INT. J. RADIAT BIOL., VOL. 14, NO. 4, 351-361

Fluorescence of y-irradiated DNA and purinesin aqueous solution

J. P. GOEDBLOEDt and J. J. VAN HEMMEN

Medical Biological Laboratory of the National Defence Research OrganizationTNO, 139 Lange Kleiweg, Rijswijk (Z.H.), The Netherlands

(Received 7 June 1968; revision received 22 July 1968)

In y-irradiated solutions of DNA a characteristic fluorescence is observed whichis not present before irradiation. On the basis of the excitation and emissionspectra, it is concluded that two fluorescing components are present. One (A) hasan excitation maximum at 310 nm and an emission maximum at 400 nm, whereasthe other (B) has excitation maxima at 280 nm and 360 nm and an emissionmaximum at 450 nm. Both components are also found in irradiated guaninesolution, with approximately the same spectral characteristics as in DNA.Component (A) results from attack by OH radicals, whereas both OH andreducing radicals are involved in the formation of (B).

1. IntroductionIf an aqueous solution of DNA is irradiated with ionizing radiation, the

DNA is attacked by free radicals produced in water. The resulting chemicalchanges in the DNA are often estimated by measuring the decrease in opticaldensity. In general, measurement of fluorescence is much more sensitive thanthat of optical density. This was one of the reasons for examining the fluorescenceof irradiated DNA solutions.

In unirradiated solutions of DNA, purines and pyrimidines the quantumyield of fluorescence is low (Udenfriend and Zaltman 1962, Bbrresen 1963 a).Moreover the fluorescence characteristics after irradiation differ from those ofthe background and seem to be due to specific chemical changes.

In the present paper experiments are described that were designed to analysethe spectral characteristics of the fluorescence in irradiated DNA and to comparethem with those of irradiated purines and pyrimidines. A short note on thesubject was published previously (Goedbloed 1967). While work was in pro-gress, Keck and Hagen (1966) reported on fluorescence in irradiated DNAsolutions, giving, however, a much less detailed analysis.

2. Materials and methods2.1. Materials

Calf thymus DNA, adenine, guanine and GMP were obtained from theNutritional Biochemicals Corporation; cytosine and TMP from Koch Light

t Present address: J. P. Goedbloed, FOM Institute of Plasm Physics, Rijnhuizen,Jutphaas, The Netherlands.

Abbreviations: TMP, riboxylthymine monophosphate Na+ salt; dAMP: deoxyadeno-sine-5-monophosphate Na+ salt; dGMP: deoxyguanosine-5'-monophosphate Na+ salt;dCMP: deoxycytidine-5'-monophosphate; GMP: (2')-(3') guanosine monophosphate.

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352 J. P. Goedbloed and J. J. van Hemmen

Laboratories; thymine from Hoffman-La Roche and Co.; dAMP, dGMP anddCMP from the Sigma Chemical Corporation. These chemicals were usedwithout further purification.

Solutions of DNA, bases and nucleotides contained 001 M phosphatebuffer (pH 71). Concentrations were calculated from known molar extinctioncoefficients. The concentration of DNA and free nucleotides was in general10- 4M, with regard to the nucleotides.

2.2. IrradiationIrradiations were carried out with 6 Co as a radiation source. The dose-rate

was approximately 80 rads/sec. During irradiation N2, 02 or N20 were ledthrough the solution.

2.3. Fluorescence measurementsFluorescence measurements were carried out with a Baird Atomic fluorescence

spectrophotometer, Model SF-1. This is equipped with a xenon light source(150 W), two double-grating monochromators and an RCA IP 28 phototube.The signal is recorded on an x-y recorder.

Fluorescence intensities, denoted by x, were obtained by comparing themeasured intensity of the emission maximum with that of the emission maximumof 1 tg/ml. quinine hydrogen sulphate in 01 N H2SO4 excited at the samewavelength. Depending on the excitation- and emission-wavelengths, theinstrumental sensitivity was about 0 1-0-5 per cent of the standard fluorescence.The emission spectra were not corrected for detector sensitivity, nor were anydifferences in shape between the emission spectra of the solutions studied and thestandard taken into account. Such an elaborate quantitative procedure was notconsidered justified, because the nature and amount of the fluorescing radiationproducts is still unknown. The small background fluorescence intensities of theunirradiated control were subtracted.

To be able to determine the shape of the excitation spectra the intensity of theexciting light was measured as a function of wavelength. A cell containing a

EWU

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'8j

C

-.

250 300 350 400 450 500

wavelength (nm)

Figure 1. Measured absorption spectrum (fully drawn) and corrected excitation spectrum(dots) of fluorescein. Concentration 88 x 10

- 7 M in water containing 01 M Na 2CO 3and 0.1 M NaHCO,

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Fluorescence of y-irradiated DNA

concentrated solution (5 x 10-2 M) of rhodamine B in ethyleneglycol absorbingvirtually all photons in the wavelength region of interest was brought into thebeam of exciting light in front of the normal sample position. A mirror wasplaced in the sample holder, reflecting the light emitted forward from therhodamine B solution into the second monochromator, which was adjusted at afixed wavelength. As the intensity of the emitted light is proportional to thenumber of photons in the exciting light, variation of the wavelength of theexciting light provides the shape of the spectrum of the exciting light, which canbe used to correct the measured excitation spectrum. As a control on thisprocedure the absorption spectrum of fluorescein was compared with its cor-rected excitation spectrum. A reasonable agreement was obtained as is shown infigure 1.

3. Results3.1. DNA

Figure 2 shows the emission spectra of solutions of calf thymus DNA afterirradiation with 94 krads under N2 for three different wavelengths of excitinglight. From the dependence of the emission spectrum on the exciting wavelength,it can be concluded that more than one fluorescing product is formed by irradia-tion. If the maximum of the emission spectrum is determined as a function of the

C 1.0-

..U(Aa) 0.8-0

0.6-ToU,0'232E 0.4

0.2-

350o 400 450 5o0 550owavelength (nm)

Figure2. Emission spectra of irradiated calf-thymus DNA excited at different wavelengths.Concentration: 34 /g/ml. in 001 M phosphate buffer (pH 71). Dose: 94 krads(N2 ). The units along the ordinate are different for the three curves.

wavelength Al, of the exciting light, the picture in figure 3 is obtained. ForAl> 380 nm the emission maximum is at 450 nm. If A is lowered the emissionmaximum shifts to lower wavelengths. For Al between 310 and 290 nm thewavelength of the emission maximum is constant at a value of 395 nm.

These measurements suggest that two fluorescing components ((A) and (B))are present with emission maxima at 395 nm and 450 nm, respectively. If A, islowered further, however, the fluorescent light appears to shift back to higherwavelengths. This could mean that at these lower wavelengths absorption oflight by (B) again dominates over that by (A).

To examine this further the uncorrected excitation spectrum was determinedat a number of different wavelengths As of the second monochromator. Figure 4

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J. P. Goedbloed and Y. J. van Hemmen

shows that the excitation maximum is consistently found at about 365 nm(presumably the excitation maximum of component (B)) if A2> 420 nm. Forcomponent (A) the results are somewhat less clear, but figure 4 suggests that theexcitation maximum of (A) is at about 325 nm. It seems that, although thespectra of the two compounds overlap, an approximate idea of the excitationspectra can be obtained by taking A2=450 nm for (B) and A =400 nm for (A).

ECE

'0.

10

0I* I0

* I

l

l

* 1

l

400 420 440 460emission maximum (nm)

Figure 3. Emission maxima of irradiated calf-thymus DNA as a function of Al. Concentra-tion: 34 /xg/ml. in 001 M phosphate buffer (pH 71). Dose: 94 krads (N2).

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2)

E 360-S 3

E 340 -E

£ 320-

0- ~ ~ e-* * 0

0 e

-- - -- -- A

0

0

36 3;0 400 4o20 40 460

A2 (n m)

Figure 4. Uncorrected excitation maximum of irradiated calf-thumus DNA as a functionof A2. Concentration: 34 pg/ml. in 001 M phosphate buffer (pH 71). Dose: 94krads (N2).

Using these wavelengths for the second monochromator setting, excitationspectra were taken and corrected by means of the rhodamine B method. Theresults are shown in figure 5 for (A) and figure 6 for (B) (together with measure-ments to be discussed later). They lead to improved values for the wavelengthsof the excitation maxima of 310 nm for (A) and show that two peaks exist in theexcitation spectrum of (B) at 280 and 350 nm. The peak at 280 nm agrees withthe tentative explanation given for figure 3.

From figure 4 it might be concluded that the excitation spectra of (A) and(B) should be better separated when taking A2> 450 nm for (B) and A2 < 400 nm

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Fluorescence of y-irradiated DNA 355

for (A). However, it turns out that the corrected excitation spectra are approxi-mately invariant when A2 is taken in the range 440-500 nm for (B) and in therange 360-400 nm for (A). The shift of the uncorrected excitation maximum of(A) when A2 is changed from 400 to 360 nm is the result of a perturbing con-tribution of (B), which, however, is strongly exaggerated in figure 4 because theintensity of the light source is much larger at 400 nm than at 360 nm. Since, onthe other hand, the detector sensitivity drops very rapidly above 450 nm andbelow 400 nm, the values 400 nm and 450 nm for A2 were chosen as most suitablefor the detection of (A) and (B).

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0

'8

.E

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wavelength (nm)

Figure 5. Corrected excitation spectra of component (A) (A2= 400 nm) in DNA, dGMPand dAMP. Concentration in nucleotides: 10

- 4 M in 001 M phosphate buffer(pH 71). Dose: 73 krads (N2 ).

1.4 -

C 1.2 u

I,0D 1.0

0.8

c

>.0.6 -MI

-, 0.4

0.2

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2s50 300 3so 400

wavelength (n m)

Figure6. Corrected excitation spectra of component (B) (A2 = 450 nm) in DNA, dGMP anddAMP. Concentration in nucleotides: 10- 4 M in 001 M phosphate buffer (pH 7'1).Dose: 73 krads (N2).

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Dose-effect curves of the two components in DNA are shown in figure 7.Along the ordinate is plotted the intensity relative to quinine (the parameter x,see § 2). To resolve the contributions of (A) and (B) as completely as possible, thefluorescence intensity was measured at the emission maxima (400 nm for (A) and450 nm for (B)), whereas the wavelength of the exciting light was chosen nearthe corrected excitation maxima (300 nm for (A) and 360 nm for (B)). Thedifference between duplicate measurements was found to be less than 10 percent, even when different DNA batches were used.

XJ ..

0.3-

0.2-

0.1-

\A (N2 )

/

,X/ *_"~O (N2 )

/' A0·~-* 48(02)

20 40 60 80

dose (krad)

Figure 7. Dose-effect curves (N2 and 02) for fluorescence of calf-thymus DNA. Concen-tration: 30 g/ml. in 001 M phosphate buffer (pH 7-1). (A) A1=300 nm,A2 = 400 nm; (B) A1 = 360 nm, A2 = 450 nm. x = max/max quinine (see § 2).

All these measurements were carried out with DNA irradiated under N2 . Ifoxygen was bubbled through the solution during irradiation, the intensity offluorescence was roughly a factor of 30 weaker than after irradiation under N2, butsignificantly higher than the background (figure 7). Flushing with oxygen afterirradiation in nitrogen did not diminish the fluorescence.

3.2. Bases and nucleotides

The results of a detailed investigation into the question which nucleotidesare responsible for the fluorescence of DNA after irradiation are shown intable 1. The table gives the x values for exciting light of 300 and 360 nm. Theconclusion can be drawn that only in irradiated purines both products (A) and(B) are simultaneously found (as in DNA), so (A) and (B) can arise from one base.The pyrimidines, especially cytosine, also fluoresce, however, the spectralcharacteristics of the fluorescence are different from those of DNA.

The corrected excitation spectra of (A) and (B) in dGMP and dAMP are alsogiven in figures 5 and 6. Both components of dAMP and of dGMP show someresemblance to the corresponding components of DNA. Although component(B) of dGMP shows two maxima at about 280 and 360 nm just as DNA, whereasdAMP does not show two well-separated peaks, the ratios of the maxima ofdGMP and DNA differ considerably. The pure base guanine resembles DNAmuch more in this respect (figure 8); the spectrum of component (B) of adenineon the other hand shows only one peak. Especially the observations in figure 8might be an indication that guanine is the base which makes the greatest con-tribution to the fluorescence of irradiated DNA.

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Fluorescence of y-irradiated DNA 357

A1= 300 nm A1 = 360 nmCompound

Amax X Amax x

Adenine 410 038 445 009dAMP 405 015 450 017

Guanine 380 0-31 435 0-12dGMP 395 034 455 034GMP 400 1 28 445 0-37Guanosine 390 0-68 445 034Deoxy-guanosine 380 0-65 455 0-28

Cytosine 400 017 415 006dCMP 400 009 415 0-19

Thymine 405 023 - -TMP - .

2-deoxy-D-ribose - - - -

DNA 400 0-22 450 009Nucleotide mixture 400 035 435 033

Table 1. Emission maxima and x values of DNA, bases and nucleotides after irradiation.

Dose: 50 krads (N2). Concentration: 10- 4 M in 001 M phosphate buffer (pH 71),

except guanine: 2 x 10-5 M. Concentration of calf thymus DNA: 31 Lg/ml. The nucleo-

tides in the mixture were present in a concentration corresponding to 31 kg/ml. of calfthymus DNA. A dash means that no significant fluorescence was observed. x = max/maxquinine (see § 2), A1 is the excitation wavelength.

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06-

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0.4 2-

0.2 -

0250 300 350 400

wavelength (nm)

Figure8. Corrected excitation spectra of (A) (A2 = 400 nm) and (B) (A = 450 nm) in DNA,adenine and guanine. DNA: 34 tg/ml., adenine: 10- 4 M, guanine: 2 x 10 - 5 Min 001 M phosphate buffer. Dose: 73 krads (N2).

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358 J. P. Goedbloed and 7. J. van Hemmen

3.3. dGMP and GMP

Figure 9 shows the emission spectra of the components (A) and (B) in irradia-ted dGMP. In the emission spectrum of component (A) a disturbing contribu-tion of component (B) is present, which cannot be eliminated, because therelative concentrations of (A) and (B) are not known. Obviously the spectrum

0C 1.2U .

~! 1.0

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0.

:C 0.5

2 0.4

-

0.2

0

wavelength (n m)

Figure 9. Emission spectra of irradiated dGMP. Concentration: 10- 4 M in 0.01 Mphosphate buffer (pH 7-1). Dose: 80 krads (N2). (A) A1=300 nm, (B) A)=360 nm.The spectrum of (A) was recorded at a greater instrumental sensitivity than that of(B).

I

B0.8-

0.6 0

0

oO 0.4-

0.2 0/Y02 /

/ /0

I -,20 40 60 80 lo 120

dose (krad)

Figure 10. Dose-effect curves (N2) fluorescence of dGMP. Concentration: 3 x 10-4 Min 001 M phosphate buffer (pH 71). (A) A1 = 300 nm, A = 400 nm; (B) Al = 360 nm,A 450 nm. x=max/max quinine (see § 2).

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Fluorescence of y-irradiated DNA

of (B) contains no contribution from (A), so it is evident that in this case (B)predominates over (A) (the scale on the vertical axis in figure 9 is different forthe two curves).

The dose-effect curves of dGMP (figure 10) show the same pattern as thoseof DNA. The slope of the curve of (A) strongly decreases at higher doses,whereas for component (B) the curve becomes steeper. This effect would beeven more pronounced if the curve for (A) could be corrected for the con-tribution of (B), since at higher doses this contribution is relatively larger, as canbe seen from the dose-effect curve of (B).

To determine which radicals are responsible for the formation of the fluores-cing product, 10 - 4 M GMP was irradiated under various gases in the presenceand absence of 10 -

3 M KI, which removes OH radicals. Addition of iodide afterirradiation did not influence the fluorescence. The results are shown in table 2.

Gas I- concentration (M) (A) (B)

N2 0 28 5610-3 2 20

N2 0 0 60 8810-3 2 31

Air 0 5 3310 - 1 1

02 0 0 410-a 1 1

N2 10-3 added after 28 56irradiation

Table 2. Effects of iodide and of various gases on the formation of component (A) and (B)in GMP by irradiation.

Concentration of GMP: 10- 4 M in 001 M phosphate buffer (pH 7-1). Dose: 20 kradsIntensities are given for (A) and for (B). The ratio of the figures for (A) and (B) has nomeaning.

4. DiscussionThe experiments show that after irradiation of DNA in neutral aqueous

solution, fluorescing products are present. The spectra can be resolved into twocomponents (A) and (B). (A) has its (corrected) excitation maximum at 310 nmand its (uncorrected) emission maximum at 400 nm. (B) absorbs at 280 and360 nm and emits at 450 nm.

The shape of the spectra, especially the corrected excitation spectra (figures5, 6 and 8), the similarity of the dose-effect curves of DNA and dGMP (figures7 and 10) and the data in table 1 all suggest that the most important contributionto the fluorescence of irradiated DNA comes from guanine. This base showsboth components after irradiation.

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It has been reported (Hems 1960, Ponnamperuma, Lemmon and Calvin1963, Conlay 1963) that after high doses (thousands of krads) two kinds ofproducts are found in irradiated solutions of purines: products in which thepurine structure is conserved (e.g. 8-hydroxyadenine) and products in which theimidazole ring is destroyed (e.g. 4,6-diamino-5-formamido pyrimidine).Because in the present experiments component (A) is already found at relativelylow doses (2 krads) and because the dose-effect curve of (A) is straight in thisrange (figure 10), it seems probable that (A) is a primary product, in which thering structure of the purines is conserved. The fluorescence of (A) would thenbe the result of electronic transitions in a purine base in which the irradiation hasintroduced small modifications. This means that we have to do with well-known n7r* and irrT* transitions.

In the literature, fluorescence with the same spectral characteristics ascomponent (A) has been described for some purines. B6rresen (1963 a) andUdenfriend and Zaltman (1962), for example, observed fluorescence of GMP inacid solution with an emission maximum at 380 nm. The reason that GMPfluoresces in acid media and not at neutral pH seems to be that a proton is addedto N7 of guanine in acid solution. As a result the lowest excited state nowcorresponds to a rrr* transition in neutral guanine. After the alkylation of N7 ofguanine this fluorescence is also observed (B6rresen 1963b). Another substancewith the same fluorescence characteristics as component (A) is 2-amino purine.For this compound in aqueous glycol solution at pH 7 a quantum yield of 0.7 hasbeen reported (Drobnik and Augenstein 1966), an extremely high value forpurines. The wavelength of the absorption maximum is 315 nm, and the emis-sion maximum is 380 nm. 2-amino purine derives from guanine by the subtrac-tion of the hydroxyl group at C6. These data support the hypothesis that thefluorescence of component (A) originates from a relatively minor modification ofthe purine bases by irradiation. Possibly component (A) is a guanine base whichis modified at C8 of N7 by the attack of a free radical.

Component (B) has its excitation maximum at a wavelength at which purinesdo not absorb (360 nm) and an emission maximum at a wavelength at which nofluorescence has been observed for purines in dilute solution. Phosphorescenceis very unlikely at room temperature and can therefore be excluded. However,Basu and Greist (1963) and Basu and Loh (1963) found a new absorption bandat 360 nm and an associated emission maximum at 450 nm in concentratedsolutions (5 x 10-2 M) of nucleosides. They ascribed the appearance of this newabsorption band to the formation of charge-transfer complexes. This suggeststhat component (B) could be a complex arising from two neighbouring nucleo-tides by radical attack.

The question arises, which radicals are responsible for the formation of thecomponents (A) and (B). From table 2 it follows that OH radicals are requiredfor the formation of (A) and that the reducing radicals H and eaq- do not con-tribute to this process. Iodide ions which react rapidly with OH radicals cansuppress the formation of component (A) almost completely, and conversion ofeaq- into OH by N2O(eaq- + N2 0 + H20 ->N2 + OH- + OH) doubles the yield of(A). For component (B) the situation is less simple. The presence of iodideduring irradiation influences the formation of (B), but not to such an extent as toprevent it completely. Oxygen suppresses the production of (B) nearly completely.Although this might be a result of the scavenging of both eaq- and H, it cannot be

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excluded that fluorescence is prevented by oxidation of base-radicals. Conver-sion of eaq- into OH by N2 0 increases the amount of (B), but does not double theyield. Therefore OH is not the only radical involved in the formation of com-ponent (B).

The dose-effect curves of DNA and dGMP (figure 7 and 10) show greatsimilarity. Both curves of component (A) are initially straight, but exhibit adecreasing slope at higher dose. This can be explained by increasing competitionof radiation products for free radicals. The fluorescence of component (B) ofdGMP is neither a linear nor a quadratic function of dose, but can be fairly wellrepresented by a sum of a linear and a quadratic term. The non-linear formationof (B) suggests that during irradiation a product is built up, which gives (B)after further reaction with radicals, whereas (B) is also formed directly. Possiblyone of these processes is inhibited by iodide. A complete explanation of the dose-effect curves must await determination of the nature of the components (A) and(B) and elucidation of the reaction scheme. Preliminary measurements of theamounts of products (A) and (B) after separation by gel-filtration of the irradia-tion products indicated that the G values lie somewhere between 0001 and 0.01.These small yields give rise to a negligible optical density. This is in agreementwith the results of Keck and Hagen (1966) which show a significant fluorescenceat doses where no decrease in optical density is detectable. Further work is inprogress.

ACKNOWLEDGMENT

The authors are indebted to Professor Dr. Joh. Blok for suggesting thesubject and for discussion on the results of the experiments.

Dans les solutions de l'ADN, irradiees par des rayons y, on observe une fluorescencequi n'est pas presente avant 'irradiation. Des spectres d'excitation et d'6mission, onconclut que deux composantes fluorescentes sont presentes.

Une de ces composantes (A) a un maximum d'excitation 310 nm et un maximumd'emission 400 nm, tandis que l'autre (B) a des maximums d'excitation 280 nm et360nm et un maximum d'emission 450 nm. On observe galement deux composantesfluorescentes dans une solution irradiee de guanine, avec peu pres les memes caracteris-tiques spectrales que 'ADN. La composante (A) rsulte de l'attaque du radical OH,tandis que pour la formation de (B) les radicaux H et OH sont impliques.

In y-bestrahlten DNS-L6sungen wird eine characteristische Fluoreszenz beobachtetdie in unbestrahlten L6sungen abwesend ist. Auf Grund der Anregungsspektren undFluoreszenzspektren wird geschlossen da3 zwei fluoreszierende Verbindungen vorliegen.Verbindung (A) hat ein Anregungsmaximum bei 310 nm und ein Fluoreszenzmaximumbei 400 nm, wiihrend (B) ein Anregungsmaximum bei 280 und 360 nm und ein Fluoreszenz-maximum bei 450 nm zeigt. Beide Verbindungen werden auch in bestrahlten Guanin-16sungen mit ihnlichen Spektrenschaften wie in bestrahlten DNS-L6sungen beobachtet.Die Verbindung (A) entsteht nach Reaktion mit OH Radikalen, wiihrend (B) von OHund H Radikalen gebildet wird.

REFERENCES

BAsu, S., and GREIST, J. H., 1963, J. Chim. phys., 60, 407.BAsu, S., and LOH, L., 1963, Biochim. biophys. Acta, 76, 131.B6RRESEN, H. C., 1963 a, Acta chem. scand., 17, 921; 1963 b, Ibid., 17, 2359.CONLAY, J. J., 1963, Nature, Lond., 197, 555.DROBNIK, J., and AUGENSTEIN, L., 1966, Photochem. Photobiol., 5, 83.GOEDBLOED, J. P., 1967, Int. J. Radiat. Biol., 12, 383.HEMS, G., 1960, Radiat. Res., 13, 777.KECK, K., and HAGEN, U., 1966, Biophysikalische Probleme der Strahlenwirkung, edited by

H. MUTH (Stuttgart: Georg Thieme Verlag), p. 112.PONNAMPERUMA, C., LEMMON, R. M., and CALVIN, M., 1963, Radiat. Res., 18, 540.UDENFRIEND, S., and ZALTMAN, P., 1962, Analyt. Biochem., 3, 49.

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