INT. J. RADIAT. BIOL., VOL. 14, NO. 4, 363-371

X- and y-irradiation of dilute solutions of chymotrypsin:the active intermediate

K. R. LYNNT and GAIL ORPENt

Division of Biology and Health Physics, Chalk River Nuclear Laboratories,Chalk River, Ontario, Canada

(Received 11 March 1968; revision received 17 June 1968)

X- and y-irradiation of dilute aqueous solutions of chymotrypsin in 10-3 MHC1 or in water produce difference spectra, over the range 210-330 m/k, qualita-tively similar to that obtained on reaction of the enzyme with hydroxyl radicalsfrom Fenton's reagent.

The protection of the esterolytic properties of chymotrypsin against ir-radiation has been measured using BTEE as the substrate and sodium formate,acetone, glucose, ethanol and iso-propanol as protectors. The results obtained,when combined with absolute rate constants available for reactions of the hy-droxyl radical, show that radical to be the predominant reactive species in theirradiation of dilute aqueous solutions of the enzyme.

1. IntroductionSome effects of x- and y-irradiation on the enzymatic (esterolytic and pro-

tease) reactions of dilute solutions of chymotrypsin (E.C. 3.4.4.5) have beenreported by Butler, Robins and Rotblat (1960), Mee (1964), Mounter (1960) andMoore and McDonald (1955), However, the nature of the active species respon-sible for the effects of the radiation has not been established. Evidence fromdifference spectrophotometry, and from studies of the effects of several protectivereagents, is presented here and suggests that the hydroxyl radical is a pre-dominant reactive intermediate in the irradiation of dilute aqueous solutions ofchymotrypsin.

2. Experimental2.1. Reagents

The chymotrypsin used throughout this work was the crystallized (3 x)protein supplied by Worthington Biochemical Corporation. It was commonlyused as received, after storage in a dry, cold atmosphere. The enzyme purified(Yapel, Han, Lumry, Rosenberg and Da Fong Shiao 1966) by gel-filtration onSephadex G-25 gave results identical with those from the commercially-available protein. The concentrations of solutions of the enzyme were checkedspectrophotometrically (Butler et al. 1960).

Benzoyl tyrosine ethyl ester (BTEE: supplied by Calbiochem) was the sub-strate (Hummel 1959) employed in the studies with protective reagents.

t Issued as NRCC No. 10411.t Attached staff from Division of Radiation Biology, National Research Council of

Canada, Ottawa, Ontario, Canada, to which address requests for reprints should be sent.

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Activity measurements of 5 x 10-8 M chymotrypsin were made with N-carbobenzoxy-L-tyrosine p-nitrophenyl ester (CTN) as substrate (Martin,Golubow and Exelrod 1959). It was used as supplied by Mann Laboratories.

The solvents, buffers and protective reagents were of analytical grade unlessotherwise noted.

3. MethodsA Phillips 300 kV x-ray machine operating at 10 mA, calibrated with a

Landswerk capsule dosimeter, or a 60Co y-irradiation unit (AECL Gammabeam150: 600 Curies) calibrated electrometrically, were used for irradiation.

The solutions of the enzyme, in Pyrex containers, were freely exposed tothe atmosphere during irradiation: mechanical stirring during the process waswithout effect on the results obtained when compared with data from unstirredsolutions. Chymotrypsin was dissolved in 10-3 M HC1 or glass-distilled, de-ionized water (pH circa 5): the results obtained were not significantly different,though more extensive changes in pH affect the irradiation of the enzyme(Moore 1955).

Difference spectra were measured on a Beckman DB spectrophotometerconnected to a pen-recorder. The instrument was equipped with a scaleexpander allowing up to tenfold amplification of the spectra. The wavelengthcalibration of the spectrophotometer was checked against the spectrum ofpotassium dichromate (Haupt 1952). Cells of 1 cm light-path were usuallyemployed, though cells with lightpath of 4 cm were also available. Whennecessary, because of the complexity of the mixture being examined (for examplewhen studying the effects of Fenton's reagent), a tandem arrangement of cellswas made, that is, two 1 cm cells were used in both the ' reference' and ' experi-mental ' light paths. Spectra were measured over the range from 210 to 380 mpt.

Fenton's reagent was prepared as described by Barb, Baxendale, George andHargrave (1951). Reaction with the chymotrypsin for 30 min was commonlyemployed: further exposure of the enzyme to the reagent had no measurableeffects on the difference spectra.

Gel-filtration was on Sephadex G-25 (Pharmacia Inc.) equilibrated withHCl (10-3 M). Columns (1.9 cm I.D. x 40 cm) of Pyrex glass were packed fordownward flow and fractions were separated on drop-counting collectors. Theprotein content was analysed using the conventional Folin-Lowry procedure.After irradiation, solutions of chymotrypsin (5 x 10- 6 M) were freeze-dried andthen the enzyme was applied to the molecular sieve in a concentrated solution.

The method of measuring the protective effects of acetone, glucose, sodiumformate, ethanol and iso-propanol, with BTEE as substrate, was that describedby Sanner and Pihl (1967). Initial rates of esterolysis of BTEE were measuredspectrophotometrically (Hummel 1959).

4. Results and discussionChymotrypsin solutions ranging in concentration from 5 x 10- 8 M to

10- 3 M were irradiated (x-rays) and residual esterolytic activity measured usingBTEE as the substrate for the more concentrated solutions and CTN for themost dilute. Exponential loss of activity was found at each of the concentrationsof enzyme investigated in that range as has been reported by other authors

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(Butler et al. 1960, Mee 1964). The relationship between values of D37 estimatedin this work, and enzyme concentration, was comparable with data reported byButler et al. (1960) in which acetyl-tyrosine ethyl ester (ATEE) was the sub-strate. Also, in agreement with those data, on irradiation of chymotrypsin at5 x 10-6 M the D37 for esterolysis of BTEE was unaffected by the dose-rate overthe range 6 x 102 to 5 x 103 R/min. It is apparent, from the results describedabove, that the behaviour of the chymotrypsin employed in these experiments iscomparable with that found by other investigators.

Difference spectra observed in the range from 210 to 380 mF, after x- ory-irradiation of chymotrypsin, or after treatment of the enzyme with variouschemical reagents, are attributable predominantly to effects on the absorptivitiesof tryptophan, tyrosine and phenylalanine. Aggregation is known to occur after

10

0

>1

o

- 10is

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(b)

(a)

380 300m A

Figure 1. Difference spectra measured for aqueous 5 x 10-6 M chymotrypsin: (a) aftery-irradiation with 37-8 kR, at 2 x amplification; (b) mixed with 10- 2 M FeSO4and 0-3 M H1O2, measurement after 30 min at 20°c, at 5 x amplification.

irradiation of proteins (Rosen 1959). Light-scattering caused by such aggregationis not a significant factor in the production of the difference spectra discussedhere, for no direct relationship (Stacey 1956) between A- 4 and the extents of themajor spectral changes could be detected. Furthermore, gel-filtration on Sepha-dex G-25, with HC1 of pH 3 as the eluting solvent, showed that even after atotal dose of 50 kR less than 3 per cent of a dilute (5 x 10- 6 M) solution ofchymotrypsin was converted into a protein of higher molecular weight (estima-tion by the Folin-Lowry procedure).

When solutions (5 x 10- 6 M) of chymotrypsin in HCl of pH 3 (or in water)were irradiated marked changes in the difference spectra were observed. Measur-able increases in absorption were found at 305 and 243 m/ and decreases at 292and 283 m/, as is seen in figure 1 (a). When irradiation was continued, these

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ac20

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00

0

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Figure 2. Spectral shifts as a function of dose for x-irradiation at 600 R/min. A, increasedabsorption at 305 mx; 0, change in absorption at 305 my plus that at 282 m; E,change in absorption at 305 m/x plus that at 293 m/t.

u

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Figure 3. Interpolated values from data of figure 2 plotted semilogarithmically in per-centage of total distortion, against dose, lines (a), (b). Line (c), fractional residualactivity after x-irradiation at 600 kR/min of 5 x 10-6 M chymotrypsin in HC ofpH 3.

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X- and y-irradiation of dilute solutions of chymotrypsin

differences attained essentially constant values, as the data of figure 2 show.Work by Barron, Ambrose and Johnson (1955) suggests that spectral changesobserved after irradiation of 10- 4 M chymotrypsinogen with moderate doses(50-75 kR) of x-rays derive largely from the effects of irradiation on tryptophan(see, also, Swallow and Velandia 1962). However, we have no evidence to showthat the effects of the lower doses which inactivate 5 x 10-6 M chymotrypsin(see below) are similarly derived. It was, then, assumed only that at the totaldoses at which the plateaux of figure 2 are achieved a completely ' distorted 'molecule exists; that is, one in which changes of structure (primary, secondary,etc.) have occurred to a degree that further spectral shifts are not apparent oncontinued irradiation. Data such as those of figure 2 may then be calculated interms of the extent of distortion per unit dose. Semi-logarithmic plots of suchdata are represented by figure 3 from which evaluations of D3 7 may be made foreach of the wave lengths examined.

Comparisonof data fromeven the fastest changing difference spectralmaximumin irradiated chymotrypsin of 5 x 10-6 M with that for loss of esterolytic activity,showed that the D37 for distortion (25 kR at 243 m/x) was appreciably greater thanthat for loss of activity, namely, 15 kR (figure 3). Accurately measurable differencespectra could not be observed with chymotrypsin (5 x 10-6 M) at the latter totaldose even using the maximum sensitivity available: tenfold amplification of thespectra and cells of 4 cm light-path. Clearly the changes at the esterolytic site ofthe enzyme caused by irradiation occur at lower doses than those in the environ-ments of the amino acids responsible for the observed difference spectra.

The data reported above were obtained from 5 x 10 - 6 M solutions of chymo-trypsin: comparable results were found when the enzyme was irradiated in5 x 10- 5 M solutions in the same solvents. However, significant changes in thedifference spectra were observed when chymotrypsin, at a concentration of10-3 M in HCI of pH 3, was irradiated with x-rays: examination of those resultsis proceeding. Effects from varying the dose rates for irradiation of chymotrypsinwere observed in the difference spectra discussed above, but were not extensive.They were not examined in detail as no effects from changes of dose rate werefound in the examination of the loss of enzymatic activity following irradiation(above and Butler et al. 1960).

Difference spectra of chymotrypsin subjected to a variety of chemical re-agents were prepared, for comparison with those obtained on irradiation of theenzyme, using the tandem-cell arrangement. Reagents such as glycerol (40 percent solution), mercaptoethanol (0.5 M) and sodium chloride (5 M) each inducedunique difference spectra. These were qualitatively distinct from those observedafter x- or y-irradiation of the enzyme to which, however, some similarities werefound when the chymotrypsin was subjected to HCI of pH 1, or to 8 M solutionsof urea. These two reagents would affect the hydrogen bonding in chymotrypsin.It is thus possible that the difference spectra observed on irradiation result, inpart, from changes in the secondary and tertiary structure of the enzyme.

Of greater relevance to our present interest, however, are the differencespectra when chymotrypsin was subjected to Fenton's reagent (Barb et al. 1951).The precise nature of the radical generated by that reagent is disputed and ispossibly dependent on the reaction mixture in which it is prepared (Shiga 1965).As there is no data directly relevant to the reaction system employed in this workwe have accepted the evidence of Dixon and Norman (1963) and Smith, Dixon

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and Norman (1963) that Fenton's reagent yields OH- and shall discuss ourresults in terms of that radical. The spectra obtained after irradiation of ehymo-trypsin and after treatment with Fenton's reagent are notably similar, as isshown in comparison of figure 1 (b) with (a): increased absorptivity again occurredat 305 m/ and 243 m/, increased transmittance at 292 and 283 m/. While it isclear that difference spectra of chymotrypsin subjected to x- and y-irradiation arequalitatively similar to that of the enzyme reacted with radicals produced byFenton's reagent, accurate quantitative comparison was not possible because ofside reactions. These were demonstrated with measurements of both differencespectra and loss of activity to BTEE following exposure of chymotrypsin toFenton's reagent. Both showed a marked dependence on the concentrations ofhydrogen peroxide and ferrous sulphate employed, even though large theoretical(Barb et al. 1951, Hart, Thomas and Gordon 1964) excesses of OH- were gene-rated. It is known (Hart et al. 1964) that peroxide and ferrous ion are scavengersfor hydroxyl radicals, and so would be in competition with the chymotrypsin.Generation of hydroxyl radicals by ultra-violet irradiation of peroxide (Hochana-del 1961) could not be employed as an alternative procedure because the u.v.light alone caused significant destruction of the esterolytic activity of the enzyme.It is of relevance that in a given series of concentrations of Fenton's reagent, bothpercentage distortion and loss of activity of treated chymotrypsin followedexponential laws when plotted against the concentration of Fe 2+ (i.e. [HO-], cf.Barb et al. 1951) used. As with the x- and y-irradiated solutions, complete loss ofenzymatic activity occurred at lower concentrations of hydroxyl radicals than didtotal distortion.

Recently, Sanner and Pihl (1967) have suggested a method for directlydetermining the nature of the species reactive with the solutes of irradiatedsolutions, using absolute rate constants now available (Ebert, Keene, Swallowand Baxendale 1965) for such ephemera as OH and eaqu.. The procedure reported(Sanner and Pihl 1967) which was that employed in the work described here, isvalid only if the protective agents act exclusively as radical scavengers. That thiswas so was shown when experimentally determined values of D37, obtained atincreasing concentrations of protector, were plotted against those concentrationsto yield a straight line (Sanner and Pihl 1967).

The reaction between enzyme (E) and reactive radical (X) is described by:XkxE + X>Einact. (1)

Assuming that the radical will react with E and with Einact. at the same rate,Sanner and Pihl (1967, and references cited therein) showed that:

G(x) D37, = (ke /ki)Eo + (kp /kiPo + (kb /ki)BO, (2)twhere E0, P and B0 are initial concentrations of enzyme, protector and buffer(or impurity) and ke, kp and kb are the rate constants for reactions of X with E, Pand B. D37 and G(x) take their usual definitions. While k i is the rate constant forinactivation of the enzyme by radical X, ke is that for reaction of the radical withboth the inactive and active enzyme.

From which it follows that:G(x)(D3 7p - D3 7) = (1 /ki)kp Po) (3)t

t G(x) does not appear in the equations published by Sanner and Pihl (1967), though itspresence is implicit in calculations made by those authors.

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where D37 is the value of D37 obtained when irradiation is performed in thepresence of the protector.

When a plot of D3 7 versus the concentration of chymotrypsin was made, astraight line was obtained over the range 5 x 10- 5 M to 10- 7 M (see above andButler et al. 1960), as required by equation (2). When (D3 7 p-D37) was plotted asa function of kp Po, figure 4 was obtained, assuming X = OH .

Some scatter of points about the line of best fit is apparent in figure 4, butmay be attributed to uncertainties in values of kp used (compare, for example,Adams, Boag, Currant and Michael 1965, Scholes, Shaw, Willson and Ebert1965, also Thomas 1965). The values of kp employed were selected so that theywere as self-consistent as possible.

5o

r0sI

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Kp Po (x 10 4 sec- 1)

Figure 4. Differences in Ds7 as a measure of protection of 5 x 10- 6 M chymotrypsin inHCI of pH 3. Protective reagents present at 5 x 10- 4 M. Symbols, and the sourcesof values of kp used, are: A, acetone (Adams et al. 1965); E, ethanol (Thomas1965); F, sodium formate (Hart et al. 1964); G, glucose (Davies, Griffiths andPhillips 1965); P, iso-propanol (Scholes et al. 1965).

As all solutions used in this work were aerated, hydrated electrons would berapidly scavenged and so cannot be the intermediate which attacks the chymo-trypsin. The presence of oxygen in the irradiated solutions necessitates examina-tion of 02- as a reactive species: at pH 3 the equilibrium between O 2- and H2 0-would favour the latter (Czapski and Dorfman 1964) though both would bepresent. Values of neither kp(O-) nor kp(HO 2 .) for the protective reagents usedin this work are currently available and so the species cannot be definitelyeliminated as intermediates. However, it seems unlikely that values of kP (0 2-)or kp(HO2-) for each of the five chemically diverse protective reagents employedshould be such that a line comparable with that of figure 4 would be obtained inan analogous plot. The data from the effects of protectors, then, in conjunctionwith those from difference spectral measurements, strongly suggests that thereactive species in the irradiation of dilute solutions of chymotrypsin is OH-.

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From the slope of the line in figure 4, ki may be evaluated: when G(OH.) = 2-6(Matheson and Dorfman 1965), k i =4-2 x 109 M- 1 sec-L. Apparently the reactionof hydroxyl radicals with chymotrypsin proceeds more rapidly than reaction withtrypsin (Sanner and Pihl 1967). From the slope of a plot of D3 7 against chymotryp-sin concentration ke was estimated as 3 x 1010 M - 1 sec.-L.

L'irradiation aux rayons x ou gamma des solutions dilu6es ue ecnymotrypsine dans l'eaupure ou dans l'acide chlorhydrique au millime de mole, produit des effets bathochromeset hypsochromes, dans la gamme 210-330 m/z, semblables qualitativement ceux qu'onobserve lors de la reaction de l'enzyme avec les radicaux oxhydriles du ractif de Fenton.

L'auteur a mesur6 le degr6 de protection des propri6t6s esterolytiques de la chymo-trypsine, l'aide de BTEE comme support, les corps protecteurs 6tant le formate de sodium,l'ac6tone, le glucose, 1'thanol et l'iso-propanol. Les rsultats obtenus, compares auxconstantes absolues des vitesses de raction du radical oxyhydrile, indiquent que ce radicalconstitue le facteur le plus actif lors de l'irradiation de la solution aqueuse dilute de l'enzyme.

R6ntgen-und Gammabestrahlung verdiinnter wasseriger Ldsungen von Chymotrypsinin 10-3 M HCI oder in Wasser verursachen spektrale Unterschiede im Bereich von 210-330 mA, die qualitativ denjenigen ahnlich sind, welche bei der Reaktion des Enzyms mitHydroxylresten aus Fentons Reagens erhalten werden.

Der Schutz der esterolytischen Eigenschaften des Chymotrypsins gegen Bestrahlungwurde gemessen, wobei BTAE als Substrat und Natriumformat, Aceton, Glukose, Aethanolund Isopropanol als Schutz benutzt wurden. Verglichen mit den zur Verfuigung stehendenabsoluten Konstanten fr Reaktionen des Hydroxylrestes zeigen die erhaltenen Resultate,dal3 dieser Rest die iberwiegende reagierende Art bei der Bestrahlung von verdiinntenwasserigen L6sungen des Enzyms ist.

REFERENCES

ADAMS, G. E., BOAG, J. W., CURRANT, J., and MICHAEL, B. D., 1965, Pulse Radiolysis,edited by M. Ebert, J. P. Keene, A. J. Swallow and J. H. Baxendale (New York:Academic Press), p. 131.

BARB, W. G., BAXENDALE, J. H., GEORGE, P., and HARGRAVE, K. R., 1951, Trans. FaradaySoc., 47, 462.

BARRON, E. S. G., AMBROSE, J., and JOHNSON, P., 1955, Radiat. Res., 2, 145.BUTLER, J. A. V., ROBINS, A. B., and ROTBLAT, J., 1960, Proc. R. Soc. A, 256, 1.CZAPSKI, G., and DOREMAN, W. T., 1964, J. phys. Chem., Ithaca, 68, 1169.DAVIES, J., GRIFFITHS, W., and PHILLIPS, G. O., 1965, Pulse Radiolysis, edited by M.

Ebert, J. P. Keene, A. J. Swallow and J. H. Baxendale (New York: Academic Press),p. 181.

DIXON, W. T., and NORMAN, R. O. C., 1963, Proc. chem. Soc., p. 97.EBERT, M., KEENE, J. P., SWALLOW, A. J., and BAXENDALE, J. H., 1965, Pulse Radiolysis

(New York: Academic Press).HART, E. J., THOMAS, J. K., and GORDON, S., 1964, Radiat. Res. Suppl., 4, 74.HAUPT, G. W., 1952, J. Res. natn. Bur. Stand., 48, 298.HOCHANADEL, C. J., 1961, Radiat. Res., 17, 286.HUMMEL, B. C. W., 1959, Can. J. Biochem. Physiol., 37, 37.MARTIN, C. J., GOLUBOW, J., and AXELROD, A. E., 1959, J. biol. Chem., 234, 294.MATHESON, M. S., and DORFMAN, L. M., 1965, Prog. Reaction Kinetics, 3, 237.MEE, L. K., 1964, Radiat. Res., 21, 501.MOORE, E. C., and McDONALD, M. R., 1955, Radiat. Res., 3, 38.MOUNTER, L. A., 1960, Radiat. Res., 12, 187.ROSEN, D., 1959, Biochem. ., 72, 597.SANNER, T., and PIHL, A., 1967, Biochim. biophys. Acta, 146, 298.SCHOLES, G., SHAW, P., WILLSON, R. L., and EBERT, M., 1965, Pulse Radiolysis, edited by

M. Ebert, J. P. Keene, A. J. Swallow and J. H. Baxendale (New York: AcademicPress), . 151.

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SHIGA, T., 1965, J. chem. Phys., 69, 3805.SMITH, L., DIXON, W. T., and NORMAN, R. O. C., 1963, J. chem. Soc., p. 2897.STACEY, K. A., 1956, Light Scattering in Physical Chemistry (London: Butterworth &

Co. Ltd.SWALLOW, A. J., and VELANDIA, J. A., 1962, Nature, Lond., 195, 798.THOMAS, J. K., 1965, Trans. Faraday Soc., 61, 702.YAPEL, A., HAN, M., LMvRY, R., ROSENBERG, A., and SHIAO, DA FONG, 1966, Y. Amer.

chem. Soc., 88, 2573.

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