THE UN I VE R S I TY OF MI C H G- A N MiCHIGA- MEMORIAL.PHOENIX PROJECT Technical IReport No~ I ERfSONANCE IN RADIEATION EFFECTS Henry J Gomon'berg%, Principal. Investigator Marvin C, Atkins William R Clenrdinning Ardath H Emimolns Julian L. GCarsoAdlon A, G-ord-uts UNLI Proj ec-t 030 49 suppor-ted by: MICHIGA'N MEM]:ORIAL —. P3PHSid-iNIX PROJS.TECT AND U' S AtJOMIWC ENERGY COMMISSION CONTRACT. OAT (II.-1) -684 adi,:ni istered byy: THE UNIVERSITY OF MICH! IG SSI RESEA:IR3C- INSTITUTE. I N.ARBOR FebruaFry 1960

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TAB3SLE OF CONTENTS Page LIST OF TABLES v LIST OF fFIGU.RES' INTRODUCTION "Radiation Damage1 I General ExperJnaen.tal Techenicu.es 2 i. STUDIES OF THE EFFECTS OF MONOCIHROMATIC RDIATIO ON SOLID SOLUTIONS CONTAINING ORG-ANIC' HATIDES 5 Res ults 6 II STTUDIES OF THE EFFECTS'OF0'OR3. AOCHOATI i DTAAIC RABIATION ON T HE CATALASE SYSTEM II Intro duct ion 1 Catalase Solutions -Diffracted X-Radiation 12 Constant Absorbed Dose Irradiations 12 Possible Effect of Second~Order Radiation 12 Damage of Caatalase Solution)s at 6 ~9 and 7. 1 kev as a Fu.ncL-ion of Photons Absorbed 15 X-ray Fluorescent Irradiation of Catalase Solutions 17 Possibility of Sharp Resonance Absorptions 1.7 Conmparison of Results -C.taI(, ase Solutions 19 X-ray Fluorescent Irradiations of Dry Catalase 19 Chromium and Nickel IrradiatiLons 21 Manganese Irradiation 21 iron Fluorescent Irradiation of Dry Catal'tLase 21 Discussion of Results-Catalase System 24 Molecules Damaged per Photon 24 Dose-Rate Dependence 26 III THE n-BUTIL BROMIDE SYSTEM 35 IV AN ORGANO-MERCURY SYSTEM 35 APPENDIX X-RAY ENERGY RESOL UTION 31 XL Il

LIST OF TABLES No. Page I Coomparison of Efficiencies of Radiation Dosimeters at Various Energies 3 II Num, ber of Halogen Atoms Produced per X-ray Photon Absorbed for Various X-ray Energies I III Dose-Rate Data'Dry Catalase Irradiations 21 IV Number of Molecules Darmaged per Photon Absorbed in Various Catalase Irradiations 25 V Destruction of Catalase Solutions as a FIunction of Dose Rate 27 VI Ratio: Dose Rates and Photon Damage Efficiencies, Catalase Solutions 27 V

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I1ST OF FIGURES No. Page I. Reproducibility of results as a function of energy for irradiations of portions of a film consisting of PS-vITDCI-C3CBr, 8 2 Damage response as a function of energy for film consisting of PS-ATD=0CHIZs -CBr 9 3 Catalase solution, loss of catal yic activity -when irradiated with selected energies of X-radiation, 13 4 Wei.ghted and total mass absorption coefficient for catalase and catalase solution. 14 5 Destruction of catalase solution with monochromatic X-rays of 6.9 a.nd 753 kev 16 6 Destruction of catalase solutions -by target X-ray fluorescent raid.iation. 18 7 Destruction of catalase solutions by X-ray fluorescent radiation. 20 8 Destruction of dry catalase by X-ray fluorescent radiation from nickel and chromium targets, 22 9 Destruction of dry catalase by X-ray fluorescent radiation from iron and manganese targets. 23 10 Destruction of catalase solutions as a function. of dose rate~ cobalt-60 gammxa source. 28 11 Catalase solution loss of enzymatic activity at 50C and shielded from light, 30 12 Catalase solution loss of enzymnatic activity at 24~C and in sunlight. 31 1.3 Damage per unit energy (in arbitrary units) as a function of fluorescent X-radiation energy. 33 14 Percent of energy a;bsorbed by a sample in 2 i Bragg angle regions centered about the Bragg a.ngle 29, 357

INThIRODUCTION Radiation effects in chemical a nd'biological systems are often correlateu with such. quanti-ties as energy lost per unit path length of ionizing particles, ionization density, dose rate,., tenperatutre, eotc h There has been liEttle w-ork particul'arly in the soft X-ray region1 on the relationship between the energy of the radiation and the effects observed. it is of i.nt.er-est, "tiherefore to determine,whether preferential or ^resonance" effects exist in rhadiation darmage, and wheheer any correlation exists betw^een the absorption spectrum of the system and the effects observedIt appears reasonable to -think tha-t preferential absorption of photons in the electroon shells of specific important atoms in a complex e mlecule ma y result in "r:adiation damnrage" in excess of tthe pr.oportional armouent of energy a, bsorbed.. As an example specific metals, such as iron or manganese, exist in only trace concentrations in certain biologi.cal systems such as cytochrome' catalase, or chlorophyll, but their action is -vita to the behavior of these systems. Preferential absorption of radiation by such atoms could lead to more significant "damage" effects than would the absorption of an e.qui.ivalent or greater amount of energy elsewhere in the complex moleculeit was the purpose of this study to explore any existing relationship between radiation at specific energies.in the X-ray.region aind some defi nable I"riadia-tijon. effects " Specifically, various-s comnpounnd.s were irradiated with monochromatic X-rays in the energy range of from- 5 to 35 kev and. -the extelnt of "rad.iation dama ge" was determin.ed, "RADIATION DAMAGE".Unf.ortunate.tly, the term'.-radiastion. damage" has been used frequently -without precise definition, Such precise definition is necessary since the extent of damage observed will depend e pon the particular ana1.ytical d.emiage tdetection method ehosen and will not necessarily take into account all radiatiomn-induced changes which have occurred in the system. For the systems described in. this report, radiation damage was defined in terms of a property of the system which. it -was thouelghrth might depend upon the energy of the impinging radiation In in iterpreting da.ta, however, it was n.ecessary to account for the fact that the ex<tent of absorption of X-radiation by a sample is a function of energy For example, if the system under study contain'ed a large fra ct iorn of attom-f s of an. e.le-mernt whos.e K absor T) pt ion, edge P was in the enersgy region investigated the mass absorption coefficient for tthe Sample would.. be app:reciably greater- at energies just above the K absorption edge than at energies just below this K edge Since te the ac-moen't of radiction absorbed will

"be a fuilction of energy, it w-as n-.ecessary- to evaluat te te data in terms of "dcamage per photoni absorbed or per ibL. nt e.nergy aebscorbed" as a fut-nction of "energy of the incident radia8tion,' Observed variations in d image per pe hoton aIbsorbed. rmust be due to some ph.enomena directly connected with the effectiveness of the absorbed energy in creating damnage, since variations in the absorption coefficient are -taken into account. GNE RAL i EXPERIENIE.NTAL.TEh.LKIQtJES The monochromat tic rZad.i.ation source utilized i.n all thnese stu dies was a. Genera- Electrice Model X bD-,5 X-ray- unnit in some studi es es sentially -onochromatic X-rays (see the Appendix) were obta..ined using a sodiuml chloride crystal as a diffracting mediumn. In other stud.ies' nearly monochromatic X-rad-i a.tion. nias obtained utilizing the fluorescent-, emission spectrum of variou.ss targets' about 85,o of the radiation being due to K tfluorescence of -the -target radiaftor, the remainder, K, The most recent stud.ies utiltized radiaiion that is more tr;ul-y monochrormatic consisting of approximaitely e 99 to 99. 5%0 K^ rad-iation obtai ned by filtering out most of the KP radiation.- emitvted, by a- fl.aorescent radiator,4 The energy dose rates imparted to samples were determi.ne d swith either a Fricke-ferrous sulfate dosimi.etern a Victoreen vModel 651 ion-chtamc.beir ra-te mete.r or the SPG No 1. X —ray counter associated with the Gener"al Electric RD-5 X-ray unit% The SPG-I counter operates in the propor' tlional' reegion. has a 65 -cm argon gas fiJ Jil.ing and. a berylli um window 0.05 in. thi ck. These counters were "calibrated" by cop.orirng their effici encies with that of the ricke dosimeter (a G value of 1 5 was used1) A comparison of the ef f:ciencies of these various dosimenters:is given in Tabl.e 1. Even uthough -thie deterction effici.encies are vastly different the ratios of efficiencies ap-pear to be rea sonably consistent snd appare.nly exhibit no large en.ergy dependenece As is evident from the diffe rences in detection efficienies' it is noit possicble to asertain the absolute energy dose imparted to a sampple. In a continuin.g e:ffo-r-t t o determine thi.s qu.antity, a total absorption thermal. d.osimeter is now beJting constru cr-ted. It will consist of a gold plate which willi in-tercep-t the X-ray- beamr, The radia-tion,: lipon conversion to heat energy, will be detected by thermistors.' thus providi.ng a direct measure of the total energy of'the X-ray beoam. 1Cottin, M. and Lefort, Mt J., "Etalonnage A.bsolu 5Du Dosimntetre.Au Sulfa-te Ferreaux, Rayons X -mous d.e 10 et 8 KeSr SU Cinar tl Ptysys 53, 265 (195 6)'At the ti.me of w-riting i:ni-'tial -tests a-t 25 nmicrowatts were su.ccessful, 2

COONPA~RION OT EFFICIENCY S OF, 3ABSIATION DOSIIiER RS AT FAnIOUS EN BRGES Target and Energy Irg^et, aria Eneergy Dose Rate*: ergs/cm/hr 3 of Fluorescent K ~__~ Rato atio Radiation key. (A) Fricke (B) X-ray counter (C) Ion ChIa'er A A Zn 8. 614 5. 67 x 10i 2 31 x 105 0407 Cu 8.00 4,91 x 10 2,32 x 105 0.472 NI 7- 47. 5 68 x i0o P.47 x 104 2042 x 105. 0o096 0.426 Co 6,93 3 03 x 105 1 16 x 10C 0583 Fe 640C 4 29 x 105 4 16 x 104 2 x 1 5 0.097 0 595 n 5 89 2.47 x 100 2.44 x 104 1 15 x 105 0.099 0 466 Cr 54-0 1.28 x 105 1,33 x 104 4 20 x 10 0 10o4 0.328 *All determined at, or corrected to. X-r t-ube operat on at 50 pkv and 40 -ma.

I., STIDIES OF THE E EFF'ECTS OF M'4ONOCHROMATIC RADIATION ON SOLID SOLUTIONS CONTAINING ORGANI. C H -ALIDES.In an attemilpt -to avoid possible lon -diff-usiorn reactions resulting from the radiation, a series of experi'ments were performed using solid solutions containing organic halidesk* The solid solutions wemre ree dpared in the form of thin plastic films 0 @25 to 0 38 ~ 0 005 mm thick and containing the halide These films consisted of three basic components Ix An organic halid.e or mixture of two organic halides~ Included. in -the studies were: CHIn C3-r4. C2EBr-5, and. C2CCl 2 A dye-'ornming substance That used. ws pp;pjp"-Mehylidynet:ris (N-~, N dAimethylani-iline) (vMiD) which upon reaction with halogen'becomes crystal violet (CV) 53 The film-forming material was polystyrene, It w-as chosen because it exhibits.mu.ch greater radiation resistance to free radical production than do the organic halides A polysityrene and MTPTD film is far more resistant to radiation. as measure, by color change, than is a film. wit.h MTD and halide In preparing the films, usuall.y 2 gm of polystyrene, 0l1 gm1 MTTD,. 3d5 in of the organic halides were dissolved in 100 ml of carbon disu.lfide T'his mixturie mwas then poured on an area about 15L x 15 cm2 and allowed -to dry, Small portions of the film. were irradiated with diffracted X-radiation of a specified energy The extent of aJbsorption of X-rays zwas d.ete:rmined with "the SPGC-2 kyrpton-filled or the SPG-L argon-filled proportional counter, associated with the instriument, Damage was defined in termns of the extent of production of crystal -violet and. thus was an indication of the extent of prod.uction of halogen atoms. The amount of crystal violet in a sample was ascertained by d.etermiinig the optical density of the film at 615 mg- An unirradia-ted. piece of the film was used ina determinin:g the "background" optical density of the film The extent of absorp-tion of crystal violet was determined by preparing solutions containin.g known ami.ounts of MT1D and 12e iMore extensive data and information can be found in the PIh-D~ thesis of JulLian L, Garsou filed in 199 with the Faculty of Scienc s' the Un.iversity of Leigen Belgium 5

RESULTS Because of the complexity of'these systems' it -was somewh.at difficult'to obtain reproducibility. Tabe II contains a slmmary of tlese initial resul ts' The reason for these variations in da-mage is not apparen.t especially since it was thought that all large sources of error were eliJm.:i-na-tec1d IIt -i s possible,3 however, that twhe irreproaouc...ibii. ty- might be due in part to so'Re unkn.own vanriable i.n the films On the basis of experience gained in the manufacLture of th else previous film samples a new series of films i wcs prepared... The preparatiionl of t1hese films appears -to some extent to be an art in itsvelf, These new filmSs appeared. to be m uch better both in q-ua..lity and uniformity. One such film, consisting of 01 gm MTD, 2 gm polystyrene. 0.3 g-m CHIs3 and. 3 32 1 COBr4, was cut into small portions and. irradiated at various energies Usually three separate portions of this one film were irradiated at each energy..Th..e results in the energy range 24 to 35 kev are given in Fig, 1. Again, there appears to'be some variation inl the results These data seer to indicate that slightly higher extents of damage occur in the region of the iodine K, energy' and. slightly loTer extents of damage in. the energy. region just above the iodine K absorption edge However), whether or not these effects are real (in view of the variations observed at any given energy) is open to ques'tiono It appears as though these data of Fige 1 could be approximated by the solid line drawn on the graph. Such decrease in d.amage with increase in energy is also observed, in the li-quid n-bu.tyl bromEid.e+ —DPPH system d.escribed in this report. It is suggested. -there that this effect co-u-ld be due to dosi-metry energy dependencies. Portions of this same film (CtI3 + CBr4) were also irradiated in the region 11 -'to 15 kevn These results are given in Fig- 2 and indicated by open circles. Again there are variations in damage observed at given energies. In. an attempt to minimize fvr-tther any possible inhomogeneities in the fim itself, a single piece of filmt prepared in this same manner was subjected. to ir-radiation in such a manner -that -the diffraction unit oscillated througe a, small$ angle and thus irradiated the film over a linear distance of about 5 -nm. ThSis linear distance -was related to the ener'gy of the impinging X-rays ~ These data, obtained by determining the optical density of this film at every 0 2,5 or O 5-rm interval~, are plotted as closed circles in Fig. 2. Here smal..l but perhaps significantr, sensitivi'ties to d.amage are observed in the energy- region of the'k, Kp, and K absorption edge of bromine Because of the uncert ainties in these d.ata. it is difficult -to conclude nlwhether any abnormal effects did occur in the is K4, or K a:bsorpt.ion eniergy regjons of these halogens 0

TABLE II NUMBER OF HALOGEN ATOMS PRODUCED PER X-RAY PHOTON A3SORBED FOBR VARIOUS X-RAY ENERG-EiES X-Ray Organic Halide Film Consti'tuentsa Energy,, 3.32 gm CBr,, e3 32 gm CB:r4. kev 7,32 gm CBE4 0.3 gm CHI3 0 g3 C gm CHI3, (b c) 3 0,24 gm 020 0 38., 0. o x 103 0.9 x 103 0. 3 x lo0 34,. 1 2 0o 8 0, 6 x lo o0 5 33.1 1.0 2.53 0.6 0.6 3350 1.1 3.1 0 6 0e5 32,2 1.1 200 LI 0 6 30 5 1, - 1.3 30,2 1.6 31.2 1.2 29 4 2 - 1.2 - 28.6 1.7 1.7 1.9 1.2 28,3 2,1 2.0 1.6 0.7 26,4- 43,2. 9 0. 7 1,,2 25.6 2.6.. 24.8 3.5 24 3 2,4 14i 1.8 25.0 2.0 7.9 22.1 23 10.9 20,9 1,8 - 18 15 153 15 138 1.4 33 0.a8 0 9 15 o0 8 3.0 1.1.1, (13'.3 l.,3 l,3.58 1.5 L1313 3- 3 1,2 - 1.2 - 6.6 - t13'2 1. 2- - 12,6 - 07 -. f(11-.9 1.7 2,6 1.4 2. 6 1311.9 2,1 2 1 1.7 1.7 11.2 2.,6 27 2.1 aTihe filmls contained, in additilon, 0,1 gm MID and 2 gin polystyrene. bTo obtain a high photon fl.ux, a large slit width was used, In each irradiation 90I of the energy imparted to the sample was in the Cpproximaite range 0,95 E to 1.05 E, whne.re E is the stated X-ray energy (see Appendix), "The characteristic energies' in keyv associated with the halogens are: lodine: K - 28.6, K~2 >5^1 52 50 K 55- 2 Brom'ine: K % 11.9, ~ 19. K0"i L 9 r 13n3 15. K50 =!1534, Kas 1 t15,5 7

Molecules of Crystal Violet Produced per 10-3> Photons Absolibed 0 0 0 0 D. P I P<-,^) r 0t H- -roJR H H (K — I - -- O'wO. H 0-1t, o U t 0 C) Pi - I I. t 000 0 /~,,.I i i'PI:'%, Ol, l (C.JO r t t- I6J r-~-i, I'' _ I~ k. I + t 0 Pi-ia 0~~~~~ I

i! I 4-1.6 0 -~ -- --- — T —--- ~ I i i I i r. I,' _ t........... I -...........................-.....................-.............. —------------—....? —-----— h -—.................... t O o 6 ~~~~~~~~~~~~~~~~~.~.._...... ~..., ~._~......... ~....I~.... _~.................................................................................... J J. ii~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 0.I~I 6 14 11 12X,0/ TV: t 6 Y 6 t V YfD.... C.:~.... C.~ ) Open.-.e s _odu.'-i t y of 4esu4ts for4 senic $t.4 o, C)D-^7(>til IirxvC~r~t (a) C~pte~l e^>lT>CJ~e~b 5*r-4eS3;rl0(3U>-tlt)-) ] -i C< (3t L~eiSU~tS t~r S~tE~tL2 a.:t-Ln os o 3..f>m (. G.- c:rc:._ rs ponse o: a si N ^ gie por-tb.eo of film to an osci]llating ~.rrad.i.n, i;-~on,,

If there are abnorlmal effects in -these energy regions, such phelno-mena wtould be unique and unexplainable in terms of current theories, To investigate rwhether such effects do indeed occur, it wras decided. to center attention on systems that, perhaps, would tend to be mor.e reproducible. The remainder of this report describes the results obtained in studies of the catalase system. the n-butyl bromide system. and a system consisting of an organic mercury compounds In the process of investigating these systems,, various techni ques, a few of which are some'what novel, have been perfected. To daten the most complete results have been obtained for the catalase system. These data, which are presented in the next section, also suggest, but -vwith a higher degree of confidence -that aobnormally large effects occur in the energy region of -the iron KC1% Kp, and K absorption edge 10

II. " S'TUDIES OF THE EFFECTS O.t: MONOCI:t0.CBROM.~{IC RAOIATV'IO0 ON THE CATALASE SYST-tJ* INTRO.t JUCTIOI N Catalase is a high-molecuia ar-.weight (225,o00) e.nzym.e whO.se most important prope-rt he aly is tcta e he a ility t c y e deconmzpositio" n of hy drogen peroxide The catal..ase molecule belongs to a general class oF biologically importrain-t molecu.-les iChose function is attributeed primar ily to the bttriace qu antity- of a metal present in. the moleculee Four iron atoms aere incorporated. in each molecule), a concentration of 0,09To iron by weigt Trhe -. bility to cause adestruction of hydrogen peroxide is attributed to tchs iron contients It was the initial purpose of this study to attempt -to determine if this assumpti-on is correct. Specifical.ly. it was dei o..ded. -to study the extent of deamage to this mIolecule per pro-ton. absorbed.in the energy region of tthe iron K absorption edge- If the iron is i.-nti-c.mately asso iated with the biological activity of catalase, itis concei vable tat a tlarger extent of damsage per proton absorbed would occur at energies i.immediCately aJbove the iron K edge because a larger fraction of the energy absorbed per molecule is deposited in the iron atoms Radiation damage -to -this molecule has been defined in this exer-iment as the loss of catalytic activity w.-th respect to hydrogen. peroxide decomoosition. Thus if -the assumption is correct9 the experimental resul.ts.would. serve as an indication of the extent of "darmage" to those iron atoms or those portions of the molecule encompassing iron atoms which are "acti'vity sites." The amount of catalase in a sam:ple, as well as the amount of catalase "which did not undergo "radiation datmage," was determined by- measurement of the postirradiation capability to cause catalytic deompositio n of hydrogen peroxideThe catalase was dissolved. inL 1/ tt h rmolar phosphate bu ffer of pH 6 8. Measured amounts of hydrogen peroxide were then added and the ttransnimittance of the mixture recorded automatically (at 2120A) for a period of about one minute'ollowing -the addition of the peroxide e Since the n.iti. rate of decomposition of hydrogen peroxide by ecatalase is a fi.rst-order reaction.2 it was possible to relate the concentration of cattalase to Lthese observed initial first-order rates. Catalase concentrations were measured by this me.thod; the uncertainty in the measured. values were ~ lO/o of -the val.ue, vMore complete descipti.ons of eperimental techniques and. data can be -ound -in the Ph.D P thesis of Ardathh Hi Emrnonsx f~iled with The U'niversity of Michigan. 1960, and available through. Un.iv'ersi.-ty Microfilm.s An.n Arbor, Michiga.n 0Dixon, MH, and Webb, E C, slme..... lew York: Academic Press, In.c 1958. 11

The experiments were essentially of two typtes: those invoving solut..uions:L. of catalase and -those invoving sannpl es of d.ry ca'talase,, CATAiIA0E SOLUTIONS -DIFFRP CTEbD X — RA1S.,ATION Constant Absorbed DIose Irradia-t-ions -A stock solution 10'7 -molar in ca-talase and 1/15 molar in phosphshte buffer was prepared. A l.i.e it. holder was constructed with *twoo sample positions; one in.terceptLed the radiation beam and the other was shielded from scattiered X-rays. SamLpl.es of 0 18 rml waVere contalined. in these positions. The holder was cooled by circulat ing 5 C'-waer and the whole assembly painted black to miinimize enzymne actL; v,..tivty loss induced by lights. With the X-ray tube operating at 50 pkv, samples wTere sulbjeclted to a selected X-ra y energy (as obt-ained bry NaCI diffraction) The irrad.al'tion at each energy was for a period such that 18 x 10 1". X-.rty photons were absorbed per cm3i P1hoton d.etection was effected by means of the X-ray counter.v The dose rate was maiantained aC 9^ 5 1.0 x 110 photons inciden-t per hour *by adjustment of the X-ray tube current. Three 0.050-ml aliquots of the irradiated and of the controlt sn aples were ther. analyzed for catalytic a. ctivi -ty Tese data are plotted in Fig. 3 There each point represents the average of the measurements on the three aliquots of a given irradiationThese data sow an inc rease in.n efe. e ct as th e photon erY y crosses -the iron K-eabsorption edge (7 11 kev). For comnparison, a normalized. mass absorption curlve for iron is indicated by the broken curve in Fig 3. Referring to Fig 4 - i t is noted. -tha-t, because of the very.low iron. concentration, the totalt mass absorption of th-e molecule and the meforee aof the solution is a reasonab1ly smoothi function in the energy region 5-12 kev- l Henrce. in termrs of d.ma.ge per photon to the caltalase solution as a whole, no abnormal effect wcould be expected in this energy range. However a larger fracction of the photons absorbed by the solution wouid -be absorbed d by the iron atoms for photon energies just above the iron K edge than for those just below this edge, Theeforre if dlam g perp photon absorbed is measxured in terms of the iron comnponent of the molecule.e a discon:tinuinty could exist. A possible conclusion, -therrefore is -that damage to calt alas e is as s.uggested, associated with the iron atoms in the molecule and that this d.age can be affected by selection of irradia.tion energy. Possible Effect of Second —rder.ad.iti on i s icrease in iam.age in the vicinity of 7 ke could perhaps be ascribed to effects of contam.nating second — order diffra'ction lines. Th.e TLa li ne of tu ngsten-, c the target nrmaterial in'the X-ray tube, is aUt 11.28 kevv. Therefore,. t j.his coul.d contribu.te a second.-o:rder line at 5.64 kev. Perhaps there exists a depend.ence of d.amage on energy that does not exhibit any discontinuit.ies If the extent of dmao increases as the energy increases an.1.28-kev X-ray wo-u..ld. ibe more effetive than- a 4 h-.kev X-ray, although the efficiency ratio is not knowN. On this o 3sis then1 one would expect an increase in dnutge o-t the 5,^-kev crysita settinn g due to the 1. 1 2 8-kev con Ltr.ibution. 12

Eaon saipl e absorbed doseJ cm — Catalase conceczra t-ion = aid2x7,O M 80 iciden-, dse ra-e 9.5Incident dose r+e X-ray0 s 5 fl-l 0. 3 6 n, - ------ - - ----- ---- \ - _ _ _ 3 00 C d ------- FV.- tV I P-3;3~~~~~~~~~~~~~~~~~~~~~~~~~~~~~3 "IN~~~~~~~~~~~~~~~~~~~~~ ~~~-3 ~~ -vJ J 20 20Q~~~~~~~~~~~~~~~~~~~~~ ~~ i t~~ ~ ~ ~ ~ ~I'B ____ 3~ t 3 ~ ~~ 33 i 1 - 0~~~~~~~~~~ Fdg., @atlase alw -on i oss Of ly -'Tctk actcivit"i when`rrad-lateat d w'th~ s — <33 o ~~~ig- 3 Ctaas slt_ -ca Vl-1 lect~ed,energiejs ofl X-rad-J'tioatN"I'Lon.e iron-mxzass ab sor-p~t onr cu~rve giiven'by b rok11-1n c u _r-v,

— ~, —(-,.-~ t.I... I I I' ^~~~~~ II0 0 K i-qx: i~f. ---' t___, 4t- K t - t I I i l ~ -I I 0 -'~ (1?..~''~~~~~s^

Tihis possibility is, ho'wever, somewhat, di.fficu.lt to accept for'three reaSons: First the actual ratio of drstage at 1 2 kev co.parei't ith 5 6 kev w ould have to be very large Accord.in g to the data, of Fig- 35 thi s is not so Second the discontinuity in Fig 35 appears to be nearer to 7 kev- evern consider t ing the large utncertainties in the rneasurementss. 1sThirdc the obser-ved data corr-relaxate reasonably Awel with the mass absorpti.on c'lurve for iron, AMlACE OF OATJLA O3SF SOLUTIOINS AT 649 A.uD 7.3 11EV AS A FUOCTION OF PHOTONi ABSORBED To minimizses any possible effects dule to second-order radiation. two additional sets of experimenrts were conducted at 6, 9 and 7.53 kev, In these rui.ns the exc itati on potential on the X —ray tube was reduced to 15 pkw-,, thus reducing any second-order contr ibutions. The di age results at these two energies as a function of total dse a e preseunted in Fig 5 The characteristic initial increase in catalase activity displayed by irradiated c at-alase solution. is also observed here.re It is possible to compare qualitative ly the data of Figs 5 an d 5 The data of Fig o5'were obtained at a constanit a~bsorbed dose of 18. 10~'L photons per cmaS. If one assumes that no dose.rate dependsence exists the data of Fig 5 a may be interpolated at the level where 18 x 10 phot(ons.were absorbed per orm The data for the 7- 5 -kev irradiations have been extrapolated (dsed c(ashed e) in terms of an exponen.tial tailing as is characteri stic of such i...rradiations:a Therefore, ait the 18 x 10' absorbed photon levelt, almostt 100)/ of the catalase activity wras- lost when irradiation was with 7e 5seY photons a3nd about -10- lost when -the irradiation -wa with 6 - -kev photons Qualitatively, "then, these data of Fig 5 are in agreement with the data of Fig 3...The data of Fig. 5, however, also p-riCovide a dir e t correlation of dtriage efficienci es at the two energies Neglec-tiLng the chacracteristic initi a in.crease in activity,, it is noted that the linear portions of these two curves, -when extraupolatedc hatve the same inLercept. indicatin.g a consistent ratio of efficiencies and the lack of a' m.ulAti-hit" effects The damage efficiencies may therefore'be obtained from the ratio of the slopes Such a com.pr..ison indicates 5Forssberg; A., "Action of X-rays on Catalase and~ Its Biological Significancen Arkid, Kemi- Miner Geol, 21A I.. (1 15)'It was noted that this activation could be duplicated bty adding micromole qouan.ti ties of very dilute hydrogen peroxide to the catealase solutions pr ior to the nalyssis in which, as men'ttioned macro amo0unts of hydrogen peroxideT were used15

0'~,. 1-~ l c Co', I i. _ p v tJ ii 04c: _ _1Ct 1 i' S 1,'I _ _ _ k, o o0, h I b(3.I<o __-_ ____.4.H' X o W W s 1! t e't X X I oH I 1 | 1 | [-:""'< D. l A i~3 IO C,~S ~ g 0 - 4 d H l I' - OnJr-'J I _r i 6 i 1 i 3 f I

rad.ciaGtion a.t 7 3 kev is about 2 9 t Gimes as effective per photon J in prodaucing damage as is radiation at 6~ 9 kev. X-RAY FLUORESCENT IkRRADIATION' OFc CA.iAISSE SOLUTIONS To o'btain higher dose rawes and thus short.er irradiation tin.es with the ex.: isting X-_ray 2eq ugipmniL.entL, a serioes of irradiations waos undertaken utilizing tule fluorescent radiation emitted by a target,, Three d.ifferent targets were used. Since tie K aradiation of the target, which consti tutes a hout i, of the tota radiathion^, was not filtered. out in these e x eriment the'se iradiations were onl..y.approximately monochromati c The three target radiators used and the energies of the emitted fl.uorescent rCadiact;ion are gi.ven below: Ka (kev) 1K (kev) Target Element 5. 85 T of Total. 15% of Total Iron 6 40 7- 06 Ni ckel 7 74 1 8 26 Manganes e 5 9 6 49 (Note: Iron n < absorption edge is at.11 ke-v ) Th'e results of these i rradiations. again showing the ch aracter isti i..i ti al activationu are give:n 2in Ftig, 6. The extent of damage praodutced per photon for:' -the catalase solutions i rradiated with nickel and manganese fluorescent radiation appear qualitativel.y, as'would be expected on the basis of data of Figs. 3 and 5" The nickel.radiation (wh< h is of gcreaster enertngy than -the iron K absorption edge) is more effel'ctive -than man P-garese r.ad iation. (energy l.ess than iron K edge in prod.ucing damage Possibi ity of Sfharp Resonance Absorptions. T -— rThe results usin r.,g i ron flu.o rescent rad.iation. are however, co-mpletely u.,ne.ixpected^ On the basis of the iro:n K absorption edge model itndi cated by the previous data, it -wourild be expet.ed thac t th.e iron radiation, being of an energy lover than LIthe iron K edge^ -would ex hiL bit ad.-ge effciency le ss than that of nickel radiation. tand of the order of that. ob served for manganese radiation. This high. efficiency of iron fluorescent rcadiation i prodducing damage in catalase solut'ions is ine xplictable in terms of existing'theories. Perhaps some type of resonarnce adiation absorption may occur wahen the iraon- on- tain ning catalase is irradiated wyith iron scfluor..sent emission irad. iaton Becau.se of the nca.turet of these results - with iron f.lu.oarescent cacada Uh-ot Ut is iportaUnt to intvestigaate aurthera this possibility oa ancreaased dtaage due to resonance radiation absorptiono, It is planned to repeat this par:ticular set of 17

:.t........ t. 100 --------- - -r -- - --- 0 -............._'~j;; 1. - 10.......1........ i 10. ~3~6q~ W —---— Ote —----- ------ _ _ _ —W -1 —'1'-'''- -1I ——'-'-'- - _ -- -- ---- 40o 1 — tl \~~~~~~" - -: --—::1 i la r i i I 1 111 1 i i i i 1 _0 _ _ _____ 0 I Fig. 6. Destruction of catalase solutions by tacrget X-ray t uo R ac Ia 1 Ln. -8.:~3b target X- ~ ABSR E i Fig~~ 6~ De'refo off ca,-hls sottin'1 %a rg Fi,5,~st uL IQ~ P oltonsLS a~e

exper)iments as well as investigate the eff:ects of other., fluorescent radiatiso..on on catalase solutions COIPARISO ODF RF'LS-ULT,A'IA-"ASE... OLUTITO Tae data of Fig. 69 for conrvenience, havre'been plotted as a function of the logarithm of the nu-.mber of pbhotons absorbed. I plotted. on a linear scale (Fig. 7), variations exist larger than that apparent in Fig. 6, However, as';rtwas -".th.e case with the data of Fig. 5, these -L.ree sets of F1ig 7 coul..d "be amproxiated b y a straight line, Th.e relative slopes then serve as an indication of the relative efficiencies of th;ae fluorescenut radiat.ion in producing daeage These dasage per photon efficiencyr ratios are approxim.atety: bNs icel Iron Iron:l~ L J -.iE1 7,6 - ~Minanganese MJn. gan.e se Nickel This nickel-.manganese ratio.'betinag a.. indication of the ratio of efficiency of daaime c of photons of energy above Uthe iron K absorption edge to photons of enterty less than this K edge, is in reasornabl.e accord with the 7.5ke'v6,9-kev data (efficiency rat-io = 29) of Fig, 5 Again -the ratios involving the iron flu ores ce-n.t radia tion suggest the presence of some -unexpec ted phenomenon, Since both thile iron rand manganese radiattion consist of photon:os whIose energy is less than thait. of ^the iron K akbsorption edge, one would expect comparable efficiencies in producing damage. As stated above, the observed. ratio of iron-mlangainese is about 6.6. X-RAY F-LUO SCt'NT IRFADIATIONS OF DRY ("fALAT..,S In an- attempt -to minimize possible effects due to free radicals product-ced in the aqueous solvent, as we]l. as to increase the cat;al.as e t"arget concentration. a series of.irradiations'was performed. using dry ca-ta$la.se as the -target material. Irradiation was'by mea ans of target fluor esceint t emission. A Fticke fer-rous sulfate dosimeter, -was used for absorbed dose and dose-rate calibreation. Af-ter irradiation, -the smSple was dissolved. in 50 ml of 1/1.5th molar phosphat' e bufChromium,, nickel' iron,. and manganese target r'...adciea, -tors wee used in these experim.ents. The chromi um radiation consists of about 85 iQ radiation of energy 5l> kev and abo-ut 15% K9i radiation. of energy 5 94 kev. T1he radiation energies for the o ther three targeets are listed on pa ge 17" Dose. —Irate data for -h.esa irraxdiations obtained. obus ing a, Fricke dosimrae'ter, aroe listed in Table III. IC9

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-LAPOTA Il DOSE-RATE J'AAD,. DRY CAT.ALSE IRBA.D...AT..0NS Target ____ Avertage Dose Rate Fluorescent Frgs A-bsorlbeda Photons Absorbed Radietor per ms-hour petr Pge-hou r Nickel 38 x 106 3e x 10. Iron 4 0 x 106 3,8 x lO^4 MaInga8nese 2, 6 x 106 2,7 x 10 14 Chro-miu-Lm. x 1t0 2 0 x.1014 Chromium and. Nickel Irrzadiatio. sb Chromium fluorescence is of energy- less than the iron K absorption edg rhereas the nickel is of energy aove the iron edge I.f these data are examned in. -terms of the logarit..hm of.the percent remar.aiing activity as a function of energy absorbed (Fig, 8), the damage response appears to'be a linear function and. it is seen. that spporeenitly the nickel. and. chromirm fluorescent radiation are equally effective per unit energy absorbed in damaging catalase I Thuus it appears that the dcsage 0esponse is not a function of energy, at least for the photon enrg ere mitted y these two radiators M anganese Irradiation.s.The irradiations performed. using mananganese fluorescent radiation are depicted in Fig. 9 by the same'two -slope curve ascribed to the iron-toarget irradiations. It is open to question whether such a two-slope representation for the manganese data is valid, especially since on.ly one datum point exists wit-h which to identify the initial "induction. period, " It is possible' therefor e, that these man.ganese irradiations result in a g p uit ergy absorbed similair to that observed with the nickel and chr omiutim radiation Further experiments will provide a'm ore exact. r'epresentation. Iron Fluorescent Irradietion of DPry Catalase -The t-wo-slope represen.tation for the iron fluorescent irrad iations (Fig. 9), ho'weverx appears bto be reala As was the case in the irraoiations of cataalase solutions he.re again the iron.K-a and K. radiation appear to pr odu e comple-tely ine- xplicable e ffec ts Radi8aion ind;uctiJon periodS,.as. app areritly presen.t in this instance, have been observed by various expeismenters working with other y systems This induction dose of radiation prior to the appearance of damage was ascribed -to a multi. —.hit effect. In view of the lack of kno-wledge of the mechanism resulting in radiatieon d.amage to catalase, it is difficult to postulaete a mechanism based. on a mul.ti -hit phenomenon. Perh aps even more sgni.icant is the steep respo us. cutrve followsing tfhe induction region i'n these iron. f'luorescence radiations, Iit is particcularly diffi cult to expllai why, i n a system for which a. gen, group of energies (n ickel 01

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and chromium radiation) shows a one.hi. t resp onse ion. fclueor escent e nergies btimiacte sensiitivity,, DISCUdSSION OF RESULTS CATAI\ELASE SYS3m..l.T.; xIn att.-teminptuin.g -to interpret fully the sigtficaiice o~ these and other ret)sul.ts, cogn.izansce miust be taken lof the cmI ber of molecules damaged per photon and the existence of any d.ose- ate d.epend.e:sncyJy Mtolecuxle s Damagged per hotn — tl a i on of the tunimber of molecrules damaged per photon will depend upon the accuracy of the dosi:Lme-try measurelments As eviden itfroni Table! an. approx mately texnfold difference I n sensitivity exi sted between the Fr i ke-ferrous su nalte and. the SPG-I. d.osim2etiry measurements. Theref ore any calcula;ions of the rncuober of caalase mlecul es destroyed per photon wouldd be onlr y apoproximiatiors' of the true value i Wn.en the total ca/bsor.pt ion thermle.el dosimeter: m e r ntiioned. earl.ier-, is co ip l s e-1t ed it shou ld be possible to deter2mine aiccurirte va\lues of these plhoton dar-lge efficirencies. On the b sirs of the present d.osi:mset-r mea msurrements, values oi these effi) ciencies havre been calculaLted fo to hs 50G d,-:mag level (Tale IV), Irn -the irradtiatio-s of catala..sa-se solutjions' i-t can'be calcui -laed from the mass absorption coefficients (se F ) tig 4-) that only one photon is a bsorbed by an. ironatof atom for prxoximte evenry 10 phottons albsorbed byA -the solution. Therefore one would not expect any observa ble difference in.A d.-sage efficiency as the ir.uon K absorption ed-ge was crossed unless the photon dam.e efficiency was of th ordr of 10''o molecules da-laged. per photon obsor'bed.. The lowest da.mage efficiency for solut.ions. as shown a Tabe V- isLe 0_L08 Therefore on the basis of conventiornal interpretationxs of photon-i do a u.age effici sncie es most of the catalae molecules are fbeing d Lac-ged as a resiunlt osf secorn.dary radiaetioninduced processes' Thus the disscon-tirrunituy at the i ron. a;bsorption edge a nd, more ixrmpo:rtant. -the appa-rentl- greater effisciency of iron fluorescent radia tion in producing damage in catalase soluto ns. besome even m.ores puzzls -ingo In the irradiation of d ry cata-lase, referring to the mass absorption d.ata of Fig. 4, it can be calculated that, a et en t ergies below the ier onr. K absorptiLon edge. one photon is absorbed by an ironr atomi for every 190 phot)cns absorbed by thel molecul e Above the iron K ed.g e h t ve ratio is I. in 244. Here again the data 0or the nickel arnd. chromium irruadia tions goiven in Tabl3e r'V ind-icate cthat mos-t of the molecules -re da..aged as a resl....t of seeond.ry energy-t ansfer processes' ior the iron ir.rad. iation.s if one exam.8.ies only the steep p:)ortion.i of lthe curve in Fig, 9, it cans be calculated tha;t at he 500 d.am ae level 2t5 molecules are destroyed per photon riabsor"bed.. If one incl.ud.es i n the calculation the photonst absorbed, duri.rg tble i ndu.e-ton period the value is 88 molecules destroyed per photon absorbed. In ei ther case these data regarding the irradia-'tion of dry.stalase are of- t uhe s."m.e ( rde:r cf magn —t-u.de in -tecrms of photon d.amage efficiencies. Theref re c'.becsu.se of the.t-ge magni t.de eo these efeicien24-i

TABLE "IV JTOBEBR OF 14.01 CULES DAMAOT.tD PER PHOTON ABS ORED IN'VARBOUS CATALASE IIhADIAJii ONS* Molecules Damaged, Per Ph.et.on Ab.. Catalase MEolecules Damagede. Per Dos ieter hon A System Photon Absorbed Used soid n Tes of Ferrous Sulfate Dos ime trry Soluti on, 6. 9 kev SPG-1 X-ray Fig. 5 Counte Solution, 7.3 kev 7 SPG-O X- rray Fag..C ountei r Solution, Iron 0.-20 Fricke 0.20 Radiatiaon, Fig. 6 Solut-ion, N.ickel.9 o,,, o 0.046 Fricke o.046 Radiation, Fig. 6 Solution, Manannese i7^ ^/. ^0 028 Fricke 0o028 Radiat'ion Fig 6 Dry, Nicke l6Lo rCcke 6 Radiation, Fig. 8 Dry, Chroniu4 1 Radiation, Fig, 8iee *Calculated at the 0o% damage level, 25

cies it again appears puzzling that two types of response should be observed for the irradiation of dry cata lase Dose-Rate Dependence.- Associate d with these observations are the possibili.ties of variations in response due to varia tions in dose rate. A seri'es of samples of cata las e solution were i rradiatbed. with cobalt- 60 g aon radiatio n The dose required to dmag oe 50/ of the catala s proved to be a function of the (lose rate. The extent of damage.o however, was foutnd to be relatively inAependent of concentration. These data ae prese::nted'in iTable V and Fig 10., It is quite possible that the difference in damage efficec1 ct r.es for the irradiation of catalase solutions at 6 9 and 7 3 kev as clompared with -the flu.orescent irradiations (see Taeble IV) could be due to a dose-rate depend ency. If we asstme ithalt the irradliaftions t 6 9 kev t.ae s.imilar in effect to the irradiations using a mangne3e target and that f;he 7^-ske'v and nicksel-starget irradiantions are also sitmilara n maye then calculate the ratios started in Table'Vl. Three observations may be Tsntvde in. regard to these data- F"irst it i is probably not vastly signific.ant th.a)t the value is in -the last'column ar not t )in sore pe rfect agreen ent a variety of exxperimental d.ata are involved in calculating these ratios. S econd the dose- t e depeTndene in this X-ray energy region is mluch.smaller.than when h irradiation is by means of cobalt60 gamma radiation (see s Fig; 10)., Third,^ the observation of -the r.ch greater d.anage response of Cata'lase solutions to iron fluoresc ent radiation cannot be ascribed to the sniail dose-rate differ ences between the iron, manganese, and nickel irradiations (d.ata given in. Fig. 6). These irrada.0tions of catalase solutions tlilizing iron fluo escent radiation wverre at a slightly higher doss re b thian those utilizing the -manganese or nickel radieation. Therefore, on the basis of a dose -rate depenendce. it would'be expexed 1t tha t'the iron irradiations would require the absorption of a. sl.ghtlly larget r n lber of photons to damage 50Jo of the molecules, an effect opposi te to that observed. Although no dose-rate-dependence studies were mae on dry catalase it is conceiva bl e that. only a small dependence would. also exist. In. sutumiary, i't appears that'ths 4eetculia.r da.mag 8res pontse of dry catalase or citalase suolution to iatron fluorescent radiaion is not a resullt of a dose- rate effect, These abnormal effects in response as well as the apparent ir on K absorption el edge eifee:t vr n cat.Faase asolutions/ 6 ppear even more pu zzling since apparent ly most of the cat alase molecules asre das ged as a result of some secorndar y pD;'ocess. Furr.ther~ s.t..d.i. es will. be p'erformed in ra.. e.or to d.etermin e i f p ossi bl.e the nature or the r esponse o ncta asc to radi ationr Oner aspect ofil these s'tu.td-~ ies'that hts been annoyi ng?. as well. as of cornce rne: is t'he time'required for a, 26

TRA LTE V DESTRUCTION ORF CAIALASE SOLUTIONS AS A FTUNCTION 0:?OF DOSE RATE (Cobal t - 60 Gammna Source) Rep Dose to Ergs/gram Dose Rate Coneent -ra-t -on~ 50% 5G/0 rep/hr mgm/L rep/hr......g......Destruction D est ruct ion 60o 43.6 1,000 1 x 105 7,800 16,,8 800 7.8 x 0o 86,00 0 57.6 6 o000 6 x lo 131,9000 100 8 ooo0 8>5 X 1o 131,000 50 "75,000 7 X5 x " 0 1,100,000 i8,8 196, 000 1 96 x 107 T-ABLE V I RATIO. DOSE RATES.AN'D PHOTON DAMAGE -FF'RICI:E-CIES, CATALASE S0OLUI IONS Ratio: Ratio: Photons Absorbed Dose Rate s to Prioduce 0 o ), D-amage Manganese 6. 9 ke v Nickel1 7. 3 keyv 27

---. —-—. — -- J 1'~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1 V~~~~~~~~~~ I~~~~ ~ ~ C) iIIII( 1 ( 1@- 4-) L —— ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ —--- _ _ 0~~~~~~~,,!,1 - ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ------------' -- ]; 0~'~ 2.:~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ — ----- - -- ------------------------— i- -- --- -- -- Cy -- -—.-............... _~ ~ _ _ _ _ _ _ _ I I iI ~ F~~~~~~ j —-----------— 5-t~~~-f-+-~~ —~ —1 — -- - ------— ~ -----— i —- ---- -i- ------ ---- -— i —---— lf~ 1_ I 0 ) ICD - -- --------------------............i.... i —------- ---- -------- -------------- -------- --------- ----'" - -- -- 103 ~~~~~~~~~i ij i i,... i I J'-,, ""<:~~~~~~:! -— t — t --------—! —---------— t —— ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ -------------- I~~~~~~~~~~~~~~~~~~~~~~~~~~) -V~~~~~~ 0 e i 1 i r 1 1 r~1 _ ( i i — L.-.i.___ _ _ _ _ _ —------ -- - i 0 I f ) ---- - -.............. i~~~~~ ~iflO / I' I fl I S,.-i-:::0_-+-.....:'.%:%:.h:::::::~:::: h:::::::::::?::t::.I::.:..i..::::?::::::::.::::::::::::::t::-. —-----------....- -- — t — — t —-I —!-~~~~~- ----- ------- -.,::-.t. —— r —:- t.~~-. —..... ——...h:.... ----- ----; —-- -- 1 i:4, t._:..................._...... --------- -— _-_ —. —------- ---.................. - I. —C —- ---— i —--—. —-. ---- i i —-'-'~gE —--- ------ - -- ------------ - iT — ~~~~~~ —-i — i ~ ~ V - i::: ~i } — t-/d U —...i —3i —::-: —-.... J~~~ j J~ J:j t ~ ~ ii:: e I h-,, ~,,.......i..........................?: — ~~$"i~ l F:- I i: ~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~9 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~.I.. 31~~~~~~~~~~~~~~~~~~~~~~~~~..,r4Q

single irradiation., Irradia- tion times of from. 110 to 50 hour:s were not u.ncom:.on. Because catalase solutions vwere found to be easily tdam,,aged "by light and heat (see Figs. 11 and 12), it was necessary to observe ext;reme ca.utioIn in the hard. - ling of the sanmpless Control samples were used in.the irradi.ations U thus hopefully" compensating for any nonirradia-;tion. effects' sutch asi o the time of smtandciL:ng The solutions were emaintained. at 50C and shiecl.edC Cfrom light dIng the i rradiation^z The samples of dry catalase were rradiated at approximaetel y roo. temperature but, again control sacmples were used. Ideally^ to minimize any possible non.radiation effects, it "would. be desi r able to irradiate the samples for much shorter periods, This wi.ld.'be possi.le if higher dose rabtes could be obtain.ed. "Unfortunate ly, hi.gheTr dose rate s are not obtainable with the X-ray u.nit used in this s tudy. It would be des ir able therefore, to use a new X-ray unit capable of a high.er emission flux. We are cu. rrently looki.ng into the possibility of such an acquia.sition. 29

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III THE n- BUJT IY BROHiDf.E SY-STEM Samples of purified licluid n-butyl bromilde containing DPPH (diphenylpicylhydrazl) a-t a concentration of 1.2 x 10io M were irradiated with. fluorescent radiation of various energies in the range of from 5 to 25 kevy the'bromine K atbsorption edge being at 13 48 kevw Damage to this systerm was determined in terms of the amount of free radicals prioduced as indicated by "loss" of DPPH scavengers The amuount of free DPPH remaining in a sample after irrdad.ialtion was determined spectrophotomi.etricallay at 5200A us-.ng a Beckman DU spec-trophotom.eter. The Beckman waas specially equipped to utilize micro —cuvettes to measure the optical density of extremely small voSlum.es (0.150 ml) of sam.ple solution, Prelimina ry stud.ies indicated'that this system is reasonably insen.sitive to dose rate and. DPPH concentration. I At twen.tyfold. increase in dose rate resulted in less than a t-wofold decrease in damage per u.u.nin.t energy absorbed. A te nf old increase in DPPH caonentration resulted. in less than a twofold increase in dramage per unit energy absorbed. Apparently, -then the DPPH is scavenging successfully most of the free radicals formed. Dose rates of I to 8 x 109 photons absorbed per second) as dete;rnained using the SPGC-I proportional co unter, were used in all irradiations, Preliminary experimental results do not indicate any large anom.a l.oous damage response at the bromine K absorption ed.ge These preliminary results may be depicted, qualitatively,'by the straigtn- line plot of' Fig. 1'5 8 13 0 — ". L1iquid nm.Ch-Br 5 U) 7" " 5 re:) -P i t fluore:t X. ti e " 5!o 1 20 2,5 key Fig. 15: Damage per unit energy (in ardbi-rry units) as a function of fluorescent.-,radtiation energy.

The reason. for the nega-tive slope in Fig 135 is not kn3iown It is posse ible that it is a r eflection of( dosimery l dependien ce Thte (possible}) slig.t decrease In. d1m.ge e ficieny at ete t nnergies greaeter than'tie brosine K g tbst ay'be due to loss of brtoine Ky an B f, lu oresce ad..at on generated., -wi thin trhe s amp1le 1t s.ho'u.ln hbe n:ot-ed hai the bsorptpion cefficierh of n-fbutyl'b Eor i d. e d.e.-. pendis la-rgely on the absorption of t e I bromine atom. Be.low 15,48 kev pproxi - mia i te/-.,y 9 ) of li1-Oe p hootc.s a0:e atbs'-)ed'- t rhe bro-mile at.oms,'whe:eas aibove.I1,".so B-e 1ke v more ham, 9.,..e t ao b sort ed.'by the bromine am tovms Thits a ch ang, ar( t;e a hbro oine K absorpti0on o~g ifnrnage per photon a bsorbed or per unit er —t - as so>0ed could also result'if i oIn o mamner absorption in the bromine K -,hell' it 1it subseol- e K flus:or escene or.An cer clecr el e ons..-o esut n themiioe p in cJt.-he pod c%:i. of an, a.ou of'bromi. lne aitoms di. f erent f ron tha t esl. ting'V fro'm, L shell a',b;,ir:sor Lion Futuore eernes will'be dir ected toward at more precise dete t S i:> of the magnit-Ltde of the dih continuityl in acesponse at the bromine.r K absorapti on edges These data rwill provide a basis on whi1ch to determine w0, 0"hether the loss of 1 orescencta radiation ~111 account for the magnitcude of the dLiscontinlity ^~

IV. ANOR -OIC SYSTEM Samples of solid a-'acet oxyme z c uri- -ir methoxy-hydroc in:naJ.ic ethyl ester were irradiated with filtered and mixed. (iKa K) fluorescent radiation in the energyregion of from 9/2 to 17 5 kevy The mercury L- LI adsorption edge is at 12,,282 kev, Damage was defined in terms of the amount of free mercury produced as a result of an irradiation-, It was obtained by' dissolvjing the compound in. absolute alcohol and centrifuging the mercury The mercur y was then dissolved, in concentrated HE2S04 -H03 and the amount of merlcury produced (ca. 0 5 mg) was determined. spectrophtometrically using dJithiizone col:plexing agents The compound R-Hg contains 40 weight percent meercury. In the energy region studied, the mercury atoms absorbe.d a minirmum of 97O of the photons. Within experi.mental error'the damage per unit energy absorbed as a function of energy appears to be linear with no disconti nui.ties at the ercury LI1i absorp-. tion edge. "55

APPENDIX X.3R:.AY FIViK'....'... S OLUTM"ION Int the.'plastic fil m and. catal ase experimr:en,-ts util;tld, iz ing di..ffract...ed LX-ra,.i.-i. tion. i:Lt was necessary to use fairl.ty, airge sl t wid.ths to obtain a dose ra-te su;ff4icietly highi sro Lsthat atn Irraac t n could be perforned in a reas na' bly slo.shntr tire. Even w$ith the sl.it'wjid] eths used, ry I-rral.ation. we:"e of e10 to 100 h oursB duration, Because of the laro'e satie and therefore large sl %t Awidht>hs used the energy spectrum imnparted Sto these sarpl es i ncludaed appriox imaRtely the raige of 09"5 E to 1.0)5 E1 where E is the energy cal.culated f'om t... X- ray BxaaRgg angle sett ing Folr cany given angle, and thus average ener;gy about 90% of the total ern ergy arriving at the sample was found to be contained in a 2 - 50" B-,-raogg an gle3 range centered. on the dsapl as tindcated by t hve data Fgiv i 1. t Pe cent of Total Energy 1 5% 2-)% I 7/a o'^ I"'''7,~;~ tO O, 1.......... 1 20 Bragg 5 3 0 70... 5 ef 20) ww 5 ww 3 ww 1q.D 1 ~57 ~... Ankgle 2 2 2 2 2 "-o 11 E ner'Y 7'T 48 7 I...e~kg ~. go" 6e 97 o6 ~,, 7 1'7 26' - spectrum.. w,w _ -1..__. W W W. _WW at certai.i.. 12s 61e2 - 1 3,29 1, 6) 3 01 i. agle snet..3 5___'9_ t, ing"s8i1J i 8 4 - o.'_o;e_^'K_ a'b_, __,_ ^'.....__........._29 ~ 7. Kiote: K abso-rpt.ion edge, kev:'rr. I - i, eror i 7m.e -:. 5 8, 13,.48 -..odne. - 3'' 7 Fi g, 1.. Percent of energy absorbed by- a sasTmple in 20'" Bragg an-.gle regions centered abou t'the Bragg angle 29. T'o obLtain more nearly rrmonochrormatic radi.atironr a,'taget fluor.es.centl ra.diation techniqule was utilized. In. these irratditations the X-rays emitted. i by a targe t radiat'Lor consisted. of primarily/ 85% Kd and 15% Ks fluorescenLt n emission. Dose rates were genercally 103 to 10(3 greater t.han that obtained. by the difLract i.on methods The K radsiation could be reduced preferentLially by interiposin'g an f.Ulter, generall.y of:-. atomic rll.nuber. one less t'habn that of the fl'.orescent:rad..iator'.. The 37

..Ky tradiation is re-duc-ed -in i"n.tensity'b a. factor of approx3imaStely three while the K, is reduced in intensity by a factor of about 50 to 100 As a result, the filtered raidation is composed of about 99 to 99~1 0 K(a rad-iation. The dose rate is reduced only by a factor of about three. Such filtered fluorescent irradiations will be utilized. in analyzing further the damage response of the catalase, n-butyl bromide and. organooe-mercury systems. In the energy region of from 5 to 55 kev, 55 K. emiss3in energies exist. It must be recognized, therefore, that the use of this fluorescent technique limits the investigations to only specified energies. For many studies' th.is numberof discreet energy irradiat.ions could, however, provide sufficient data with which'to investigate the:damage response of a system. More important. the existence of Ka and K5 resonance radiation effectsu as suggested by the cata lase system data, would be apparent only when irradiation was by means of target fluorescent radiation. 38

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