Title Some Substances of Biological Interest Studied by ... · being 3 x 10' r per minutes. Total dosis of gamma-ray irradiation were 104- .-10' r. Irradiation was made under vacuum

Title

Free Radicals of Gamma-Ray Irradiated Amino Acids andSome Substances of Biological Interest Studied by ElectronSpin Resonance Absorption. (Special Issue on Physical,Chemical and Biological Effects of Gamma Radiation, II)

Author(s) Imai, Yasuo; Inouye, Akira; Sugibuchi, Kiyoshi; Hirai, Akira;Toyoda, Sadaharu

Citation Bulletin of the Institute for Chemical Research, KyotoUniversity (1961), 39(2): 138-152

Issue Date 1961-03-31

URL http://hdl.handle.net/2433/75795

Right

Type Departmental Bulletin Paper

Textversion publisher

Kyoto University

Free Radicals of Gamma-Ray Irradiated Amino Acids

and Some Substances of Biological Interest Studied

by Electron Spin Resonance Absorption.

Yasuo IMAI, Akira INOUYE*

Department of Physiology, Faculty of Medicine, Kyoto University

Kiyoshi SUGIBUCHI, Akira HIRAI**

Department of Physics, Faculty of Science, Kyoto University

and

Sadaharu TOYODA*** Resources Research Institute, Kawaguchi, Saitama

(Receiued September 24, 1960)

In this experiment, investigations on the ESR signals of the gamma-irradiated amino aids, peptides and proteins are chieifly made, the results obtained being as follows :

(a) ESR absorption of 104-107 r irradiated amino acids, peptides and proteins show characteristic curves respectively.

(b) The signals of those which contain sulfhydryl or disulfide groups show essentially the same pattern as those of S' or S-S.

(c) The concentration of the free radical electron in the irradiated protein (about 10L8 spins per gram) is lower than those of amino acids and peptides (abont 1018 spins per gram).

(d) When irradiation was made in the presence of oxygen, the signals of irradiated protein show marked difference from those of irradiated in vacua or in the presence of

nitrogen. (e) The protein irradiated in the presence of water shows rapid decaying ESR signal.

(f) UV irradiation also produces in protein and nucleic acids considerable amount of free radicals.

Some discussions were made on the radiation damage of the biologically interesting materials from these reults.

INTRODUCTION

As pointed out by Ingram", electron spin resonance (ESR) absorption is one of the most direct method for studying the breakdown processes in living system

produced by irradiation and a systematic investigation of radicals formed by irradi-ation of substances of biological interest seems very important to obtain basic

physical knowledges in this field. Indeed, some studies-0 along such an investi-gative approach have already appeared. We have also sttempted to observe the ESR spectra of gamma-ray irradiated amino acid, peptides, proteins and nucleic

k J~ 7i, J1= L ;" l Dill Ii , =V- )1-

*** _...h111'r~

(138 )

Radiation irrduced Free Radicals

acids to examine the nature of radiation-induced free radicals of these biologically

important substances. This brief report is chiefly concerned with such an observa-

tion of ESR spectra, its detailed account will be published later. To determine the

exact trapping portion or distribution of unpaired electron of free radicals, however,

observations on the single crystals are necessary. These experiments are now in

progress on some compounds in our laboratory.

METHODS

Samples tested : Crystalline bovine serum albumin was Armour's product, while

human serum albmin was prepared and crystalized by Cohn's method". Samples ,of DNA and RNA were prepared from pig's semen" and -calf liver microsome'>,

respectively. Other samples were commercial ones, mainly obtained from Azino-

moto Co. Irradiation : The samples, whicn were in a powdered form, were sealed in the

long glass tube evacuated to 10-'mmHg and irradiated by 2 kilo-Curie Co" gamma-ray source at the Institute for Chemical Research, Kyoto University, its intensity

being 3 x 10' r per minutes. Total dosis of gamma-ray irradiation were 104- .-10' r.

Irradiation was made under vacuum to avoid the possible oxygen effect, except

that albumin samples were irradiated in the presence of oxygen or water to observe their effect upon free radical formation.

ESR absorption : After irradiation, one end of the glass tube was annealed to

remove undesirable signals from free radicals yielded in glass and then a sample

contained in the other end was transfered to the annealed portion. For strong signals from some amino acids, the background signals from the glass tube were

negligible. For weak signals, however, such a background signal should be taken

into account. Hence the sealed glass tube was opened, the sample was transfered

into another unirradiated tube and ESR signal was observed. No appreciable oxygen

effect was observed in our all sample tested at least immediately after exposure

to air, but considerable changes in the ESR signal was obtained several hours after

exposure to air (see leucine in Table 1).

ESR spectra was observed by a hand made ESR apparatus in earlier experiments.

But Varian V-4500 spectrometer at Resources Research Institute was chiefly used

in later observations because of its stability to compare the relative characteristics of all tested samples. The conditions for signal measurement were tried to be the

same for all samples compared as far as possible, which were as follows:

Microwave frequency9450 MC/sec

Modulation frequency of magnetic field 400 C/sec.

Its amplitude 3.5 Gauss for nearly all samples.

12 Gauss for samples of weak signals.

0.22 Gauss for samples of strong signal with sharp h. f. s.

Microwave r. f. field H ==about 0.1 Gauss.

Field sweep late22 Gauss/minute. Integrating time constant 0.3-0.8 sec.

Temperature15°C.

( 139 )

Yasuo IMAI, Akira INOUYE, Kiyoshi SUGIBUCHI, Akira HIRAI and Sadaharu TOYODA

Table 1. Amino acids, peptides and protein mainly consisting of aliphatic carbon chain irradiated by 107r gammaray in the absence of oxygen.

A : possible trapping portion of unpaired electron, and breaking positions are shown by a dot and broken lines respectively.

B : The concentration of unpaired electrons per gram sample . estimated by comparing them with that of the standard carbon radical.

C: The first derivative EPR signals. The difference between the lines in the recorder chart is 22 Gauss each .

D: Integrated absorption signals, the position of g--:=2.003 being indicatea by the arrow , (A)(B)(C)(D)

/41—, —111111 —IIIM ( 1 ) —IllIIIIINMII

+' -- 1111111111111INININI ,.1NIIIIIINNINNIMI (.12-1-COOH2 x 1019 ---._. MONNIMNIMIINI

--

2NH2MIIIIIIIMMIMIMI___,. ,'r, 11111111111101111MINS0 Ga as ' " " IIIIIIMMINIMIIIIII

1 1111111111111101111M ------------------------------------ X 30110111111101111.1

IMIIIIIIIIIIIMMOMM (2)' =111====g111 — iirmilwelwal _ ,

I +mormigaw== cfp2-COOH - =1:2111nr= 1 ;

Ntli3x1019 "1.---M=1:11==parm.— 1 1Mon=11IMMIIIIII ,- - CH3-COI---rgridiallniall-20 Gauss

:-W:,^.,-1IiI, W 21-1

I— =me . . , Inil (3)2 .C1-1/-COOH iMIMI. , ' 11111111111U^, mrim=usum. , 1 12-__

NH-1===== —MTEMIMMI CO',_ammiltmarsomm

,

I%gx10i9iMilifir^WATE^ C 112—N / 1==1:117:=Mmer

l',.PRAHAMMUMIMI'lmm Co) - Wriln1=1111111=111111 - I I /1+G==ainini- NH

24-.C1-1/2, ..Alimmuninsor

i--------------------

20 Gauss _ 1===== _ . ( 4 )9 9===== --

CF13• I+INNIIIIIIIIIIIIM= -- `CH-CII2-CH-COOH1 t GH3/I1=1111181111211:011115,..:

NH.........MS6.1611.""", Ox 10'9 0=== ii,.1E— COZ w===rEti

II + NH2H-CH 2MIN----VEL‘ 'r ''.1--H__ semi /120 Gauss------- I.mum'- -,',.-

k------------------------^MS__; (5)R0 CIilia: /\N/

1' 11111111111M1_ H2 x 1B> ----- INNIMM—

. O•- —9 —; Mfflikailw um 20 Gauss--------- H I'—''''' =ram=

1 C 4 1;14 mammoni ,_._..... 11 silk fibre

R H. am

------------------- Min - ----- au (6)

Elf -- i OM Silk fibroine powder.9.sbk,.^ f ' MEI a:---=MN - -

_ ..,

_ I 1.0.V.14 aSTRIlairL j, 1 _pi.Mailii,,[I___1

___1...:____i_.____

+

( 01

a


-

_-_

111111110111111111111.11111111111•111•1111 111=11•11111111111 •1111•11•111111111•1411111111111•11U1111,

I+=...r ==A_I CH3—'H=CCH+OOOH6x 101- immi~0~e a~Bw'~~

mCCCni OH ; NH2•VASUM N

1

FAMMEMOMMOIMINIA\ ------------------------- rillIMINIMINEME

(8)mu®iii

rau.J2QGa uss CH3''= ,I,1~ CH3~CH_-F--iOOOH6 x 1°19~'u+I

J NHz.- -9=11.4r..:=, faanmszoimil= ----IHNIERIMAINCI Mil

IIMEIIIMIIMMMMOMAIIIIII CH3I+ ®®ME CH3/CH—GH2—cH—COO11®®^'i7/ =~--===1

NH4 x 101 FO-Mm®R IW=•_ Cr,'_t-~ir~ grE 20 Gauss

H3C' ̀ 0'''s .+~.~am+==s==

(10)----------------------- eM~aroINIIIIIIII•MMIIIll= 'CH31 +~~wae.i~~ `CH—CHz=CH-rOOOH®a*®• CH3/ f6 x 1018mivizent~i~. NH2m-I w ,'g~=

—C-O-O. -w .;r 1am20 Gauss

(11)m...WOn •5UUUMIME=~~. s31 .

CH3\1+pr!o)7-

CH3~H—CHz=CHCOOII101('~~!~in vacuo NH2i_,_>~~u~^^

NMIWIN MIMI

----------- ME=_,` 4 hrs in air (12)101:1111111

IMMINI MIN:1==® 20. Gauss \

NH2COOH!~!^=~r...^vri..~i. +

INHa- -CHCHz-CHzCHI+ T--7 x10A ^~~CS~U®

NH21111111111114111111111==.01 1_-

40 Gauss

(141)

Yasuo IMAI, Akira INOUYE, Kiyoshi SUGIBUCIII , Akira HIRAI and Sadaharu TOYODA

Net _ (13) 'I,----1 '-

' +

1: ___Lc -11111-1 AI /1111111101111111.___\ I CH3—CH4-COOH 5 x1019 ' -0101111111MINI -\____I I 1 ̂ R -- --I --'. NH211111111111111lar .____^ I 1 ' PIIPNIMRMIIMIIIW

J \ \ '4“.8 $10.01la . 4 --] s— -,

—11.111111EMONNINi '20 Gauss \ (14)mian=,..,\ +Eli .... sm.

CH3-,1__....r..., tir•la.,)''' ,H+COOHA4141=111Vmin^ II--111111.11/%10MI 11111111111•11111•1111111I NH4 x 101, mar ,,,rm—ra -

Islimasidomaito mrpriniummairsamrs,l'[ H3C-0-0r -Wilia911=1111111110-1i\\ mm.rmmimmi/ ." V ^

-------------------------------------------------- c 32— in mum,,GaussLoGauss _____ (15)^+.

,--4-.P i Clia—CFIr1 It-COOH /--------.,__ I

1 1', r , NH t__-__1___,.. 1, 1rJ

1 5 x 10,9-------.'— 0----C—H—CH31ER .1;.(d 4 YI , i I-------------------------------------------I 4. ,host,a_ 1+IlLetoi NH2

71 --------'40 Gauss

(16) CH3—.6H-Ir-000H

NH L — — 1 ^4.16IFAIMII I3 x 10'9 --1' •11111111111Offaiw-

o-c-ub 1

+ I----:'°1 =MINIM" - NH2 ----- ._ 111111111110111111111111 ------------------------------------------------------------------------------------- No 211111111111111111j

14-- -W11011==11101 -`—' (17) EMI-01lMIIIOIIIIIIIIIII - 20 Gauss CH3—H-It-COOH___ +1 1111111=11111111311111111

NI:1 t---- --• 11111111NR=111111111111 ------ I :--111111=111111

0=C-i-CH22.3 x 1019 INIIIIIIIIIO101=1,—---- i 1

iNH._REIM1.^011.•_ .6;1, „•,,filE , .--C-61124h,.

t- +-II.-- NH2-

20 Gauss

( 142 )


(17)

,---I--II1--1 .7.—

t

---1 COOH ---.MU \MI=•

man!MUM cHaH2-cH2-CH2-CJ10,9 H4 xMa-1.1 ,, i,K111111111 ,-K--•-[- NI-12NH2'WA all , 1111111111111111111 ,..__, &17.1i' 40 Gauss

______-------------------------------------_ ERN= iMINIM, 111=MILIMINIIIIIIIIMIONIMINIIIIIMMINIMI ===1111111111011111•111111111111111111111111111111111111111111^ (18)gutmemminsmirommEN

,, amm="1.1..mmommume... _ ,CH2-6H-li--000HNNW ^ .I ---1--'

OHH2 2.3 x 1019102.e.....--.+=312......IMIEl=m3....marikalim.=

N

, - t --• ___INRIMMIIIIPANNIMINIM 414-d-,m,,I.,1;_ rmaW*Pall111111111111111111111111111 1 ____471,/, _•111111==.... , --"--' — dININIIIIIIIIMISIMINIIIIIIMIIIMIIII ------- . IIIIIIIMNMIUIIIIIUUIIIIIMIIIMIMMIIZIMMMI

Rimiropuming1111111111, IMIIMMEMINI --------------------- ----==111=11111 ___„,. (19)20 Gauss combine with adjacentMN A ILINMEIMIIMIL—---1

molecule ....• mmumaiunini . CH2I-00014an--fy 11,9,(10,„Orwit-MILISIMMIZETWA..-

M.m0111111211111111 H2N-LCH-LCOOH I' 14"IM=.1=MELITIIMISINI: 6674.,mmumminmommi

. .._ ICI --==1:1111111111111 _------

(20) .„ 1lI MEM -2o Gauss

11 ----''' r 11111111111111111111 CH2-CH2-2-CH-F-COOH-T—H` 11E1111111111= --1---J2 x 102-

,...„ffirmimm S-CH3 NH2_'.Ain --MiAll 111M 1

i----r Ell %, ; . __---------^. . \

. !Ili...4 11111111111111+:,• ,20 Gauss

(21)liriallEMEN Cr

+

.I CH-CHEnionowinumma 1marrammonma-- ----- S-CI-13 NH1019=~-~ami~~®° --

onmEasomark- - - C0IMMIIIIIIIIIMMIM '

CH3IMPIONIOVIIIMMII:----1 ---1---1 --. --1 1

(22)I - .'1.1';.20; Gauss _.1._.',.=!,_1_... -..1: -^ I ..2 Mil - •

,4,' •-11,-14_11_:.-i 1+ d..IL-i.::......_1._4_ ."1_-_1_il. j_i_:. 11 .1ti---•1A

1

,CH2-OH24COOH-.-.114-.' .1'.--,--1A-----F7L'.-

,1----J1.8X1019:-I: I....11.11't{,..I-I.-- NH2+-CH-I-COOH ,

...___,

:-D-:-. kr.-.1.2urdir.r.a_ ...,ElirsliMMIM

- '_. 1.t Ati.',11-rr.., , elemillini - .111111111111=1111•1•111 -7 -- o ' - .1.11111/11111111•• -----

r_„_ _ _

I

(143)

Yasuo IMAI, Akira INOUYE, Kiyoshi SUGIBUCHI, Akira HIRAI and Sadaharu TOYODA

RESULTS

Amino acids and peptides . ESR absorption signals recorded were presented in

Tables 1, 2 and 3, in which a supposed (probable) position of unpared electron of

free radicals produced by ionization, decarboxylation and deamination as suggested

by previous investigators" is shown by the dot, while position of breakdown of bond is

represented by broken lines. From these results, ESR signals seem to be classified into three types according to g-values, over-all splitting and line width.

1. Aliphatic amino acids and peptides composed of them. Their g value is near 2.00 over-all splitting is more than 90 Gauss except glycine and its peptides

and line wibth is also wide.

2. Amino acids and peptides containing aromatic carbon ring. Their g value

is also near 2.00, but over-all splitting is narrower than the former (less than 80

Gauss) and line width is narrower.

3. Cystine, cystein and peptides containing them in types of sulfhydryl or

disulfide groups. Their g-value markedly deviates from 2.00, asymmetry of absor-

ption curve is remarkable. It seems worthy to note here that doublet splitting of peptides containing glycin

summarized in Tables 1-2, 3, 4, 5, 6 is different between the small molecules and the

high polymer, unpaired electrons in the former case mainly localized on carbon

atom coupling with one bonded hydrogen nucleus and in the latter mainly localized on an 0 atom experiencing dipolar interaction with bridging hydrogen nucleus,

when dipolar broadening and inhomogeneous broadening are taken into account,

and that peptide bonds are considerably resisting for radiation damage as shown in acetylated amino acids and peptides.

Protein. The results obtained are illustrated in Tables 1-5 6, and Table 4.

The signal of silk fibre was observed on the portion perpendicular to the static

magnetic field. Comparing the absorption pattern of fibroin powder, orientation-

dependet doublet was observed in the former.

Radiation effect on bovine serum albumin, human serum albumin, fibrin, fibroin

and silk fibre itself were observed. Cystine and cysteine content in these samples

IV

1101010'

Fig. 1. Corelation between the dosis of gamma irradiation and the signal height of protein.

Absissa : dosis of gamma-rays in kilo-roentogen. Ordinate : heights of ESR signals.

(144)


Table 2. Amino acids and peptides containing aromatic carbon ring. A, B, C, D are the same as in Table 1.

(A)(B)(C)(D)

ri'------------1111111•1111111111111M11= —-- -i•'4 --11111111111111111111MINI

( 1 )EMI 1111111=== + CHimaripm.= II

"

HC • C-C-CH3-CH-COOHraginlaisEME.

1 11 11I1.4 x 1018 __ _ - =MEN IIC C CH NH2waim=

/ \/ CN''' '' /--1 --=1/Sell= H- ---- 1 =

I - =1:11011MIN ( 2 )- 1111111=--- ' ,,, _+110122111:11 i'\ - cFr

\ ,±WWWW"M=1111111 ------ I \ FIC •C-C-CH2-dI-COOH10111=5.111---In i\

1itIII1. stimmum HCcCHNH6 x 1018.01==isow- /\/+I11111=11111EW1121--I CN..0-.-C HH•---1iiiiiMMINM1111111111111i

CH3=:1111111111111111r

(3)= Immallimme

mumum.maffs HO-HC -CH2111111111=-MINIIIM

I I; 1.3 x lips1111111===miN,•=as H2C•C1H-COOH -11111111===ffra= \/ I+ NH,,_wxm....h„iiiiimm...=

.M.MEMINIMIE ___----.12111---.--1111111111

N'M

CH

EI (4)--1111111-M---11=1:1111111011111

-111111WIWir HC/CHCH3-1111INAIW1111M --- 1 i-- It__00.7300 NMI-1 CH . CH C=O-11Nr=a11110111811111, 1.8 x 1018 / / _811:1=1M,IMP. ,i C NH

1III ,wi CH2-C1H-COOH1°1^ . , --

-----J ( 5)„; NIMBil&p.immtiummunig ,---

H2C---CH2 1A ill r .r-r A1116/11 MEI - ' r-r -sissmumme H2c CH—COOH1'1 1,3 x 1019/.-MERU AM—

\-- ' _ N i8-- --- 1 ̂ II JEW MIN.,..",/k __ _ , ,_' I arsummaim H^ [ ,,,, .3, ., low 1.,_--,- ••• IIIIIIIIIIIIIIIII

,-,_ IMMO -- , ' MIIIMIIIMMIIIIIIIIIII

(6), - IIMMI111/1111111___.I _-., 444 -FAMMIUM1111111

CH4.,,_ _ - IIIIIMMEME -- I - ME=1

\' ____J„AMEMEIMENI HC•CH - 1 —111111111111111111111111111=101/

I 11—I,- / 111111111M111111M1, }IC CH/ ...... . ; IMMINI1111111_11 _mr__.= V ! NH,7.5 x 1018 - - ' MIIIIIIIMI .,--=,-.-1_1' IIIIIIMMIIIIIIIIM -----

.- - In- ' Matala10111111/1///AMME' 1 L4_..,, -..,,--J- ---,t,__-= MIMAIIIIIIIIIIMIN CH2----CH4-COOH,__I,__I_1111116m10.111 Ir±i____.i„ 111111•11111=1

l'/-- /_-__--r — Imi^1

11- -,-: , ' - ; 11==--- 1I_ _ E--- ------ 4 4_, afilonsmomm 20 Gauss

( 145;

Yasuo IMAI, Akira INOUYE, Kiyoshi SUGIBUCHI, Akira HIRAI, and Sadaharu TOYODA

I------------- I- OH---------- l ------------------------

\ji1^ C HC CH 1s-----I--------------

HCCH '1.7 x 10'8 AII CNFbs

CH>CIIiC00H f — — — , I ---- 20 Gauss

(8) CHs--- r— CH . C;H—NH2 °_.-------t1 i-_,~1

N 1`—5------------

1

CHCIICOf~-----?r4 -1 s---------- 4{a_ CH CH NH ,i1,11 i'~i1Vy.t

CH -----{ rr Ie'aFLLq, uj CH2— - Cd-I-000H s,t,% -~It .7 ;,~,*I'

T g----------1---------------i -- I , —I I I i

(0)_----- CH

N---------- 44: ---------- CH ' C—C—CII2—CH-000HiI~YbI ~t, , ,ir, 1

\H C/\/ NH CH C.1,k-,)'',-7-'

CH NH....0=_C + Iit `-P, CH2CIx IcCt. ".WNW,

(10)-------------!.---------11; 1 ! ,1411 II 'if--------------11------1

CH—C—CH2—CH/COOH :! 1 _,tlL'ov,,III !-----------

I-IN N ' II/42 --1 z;-11----------~r.>~^~ CHz&7zxe-----------------}11 --f-\1

,1

are graded in several to zero percent (bovine serum albumin human serum alb-

umin>fibrin>silk= 0.)

The pattern of ESR absorption curve was not affected by change in dose of gam-

ma-rays, only effect being increase in the signal height (Fig. 1). Such a circumsta-

nce is quite similar to the dose-effect of amino acids.

DISCUSSION

Aliphatic amino acids as well as peptides and silk protein mainly consisting of

aliphatic carbon chain give, as illustrated in Table 1, widely spread signal (90-150

Gauss) with g value of approximately 2.00. Such a pattern would be interpreted

by unpaired electron of free radicals trapped on the carbon atom which has dipole-

(146)

Radiiation irrduced Free Radicals

Table 3. Cystine, cysteine and peptides containing them.

A, B, C, D are the same as in Table 1, except for the numbered arrows presented in the absorption curve of reduced glutatione.

1, 2, 3, 4 indicates the positions of g-.2.04, 2.027, 2.017 and 2.003 respectively.

(A)(B)(C)(D) -------- - - - — mar----

( 1)

---

I---MI= id" I a :1111111= --- CII2-CH-COOH111111111111:111111:1 :III --

1,-, I. .Sp:NII21.6xICIi9=MAIM-..IN=---20-dauss`e------ iii===lE111MIERI _J L------IIMMEW7---2 "T"Mwri:

=ME& ' ' '---IIMMLIM MW._ .

,-,--=MI=/ .IMMIIIINIWAI ----------IOM INEEMIllsim

,----- ( 2 )NE----I- IMB

.

CI I2-CH-COOHI-1:511111VIM-20 Gauss21, iI'diENNIII=i11111' 5.1+ NH23 1.1.11.5 x 1019IliniglEMEILIVMS'I'l 51 NH2 11111111=Bitanlif,-1---: 1 1 — IFIVG2111101111111511 _

CH2 -CH-COOH iI. =111111110111111111=E- _ } it---COMMIUMMOINIM IMMO---__M- i4

(3)_--,-- 1--r-mirrimm—ms.m._- Immi_

CF1-'111Imorimmi—win_1 ._

1--III=====--1 wIm.....mmin....m. N'0-CH

i

CH-liC2,. 1I3x 10,9_-..ammemoolgannuum.40Gauss

1

C112ammulimpz_____ 1-------------------- CO -NH-CH2-COOH 1 MANCEMILIIDINAM CHNH2WOMiall=lialMMEININ

I COOHrallama.111111111111 . - _ 1grarmr=== ( 4 ) 1

Si+IMIIIIMMMIMIl OOH1111===11111111 1CIPM---MIIIMISIMMIR %iNI-12 -CH

II1 x 1019 W.--Zalk==ilin CH°. CHMEMEILTINWITZETI

II4' NMI - -- IMICSIMMIN CHNI I-------CO CH2"I- -MilMr _MEN 1- '' ^ i''' CO-NH-CH 2C0014I---- .IMMILTAIN -----.---1111111111111110=140 Gauss

( 5 )., -'1 -S7*0 S-^1111 -I-f-1"- —swim= 1/10II,---;- -TINIMMEMI

N -FI 0=—1:MIIMEMINNI /a= 2ri f ijd'gMNIMMISIMI __I=' =mu Imo , \ mowI -- — =

, --------- -----

/ \

7 \

i

40 Gauss

1 --

( 147 )


Table 4. ESR signals of 107 irradiated protein in the presence or absence of onxyge and water.

io. 13 (PI

(1) „.‘,~i=mum -

bovine serum albuminSr~}---- irradiated in vacuo

M111=1 MO ,aq<b.thgU;Gam

2

bovine serum albuminY..: ; ®a 1. 107 r irradiated in

irradiated in the presenceim'-"a

vacuo. !x2

.var irradiated in of oxygen~_cuo.

'

--•-_i®ti~yvacuo. -. :1~i._..”_®~=3. 107r irradiated in 511Imi

saoxygen. utiz 'B

, MIN'11111110111,

(3)~~sEMI -

bovine serum albumin'®c EMI i

rradiated in the presence ;

of water~

va---MEM NEM

--- m

(4)1111111=

---- Malii=- sample tube (glass)A „,.

bakgroundill= 211m,fiiH

-=~=Ewa

--1

(5) lonnu ~- fibrin irradiated in vacuos®~ MMIIIIIII

1

m-,awa—

= -- :°e:~.~

(14b -

Radation irrduced Free Radicals

dipole coupling with adjacent protons.

Signals of irradiated glycine and its peptides are somewhat narrower (about

50 Gauss), the splitting width of their triplet and doublet being about 22 Gauss

which is not so unreasonable for CH2 and CH radicals.

The s orbital is spherically symmetrical, so that its wave function at the H

nucleus does not vanish and orientation independent (Fermi type) coupling which

is directly proportional to the density (10h*)o at the nucleus arises.

Since the doublet caused by pure s-state electron of H' has spacing of about 500 Gauss'', it is understandable on the case of amino acids and peptides having

narrower spacing than that of pure s-state electron that the unpaired electron has

the contribution of p character and considerable exchange with adjacent bonding

electron at this temperature (15°C).

In other words, if we consider the unpaired electron possesses carbon or-orbital,

metyl group or others having two hydrogen symmetric to this z-orbital give addit-

ional coupling of hyperconjugation (alanine, threonine, valine etc.).

It might be said, therefore, that the decarboxylation, deamination and CH bond

breakage are the main damage of aliphatic amino acids or peptides produced by

ionizing radiation as suggested by previous workers'", and peptide bond is conside-

rably resistive when the results of irradiated glycine-silk series and others are taken

into account.

The ESR signals of the amino acids and peptides containing ring carbons are

different from those of aliphatic carbon chain. The narrower spacing might be caused by motional narrowing of ring 7r-electron system but for peptides which

has long chain (acetyl tryptophan, alanyl phenylalanine). In the latter case, ESR signals of free radical electron in ring system are superimposed on those of aliphatic

chain.

The influence of hydroxyl group is shown in the case of phenylalanine and

tyrosine. Since or-electron density in the aromatic ring shifts to the hydroxyl

oxygen atom by its high electronegativity, namely density of the free radical

electron is lower in side chain carbon atom than ring carbon and this effect would

produce only weak hyperconjugational interaction of 7r-electron with side chain CH2, it seems not so unexpectable that ESR signal of irradiated tyrosine is mono-

tonous singlet.

Prolyne and hydroxyplolyne are somewhat different because these have no

conjugated double bond and no mobile electron. Since the n-orbital of unpaired

electron is aproximately vertical to the ring plane (Table 2-4) and wave function

of -CH bond of adjacent -CH2 group is symmetric with the orbital of unpaired electron, it would be expected that hyperconjugational interaction with these two

proton produces triplet line in the case of prolyne but singlet in hydroxyplolyne by high electronegativity of oxygen atom.

Sulfer containing amino acids, peptides and proteins show the same character

as the polymeric sulfer radical whose ESR absorption are also studied by Ingram"'

in dilute oleum.

As shown in Table 3 ESR signals of these samples have widely spread (120

Gauss) asymmetric curves. According to Ingram's results made by radiation with

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d ® --

trmmanniviguannumismammunmat

ananniammornomos _L

Fig. 2 ESR signals of 106r irradiated DNA and RNA Arrow indicates the position of g~2.003.

It° ------- lac up 1 [firt 111ME=18— -;

, , MEI I 51 N 4111 1111111111M1 ' 1111iIIMEM~~ohmmammas ®--

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I~~C-

. 44.4161111

11=111111g=Mal ' '111111 Flg. 3 ESR signals of UV irradiated DNA and fibrine in

the presence of oxygen for 5 hours.

different microwave length, these complex curves are not hyperfine structure but

caused by different g values. The g-values observed in the present experiment

were 1 : 2.04, 2 : 2.027, 3 : 2.017, 4 : 2.003, respectively, their agreement with those of

polymeric S radical in dilute oleum (20% SO3) being fairly well. This concept was also assured by another experiment with single crystal of

cystein, since g values was orientation depeudent. (to be published).

These data indicate that unpaired electron produced by ionizing radiation loca-rizes at the lone pair orbitals in -S or -S-S-. The results obtained by irradiation

studies on the aqueous solution indicated that in -SH or -S-S- containing amino

acids and peptides chemical degradation such as deamination is much smaller than

those containing no sulfhydryl and disulfide groups, except for such a reaction of oxidation of -SH and -S-S-1='.

It may be said, therefore, that -S or -S-S- group regarded as electron reserver

by Gordy protect molecules from the degradation such as deamination, decarboxyla-

tion or ionization produced by ionizing radiation, though some doubts remains

concerning its mechanism postulated by Gordy").

Proteins : -

As shown in Table 1-5, Tables 3-5, 6, Table 4-1, ESR absorption of silk fibre

shows orientation dependent doublet which is ascribed to an odd electron, localized

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on an 0 but experiencing direct dipole-dipole interaction with the bridging proton

in the ionized structure, as suggested by Gordy et al."),

O

/\N/C\ H

O\N+/ H

while that of silk fibroin powder gives only asymmetric singlet for random orien-

tation. In contrast to the above two, ESR absorption of irradiated proteins conta-

ining sulfhydryl and disulfide groups gives mixed patterns of two species of radicals depending on the cystine and cysteine content (-S. or -S S- and 0' interacting

with hydrogen nuleus by direct-dipole dipole coupling).

Yielded free radical concentration was lower in porteins than amino acids and

peptides (probably by radical recombination), and its decay time was also fast in

protein. The effect of oxygen and water observed with bovine serum albumin is shown

in Table 4.

ESR signals of irradiated protein in the presence of oxygen (Table 4: 2) is the

same as that of oxide radical observed on Teflon"). It may be said that one of the oxygen diradical electron combines abruptly with induced free radical at any

position, and that free radical electron localizes on the secondarily combined oxygen atom.

In the presence of water, the ESR signal decays very rapidly by chemical degra-

dation through reactions with water molecules. (Table 4-3). In this case radiation

effect on protein is mainly that of irradiated water molecules15'

Nucleic Acids : ESR signal of gamma-ray irradiated DNA, RNA and inononucl-

eotides are investigated by Gordy et al1". We also examined 106 r irradiated DNA

and RNA (Fig. 2), and results are the same as theirs. However, it may be worthy to

note here that 5 hours illumination by 500 W high pressure Hg-lamp produces

free radical considerably, which seems to be same as the radical produced by

gamma irradiation (Fig. 3).

ACKNOWLEDGEMENT

The authors wish to thank Prof. Sakae Shimizu of Kyoto University for his kind permission to use the irradiation apparatus, and express their gratitude to Mr , Yasuyuki Nakayama (Institute for Chemical Research, Kyoto University) for his

cooperation in the Co" gamma-ray irradiation.

REFERENCES

(1) D.J.E. Ingram, "Fee Radical as Studied by Electron Spin Resonance," London, Butterworth Scientific Publication (1958).

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(2) Bacq and Alexander, Rev. Mod. Phys., 31, 273 (1959). (3) P. Howard, Flanders, Adv. in Biol. Med. Phys., 16, 533 (1958). (4) V. P. Bond, E. P. Croukite, Ann. Rev. Physiol., 19, 299 (1957). (5) E. J. Cohn, L. E. Strong, W. L. Hughes, D. J. Mulford, M. Melin and H. L. Taylor, J.

Am. Chem. Soc., 68, 459 (1946).

(6) E. R. M. Kay, N. S. Symmons, and A. L. Dounce J. Amer. Chem. Soc., 74, 1724 (1952). (7) D. H. Benjamin and P. Doty, "Microsomal Particles and Protein" Synthes., papers presented

at the First Symposium of the Biophysical Society, at the Massachusetts Institute of Technology, Cambridge, February 5, 6 and 8, 27 (1958).

(8) C. R. Maxwell, D. C. Peterson, and Shapples, Radiation Research, 1, 530 (1954). (9) B. Smaller and M.S. Matheson, J. Chem. Phys., 28, 1169 (1958). (10) W. Gordy, W. B. Ard and H. Shields, Proc. Natl. Acad. Sci. U. S. 41, 983 (1955). (11) D. J. E. Inrgam, J. Chem. Soc., 2437 (1957). (12) W. M. Dale and J. V. V. Dais, Biochm. J., 48, 129 (1951). (13) W. Gordy, W. B. Ard, and H. Shields, Proc. Natl. Acad. Sci.U.S., 41, 983 (1955). (14) W. B. Ard, H. Shields and W. Gordy, J. Chem. Phys, 23, 1727 (1955). (15) J. Weiss, Nature, 153, 748 (1944). (16) H. N. Rexroad and W. Gordy, Proc. Natl. Acad. Sci. U. S., 45, 257 (1959).

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Title Some Substances of Biological Interest Studied by ... · being 3 x 10' r per minutes. Total dosis of gamma-ray irradiation were 104- .-10' r. Irradiation was made under vacuum

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