b l y due to radicals l o c a t e d on the arginine residues. If radicals in p r o t e i n s were f o r m e d at r a n d o m on the different amino acid residues, the resulting l o w - t e m p e r a t u r e E S R s p e c t r u m w o u l d be the s u p e r p o s i t i o n of several s p e c t r a a n d one w o u l d e x p e c t the result to be a b r o a d single line with little hyperfine structure. H o w e v e r , in practice, t h a t o b t a i n e d from zein a n d silk, consisted of c o m p a r a t i v e l y n a r r o w single lines, a n d from gelatin a seven-line s p e c t r u m . This suggests t h a t cont r a r y to the p r e v i o u s p o s t u l a t i o n of r a n d o m d a m a g e of a p r o t e i n molecule 2 a considerable r a d i c a l localisation occurs at 7 7 ° K a n d t h a t the radicals are formed on one or two specific a m i n o acid residues, which in these cases are not the sulphur a m i n o acids.

Formation and reactions of sulphur radicals Since in a given p r o t e i n the p r i m a r y s u l p h u r radical was not o b s e r v e d in a Biochim. Biophys. Acta, t o 9 (1965) 2 o 4 - 2 1 3

RADICAL REACTIONS IN IRRADIATED PROTEINS

211

m e c h a n i c a l m i x t u r e of p r o t e i n a n d a s u l p h u r c o m p o u n d , it is u n l i k e l y t h a t it is f o r m e d b y t h e direct a c t i o n of r a d i a t i o n on the a d d i t i v e . I t m u s t be f o r m e d b y t h e m i g r a t i o n of ions, e x c i t a t i o n energy or h y d r o g e n a t o m s to the a d d i t i v e . I n view of the increase in t h e G-value for radicals when t h e p r i m a r y s u l p h u r r a d i c a l was observed, we p o s t u l a t e d 1,13 t h a t this radical is p r o b a b l y f o r m e d b y the m i g r a t i o n of e x c i t a t i o n a n d i o n i z a t i o n energy to t h e s u l p h u r a t o m a n d the f o r m a t i o n of an ion there. This migratiort was affected b y t h e ionic s t a t e of t h e a d d i t i v e since it w a s n o t f o r m e d on m e r c a p t o e t h y l a m i n e - H C 1 b u t was f o r m e d on free base m e r c a p t o e t h y l amine. I n p u r e s u l p h u r - c o n t a i n i n g proteins, there is evidence t h a t the radical, - C H 2 - S " is f o r m e d b y t h e r e a c t i o n s of t h e p r i m a r y s u l p h u r r a d i c a l on w a r m i n g from 77 ° K (ref. 14). I n gelatin or s a l m i n e - s u l p h u r c o m p o u n d m i x t u r e s in which a p r i m a r y s u l p h u r r a d i c a l was observed, it is p r o b a b l e t h a t it d e c a y e d to give a CH~-S" radical. H o w e v e r , t h e CH2-S" r a d i c a l was f o r m e d in those m i x t u r e s in which t h e r e was no p r i m a r y s u l p h u r r a d i c a l at 77 ° K. Also we could find no correlation b e t w e e n t h e size of the - C H e - S " signal a n d the s t r e n g t h of the p r i m a r y s u l p h u r r a d i c a l signal. W e therefore conclude t h a t the - C H 2 - S " radicals in these m i x t u r e s were f o r m e d b y two m e c h a n i s m s - - t h e d e c a y of t h e p r i m a r y s u l p h u r r a d i c a l a n d the m i g r a t i o n of p r o t e i n radicals to the a d d i t i v e as has p r e v i o u s l y been discussedT, 8. I n a n u c l e o p r o t e i n - m e r c a p t o e t h y l a m i n e . HC1 m i x t u r e , the o n l y effects of the a d d i t i v e were an i n c r e a s e d d e c a y r a t e of D N A radicals a n d a b u i l d - u p of - C H 2 - S " radicals. These effects could be s a t i s f a c t o r i l y e x p l a i n e d b y p o s t u l a t i n g a m i g r a t i o n of D N A radicals onto t h e a d d i t i v e 7. I n the s y s t e m s s t u d i e d here there is a n a d d i t i o n a l p h e n o m e n o n t h a t is n o t a c c o u n t e d for on this simple scheme, t h a t is the acceleration of the r a t e of t r a n s f o r m a t i o n of the initial radicals to t h e " d o u b l e t " radical. I n h y d r o c a r b o n s it has been f o u n d t h a t t h e r e a c t i o n of a h y d r o c a r b o n r a d i c a l w i t h a m e r c a p t a n i s reversible 15 R" + - S H ~ R H

+-S"

Our results can be a c c o u n t e d for b y assuming t h a t a s u l p h u r radical can react w i t h a p r o t e i n b y a b s t r a c t i n g a h y d r o g e n a t o m from a glycine residue to give a " d o u b l e t " t y p e radical. This gives us the following r e a c t i o n scheme. RII . . . . --~ R" 1 R" 1 -> R" 2 R.1 + -SH-+ RH + -S" RH + -S'--~ R'~ + - S H 2 (-S') -->- S - S R" + - S ' - + R S-

inilial radical formation migration to a glycine to give "doublet" radical -CH2-S" formation "doublet" radical formed radical decay radical decay

(i) (2) (3) (4) (5) (6)

The a d d e d s u l p h u r c o m p o u n d t h e n acts to c a t a l y s e t h e m i g r a t i o n of radicals to t h e glycine residues. The final c o n c e n t r a t i o n of - C H 2 - S " radicals o b s e r v e d will d e p e n d m a i n l y on t h e r a t e of R e a c t i o n 4. I n c o n f i r m a t i o n of the a b o v e scheme our results showed t h a t in those cases where t h e " d o u b l e t " r a d i c a l f o r m a t i o n was fastest, t h e final c o n c e n t r a t i o n of s u l p h u r radicals was lowest (e.g. c o m p a r e gelatin plus m e r c a p t o e t h y l a m i n e . HC1 a n d plus m e r c a p t o e t h y l a m i n e ) . I n zein t h e a d d i t i v e molecules m u s t have m o r e m o b i l i t y , since, despite the fact t h a t t h e a d d i t i v e s h a d a m a r k e d effect on t h e r a d i c a l reactions, no - C H 2 - S " r a d i c a l s Biochim. Biophys Acta, lO9 (1965) 2o4-213

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were observed (although HENDRIKSENet al. s found a transient appearance of-CH2-S" in zein plus mercaptoethylamine), therefore Reactions 5 and 6 must be faster than in salmine or gelatin. The ionic state of added mercaptoethylamine affected these reactions. The increased protein radical decay in the presence of mercaptoethylamine as compared to mercaptoethylamine-HC1 showed that with the free base Reaction 3 was more rapid. However the free base gave a slower transformation to a "doublet" radical and a larger -CH 2-S" build-up showing that Reaction 4 was slower for the free base. In the case of the disulphide, cystamine, the initial reaction presumably was R"1 + - S

S-~R

a-S

+-S"

(3a)

Thereafter the reaction scheme would be similar to that for the sulphydryl. Again Reaction 3a was faster for the free base than for the hydrochloride. Protection by sulphur compounds It has been postulated that the protection against high energy radiation afforded by mercaptoethylamine can be accounted for by a "repair" reaction of the type 3 above6-S'16. This mechanism holds well for nucleoprotein, but the results reported here demonstrate that the situation in the case of proteins is complicated by the back-reaction (4) of the sulphydryl radical. This reaction may nullify the effect of the repair reaction and would reduce the amount of protection given by mercaptoethylamine. In such a case the amount of protection observed would depend on the relative rates of Reaction 4 and the decay Reactions 5 and 6. Similar reactions with mercaptoethylamine have been observed in irradiated lyophilised bacteria and we have also studied the role of oxygen 17. These latter results will be reported in a further paper in which the full biological significance of the results will be discussed. CONCLUSIONS

(I) On irradiation of a dry protein, the initial radicals are not distributed at random but considerable radical localisation occurs. In the case of sulphur containing proteins, unpaired spins tend to be localised in the cystine residues. (2) In the presence of sulphur compounds (cystine residues or added sulphur compounds), the ESR spectrum of an ion located on a sulphur atom can be observed after irradiation at 77 ° K. There is also an increase in the total number of unpaired spins formed. (3) At room temperature there is a migration of radicals to the glycine residues. (4) In the presence of a sulphydryl or disulphide, there is a migration of radicals to the sulphur atom of the added compound to give the radical, -CH~-S'. (5) The radical -CH~-S" can react with the glycine residue of a protein to give a radical there. (6) These reactions are affected by the ionic state of the additive. ACKNOWLEDGEMENTS

The authors thank Dr. P. ALEXANDERof the Chester Beatty Research Institute, London and Professor A. Cr~ARLESBYfor their interest in this work and for the valuable discussions held with them. B. B. SINGHacknowledges a research fellowship from the Colombo Plan authorities. Biochim. Biophys. Acta, lO9 (1965) 2o4-213

RADICAL REACTIONS IN IRRADIATED PROTEINS

2I 3

REFERENCES I 2 3 4 5 6 7 8 9 io It I2 13 14 15 i6 17

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