Photochemistry and Photobiology, 1975. Vol. 22. pp. 163-167.

Pergamon Press

Printed in Great Britain

ULTRAVIOLET INACTIVATION OF PAPAIN* J. F. BAUGHER and L. I. GROSSWEINER Biophysics Laboratory, Physics Department, Illinois Institute of Technology, Chicago, Illinois 60616, U.S.A. (Received 16 April 1975; accepied 25 J d y 1975)

Abstract-Flash photolysis transient spectra (A > 250 nm) of aqueous papain show that the initial products are the neutral tryptophan radical Tip (&, 510 nm), the tryptophan triplet state 3Trp (A,, W n r n ) , the disulfide bridge electron adduct - S S (Amax 420nm) and the hydrated electron ea;. The -$Syield was not altered by nitrous oxide or air, indicating that the formation of this product does not involve electrons in the external medium. The original papain preparation was activated by irradiating under nitrogen. The action spectrum supports previous work attributing the low initial activity to blocking of cysteinyl site 25 with a mixed disulfide. Flash lamp irradiation in nitrogen led to acfivation at low starting activities and inactivation at higher starting activities, while only inactivation at the same quantum yield was observed with air saturation. The results are consistent with photoionization of an essential tryptophyl residue as the key inactivating step.

INTRODUCTION

The flash photolysis technique is being employed to investigate the UV inactivation of enzymes, with the objective of relating the initial photochemical reactions to biological and chemical endpoints. In previous work on hen lysozyme (Grossweiner and Usui, 1971) and bovine trypsin (Kaluskar and Grossweiner, 1974) the following transients were identified at time delays of 5-25 ps after the flash; the neutral tryptophan (trp) radical from photoionization and depro510 nm; tonation of the indole ring N atom [A, Santus and Grossweiner (197211, the disulfide bridge 420 nm; Adams et a/.. (1967)], electron adduct [A, and the hydrated electron [Amax 720 nm; Hart and Boag (1962)l. In RNase A and bovine insulin (Volkert and Grossweiner, 1973) with no trp, the phenoxyl radical from tyrosine was observed with the electron co-product [La,410 nm; Grossweiner et al. (1963)l. The trp radical (Tip) was observed also in human serum albumin and bovine carbonic anhydrase accompanied by the disulfide electron adduct (-Ssl-) in the former and the tyrosine oxidation product (Tjlr) in both cases, and Tjlr was identified also in calf histone and with no tryptophan or sulfur (Kaluskar, 1973). The postulated relationships between the initial processes and inactivation were based on comparative flash photolytic results obtained with the aromatic chromophores, quantum yields and available information about the structures. The photolysis of an essential trp residue was proposed as the key inactivating step in lysozyme, while specific indirect mechanisms were suggested in trypsin and RNase A in which the aromatic residues are not

essential. The present work on papain represents the extension to a proteolytic enzyme with an essential sulfhydryl group. Papain has the unusual property of being activated when irradiated with UV light (Bersin, 1933).The effect is related to blocking of the essential sulfhydryl group in the usual preparation of Kimmel and Smith (1955). Treatment of the starting material with a thiol reagent leads to about 0.3-0.6 moles of free SH and a comparable specific activity relative to highly purified preparations (Sluyterman and Wijdenes, 1970). This process has been identified with the unblocking of the mixed disulfide in the active site by reaction with cysteine Papain-S-S-Cy

+ R-SH -+

Papain-SH

+ Cy-S-S- R (1)

The enzyme fraction which does not become active has been attributed to the presence of irreversible blocking agents such as the sulfinic or sulfonic acids (Glazer and Smith, 1971). Dose and Risi (1972) found that the specific activity after photochemical activation was lower than obtained by the pre-irradiation thiol treatment indicative of simultaneous inactivating photochemical processes; however, the quantum yields for residue destruction did not lead to a specific mechanism. Their results are extended with flash photolysis measurements in the present work and compared with new quantum yield measurements for activation and inactivation. It is concluded that activation involves photolysis of the mixed disulfide and inactivation is promoted by photoionization of an essential trp residue.

* Supported by the US.Department of Health, Education and Welfare on NIH Grant GM 20017 and the U.S. Energy Research and Development Administration on Contract No. AT(l1-1)-2217.This is COO-2217-13. I63

MATERIALS AND METHODS

Papain from Worthington Biochemical Corp. was crystallized twice by the method of Kimmel and Smith (1954)

J. F. BAUCHERand L. I. GROSSWEINER

164

and prepared as a suspension of crystals in 0.05 M sodium acetate. This material was activated by treating with dithiothreitol (DTT) followed by separation on a Sephadex (3-50 column. The activity was determined with the N carboxyglycine p-nitrophenyl ester assay technique of Gaucher et al. (1971). The average value of specific activity obtained was 10 2 p M of ester hydrolyzed per mg of papain per min. Sulfhydryl assay with p-(hydr0xymercuri)benzoic acid (Boyer, 1954) showed that the average sulfhydryl content was 0 6 f-0.2 mol per mol papain after DTT activation. The flash photolysis spectra were taken on Kodak 103-F plates using a Bausch & Lomb “medium” quartz prism spectrograph. The 20 ps photolysis flash was provided by 2 Xenon Corp. Novatron type 188A xenon lamps operated at 450 J input and the 10 ps spectroflash was obtained with a 65 J input xenon lamp. Samples were contained in a 20 cm double-walled quartz cell with glacial acetic acid in the outer jacket to filter wavelengths < 255 nm. Actinometry procedures and other apparatus details were described by Kaluskar and Grossweiner (1974). Action spectra were obtained with the apparatus of Tien (1973), in which light from an E.G. & G. type FX-6A Flashtube operated repetitively at 50 s-’ was filtered by a Beckman DU quartz prism monochromator. Samples were held in a thermostatted 1 cm quartz cuvette during irradiation and assayed immediately. Each set of flash spectra in Figs. 1-4 is the average of 4 runs.

A00

[

1

WAVELENGTH Lllm)

Figure 2. Transient spectra from flash photolysis of 175 p M trp at pH 7; 5 ps delay, Kodak 103-F spectroscopic plate. (a) N,O saturated; (b) air saturated. The difference spectrum (-----) is attributed to the trp triplet state ’Trp.

dent on the papain transients in deaerated (Fig. la) and NzO saturated (Fig. lb) conditions but not with air saturation (Fig. lc). The results obtained with mixtures of chromophoric amino acids equivalent to 15 jdvf papain (5 pts trp 4- 19 pts tyr + 14 pts phe + 4 pts cys) in Fig. 3 show the same initial products plus the tyrosyl residue Tyr bands at 410 nm and 390 nm. The 3Trp band is quenched by cystine, as evidenced by the data in Fig. 4 obtained with tryptophan and cystine alone. Comparison of Figs. 1 and 3 leads to several conin papain clusions. The strength of the -&&band RESULTS AND DISCUSSION is not altered by the presence of O2 or N 2 0 indicatFlash photolysis results ing that the disulfide adduct is not generated via e,g. Transient spectra from flash photolysis of dilute Surprisingly, this is the case also for the amino acid papain solutions at 5 ps delay are given in Fig. 1. mixture where air quenches the cystine adduct (Fig. The experimental spectra (points) have been resolved 3c) but not N,O (Fig. 3b). Bent and Hayon (1975) by fitting with the - S S - spectrum of Adams et al. attribute this reaction to the triplet state (1967); the e;, spectrum reported by Michael et al. (197l), and a new Tip absorption based on the data - S S - -+ -SS (2) ’Trp -SSobtained from aqueous tryptophan under air-satuwhich is supported by the present data in Fig. 3b ration to suppress the overlapping ea; and triplet where N,O scavenges eiq and the -SS product state (jTrp) absorptions. The latter has been resolved is formed while 3Trp is quenched. The Tyr absorption as the 460 nm band in Fig. 2 by subtracting the tranis not detectable in any of the papain spectra, which sients obtained in N 2 0 and air saturated solutions, can be explained by energy transfer from tyrosine to in agreement with the earlier identification (Santus trp in the enzyme but not in the mixture (Kronman and Grossweiner, 1972). The 3Trp absorption is evi- and Holmes, 1971). The approximate initial photo-

+

I

0.31

400

500

600

700

WAVELENGTH (nm)

Figure 1. Transient spectra from flash photolysis of 15 pM papain at pH 7; 5 p s delay, Kodak 103-F spectroscopic

plate. The experimental data (points)are resolved into the absorptions of e;, (....), 3Trp ( x x x x ), Tip (----) and -SSr (-----) the sum of which are shown as (--.--.--.-). (a) N, saturated; (b) N,O saturated: (c) air saturated.

lysis yields are summarized in Table 1 based on available extinction coefficients: €(e,;) = 1.8 x lo4 at 680 nm (Fielden and Hart, 1967); E(Tpr) = 2.8 x lo3 at 410nm (Feitelson and Hayon, 1973); E(-SS) = 9.4 x lo3 at 420nm (Adams et al., 1969); €(Tip) = 1.8 x lo3 at 510nm (Redpath et al., 1975) (all mol-’ cm-’). This Tip extinction coefficient based on a pulse radiolysis technique is considerably smaller than previous estimates of 1 x lo4 (Grossweiner and Usui, 1971) and 5 x lo3 (Pailthorpe et al., 1973). The revised low value is supported by recent laser flash photolysis measurements on aqueous tryptophan (Bryant et al., 1975) in which the initial eiq and Tip yields were related in air saturated solutions at the time of the 20 ns laser flash. Bent and Hayon (1975) showed that the photoionization of aqueous tryptophan is monophotonic and does not take place via the 3Trp state. The relative Tip yields in air saturated papain and the mixture in Table 1 inaicate that about 2.2 trp

165

U V inactivation of papain Table 1. Flash photolysis initial yields Relative Initial Yields*Photolyte*

15

IIH Papain

mino acid mixture'

Condition

Tip

Tyr

3Trp

-&

e-

NZ-sat

2.9

0

yes

0.43

0.27

NZO-sat

2.8

0

yes

0.40

0

air-sat

2.2

0

no

0.37

0

N2-Sat

7.8

3.7

no

1.06

0.54

NZO-sat

7.8

3.7

no

0.96

0

5.0

3.7

no

0

0

air-sat

aq

+

* pH 7 in 0.01 M acetate buffer. t 75 p M trp + 60 p M phe 60-pM cys. 1Approximately pmol at 5 ps delay after 450 J flash; the absorbed dose was about 120 peinsteins per flash. residues in the enzyme are photolabile. This result were destroyed per molecule inactivated in 254 and is based on the assumption that the individual trp 313 nm (Dose and Risi, 1972). On the basis of these residues in papain can be separated into two groups: results we postulate that the photoionized trp residues the photolabile residues that photoionize at the effi- observed by flash photolysis are Trp 7 or Trp 69 and ciency of aqueous trp and stable trp residues. The Trp 177, where permanent oxidation of Trp 177 is photodynamic oxidation studies of Jori and Galiazzo a possible inactivating step. The comparison of the SSand Tip yields in Table 1 shows that only (1971) show that papain is rapidly converted to an active product in which Trp 7 and Trp 69 are oxi- a small fraction of the ejected electrons are captured dized to N-formylkynurenine, indicating that these by disulfide bridges through a process not influenced residues are exposed and not essential. Prolonged by external ea; scavengers. A similar effect was irradiation led to the oxidation of Trp 177 and an observed with lysozyme (Grossweiner and Usui, 1971) inactive product with no destruction of tyrosine, histi- and trypsin (Kaluskar and Grossweiner, 1974) where dine, or half-cystine. These results strongly suggest it was postulated that a part of the disulfide adduct that Trp 177 is essential and at least partially access- is formed by a short-range, intramolecular electron ible to the external medium. The X-ray structure of transfer. The indole ring of Trp 177 is close to Cys crystalline papain (Drenth et al., 1971) shows that the 2 6 6 3 at the edge of the active site crevice where this Trp 177 side chain is buried but located within a process may take place via reaction (2). Alternatively, few angstroms of cysteine 25 where it might partici- Trp 177 or buried Trp 26 might transfer an electron pate in the esterase activity. However, spectral studies to the mixed disulfide at site 25. On the other hand, on papain (Weinryb and Steiner, 1970) as well as the the triplet state does not appear to be involved in photooxidation results indicate 2-3 trp residues are the inactivation process because the Tip initial yield exposed in the aqueous enzyme. About 3 trp residues in papain is independent of 3Trp (Table 1) and the inactivation quantum yield is the same in air and in nitrogen (see below). Photochemical results The action spectrum for activation of the original preparation (before DTT treatment) shows that wavelengths near the cystine absorption region at 250 nm are most effective (Fig. 5). The data are not in agreeA0.D.

400

500 WAVELENGTH (nm)

600

,

I

700 WAVELENGTH (nm)

Figure 3. Transient spectra from flash photolysis of amino Figure 4. Transient spectra from flash photolysis of acid chromophores equivalent to 15pM papain at pH 7; 122@ trp in the presence of 170@ cystine at pH 7; 5 ps delay, Kodak 103-F spectroscopic plate. The resolved N, saturated, 5 ps delay, Kodak 103-F spectroscopic plate. components are designated the same as in Fig. 1 plus Tfr The resolved components are designated the same as in Fig. 1. (....,) . (a) N, saturated;(b) N,O saturated;(c) air saturated.

J. F. BAUGHERand L. I. GROSSWEINER

166

I b\

I

I , 2 50

J

I

270 WAVELENGTH

2 90

(nrn)

Figure 5. Action spectrum for activation of 18 pA4 papain at pH 7 under N2 saturation. The solid line is the ‘least squares’ fit of the data to Eq. 3. (10 runs per point)

N, saturated solutions were activated at low starting activities and inactivated at higher starting activities (Fig. 6). Dose and Risi (1972) obtained the equivalent result by starting with the inactive material and exposing it to increasing irradiation dose, in which case the change from activation to inactivation occurred at about 13% activity (relative to full activation by DTT as 100%). The flash data have been interpreted by postulating that activation is promoted by photolysis of a disulfide group while inactivation involves light absorption in tryptophan. Extension of Eq. 3 leads to 4 a

=

(1/4)(?a - Yehcysfcys - (1/5)Yc%rp.Lp (4)

where ye is the fraction of active enzyme and ql,, is ment with Dose and Risi (1972) who reported a con- the probability that a photon absorbed by a tryptostant value of 4a = 0.022 at 254 nm, 280 nm, and phyl residues leads to inactivation. It is convenient 313 nm. The same workers found that between 1.0 to express Eq. 4 in terms of the specific activity A (at 280 nm and 313 nm) and 1.4 (at 254 nm) cysteine residues are released per act of activation. The solid line is the “least squares” fit to Eq. 3 assuming that disulfide photolysis is the key step where A,,, is the maximum activity obtained by pre= (11’4) YaVcyf,ys(l) (3) irradiation treatment with DTT and A , is the intrinsic activity of the active component. (This analysis where fc,, is the fractional light absorption by cystine assumes that the inert fraction acts only as an ‘inner (estimated from the amino acid mixture with 4 cys- filter’ and does not contribute to the photochemical tines per papain equivalent, ya is the fraction of acti- processes). The linear dependence in Fig. 6 in which vatable enzyme, and qcysis the average quantum effi- the lines for air saturation and N, saturation have ciency for disulfide destruction in papain leading to the same slope agree with Eq. 5. Taking A,= activation. The analysis gives y,, vcys= 0.34 and 10(+200/,) as measured and A , -24(f2Pk) gives qcYs= 0.8 0.2 taking the maximum active com- 4, = 0.0020( 20%) and 4, = 0.008( k25%) for actiponent as 0.4 of total enzyme. The quantity qcys vation and inactivation by the broad-band irradiadefines the efficiency by which a photon absorbed in tion. a single disulfide bond of the activatable enzyme leads The inactivation quantum yield 4e is in reasonable to the active form. Comparable high quantum yields agreement with the monochromatic irradiation results for cystine destruction have been observed in many of Dose and Risi (1972); 0.006 (254nm); 0.003 (280 proteins (e.g. the review of Vladimirov et al., 1970). nm); 0.007 (313 nm). However, 4a is about 10 times Typical values range from 0.2 to 0.6 based on light smaller than their wavelength-independent value of absorbed by cystine. Some workers (Risi et a[., 1967; 0.022. The large difference from the present results Dose, 1967) interpret the high photolysis yields as in- cannot be attributed to actinometry or assay prodicative of sensitized cystine destruction by aromatic cedures in view of the good agreement of the respecresidues. However, recent studies on cystine photolysis in aqueous solution (Dixon and Grant, 1973) show that typical low quantum yields -0.12 (pH 1, deaerated) are strongly enhanced in the presence of alcohols, suggesting that the relatively low cystine destruction yields in water are due to fast back reactions. It is possible that back reactions are inhibited also for cystyl residues in protein by local conformation changes after photolysis of the disulfide bond. The irradiation of partially activated papain was carried out with preparations ranging from inactive to specific activities about 5 pM/mg.min where fully active, high purity papain corresponds to 21-27 p M / mg.min (Lynn and Louis, 1972). The fractional A @M/mg.mln) absorptions with broad-band, flash irradiation were Figure 6. Effect of starting activity on ‘activation’ quantum 62% in tryptophan, 35% in tyrosine, 2.6% in cystine yield of 18 ptM papain at pH 7 for flash lamp irradiation. and 0.5% in phenylalanine. Only inactivation was N 2 saturation; 0 air saturation. The lines are ‘least squares’ fits of the data to Eq. 5. observed by irradiating air saturated solutions, while

4i

I

J. F. BAUGHER and L. I. GROSSWEINER

tive inactivation yields and probably involves differences in the ‘as received’ papain. The slope of the line in Fig. 6 for air saturated solutions gives qt,, = 0.10 +_ 0.03 as the contribution of absorption by one trp residue to inactivation of the enzyme, assuming that all trp residues absorb equally. The initial photoionization quantum yield was about 0.02 (Table 1) or 0.02/0.62N 0.03 based on light absorption in trp residues. Assuming that 2 trp residues are photolyzed with equal efficiency and the light absorbed by the other 3 residues is dissipated, the average photoionization efficiency per labile residue is 0.03 x (5/2) -0.1 in agreement with the assumption that photoionization of one essential trp residue leads to inactivation.

167

In a study of radiolytic inactivation of papain using radical anions as selective probes for specific amino acid residues, Adams and Redpath (1974) concluded that at least one trp residue is involved in the catalytic and binding functions. The parallel to the present investigation is particularly apt in view of the recent finding that the radical anions employed, Br, and (CNS);, oxidize trp to the same Tip product generated by UV photoionization of trp (Redpath et al., 1975). Acknowledgement-The authors are pleased to acknowledge the assistance of Stephan Wilkus in connection with part of the experimental work.

REFERENCES

Adams, G. E., G. S. McNaughton and B. D. Michael (1967) The Chemistry ofIonization and Excitation (Edited by Johnson, G. R. A. and G. Scholes) pp. 281-293. Taylor & Francis, London. Adams, G. E., R. C. Armstrong, A. Charlesby, B. D. Michael and R. L. Willson (1969) Trans. Faraday Soc. 65, 732-742. Adams, G. E., and J. L. Redpath (1974) Intern. J . Radiat. Bid. 25, 129-138. Bent, D. V., and E. Hayon (1975) J . Am. Chem. Soc. 97, 2612-2619. Bersin, T. (1933) Z. Physiol. Chrm. 222, 177-186. Boyer, P. D. (1954) J . Ant. Chem. Soc. 76, 43314337. Bryant, F. D., R. Santus and L. I. Grossweiner (1975) J . Phys. Cbem. 79, (in press). Dixon, C. J.. and D. W. Grant (1973) Photochem. Photobiol. 18, 387-391. Dose, K. (1967) Photochern. Photobiol. 6, 437-443. Dose, K., and S. Risi (1972) Photochem. Photobiol. 15, 43-50. Drenth, J., J. N. Jansonius, R. Koekoek and B. G. Wolthers (1971) The Enzymes Vol. 111 (Edited Boyer, P. D.) pp. 485-499. Academic Press, New York. Fielden, E. M., and E. J. Hart (1967) Eans. Furaday Soc. 63, 2975-2982. Gaucher, G. M., B. L. Mainman, G. P. Thompson and D. A. Armstrong (1971) Radiation Res. 46, 457-47 5. Glazer, A. N., and E. L. Smith (1971) The Enzymes, Vol. 111 (Edited Boyer, P. D.) pp. 501-546. Academic Press, New York. Grossweiner, L. I., G. W. Swenson and E. F. Zwicker (1963) Science 141, 805-806. Grossweiner, L. I., and Y. Usui (1971) Photochem. Photobiol. 13, 195-214. Hart, E. J., and J. W. Boag (1962) J . Am. Chem. Soc. 84, 40904095. Jori, G., and G. Galiazzo (1971) Photochem. Photobiol. 14, 607-619. Kaluskar, A. G. (1973) Ph.D. Thesis, Illinois Institute of Technology. Kaluskar, A. G., and L. 1. Grossweiner (1974) Photochem. Photobiol. 20, 329-338. Kirnrnel, J. R., and E. L. Smith (1954) J . Bid. Chem. 207, 515-531. Kronman, M. J., and L. G. Holrnes (1971) Photochem. Photobiol. 14, 113-134. Lynn, K. R., and D. Louis (1973) Intern. J. Radiat. Biol. 23, 477-485. Michael, B. D., E. J. Hart and K. H. Schmidt (1971) J . Phys. Chem. 75, 2798-2805. Pailthorpe, M. T., J. P. Bonjour and C . H. Nicholls (1973) Photochem. Photohiol. 17, 209-223. Redpath, J. L., R. Santus, J. Ovadia and L. I. Grossweiner (1975) Intern. J . Radiat. Biol. 27, 201-204. Risi. S.. K. Dose, T. K. Rathinasamy and L. Augenstein (1967) Photochem. Photohiol. 6, 423-436. Santus, R., and L. I. Grossweiner (1972) Photochen~Photobiol. IS, 101-105. Sluyterman, L. A. Ae, and J. Wijdenes (1970) Biochim. Biophys. Acta 2W, 593-595. Tien, F. T. (1973) MS Thesis. Illinois Institute of Technology. Vladimirov, Yu. A., D. I. Roshchupkin and E. E. Fesenko (1970) Photochem. Photobiol. 11, 227-246. Volkert, W. A,, and L. I. Grossweiner (1973) Photochem. Photobiol. 17, 81-90. Weinryb, I., and R. F. Steiner (1970) Biochemistry 9, 135-146.

Ultraviolet inactivation of papain.

Photochemistry and Photobiology, 1975. Vol. 22. pp. 163-167. Pergamon Press Printed in Great Britain ULTRAVIOLET INACTIVATION OF PAPAIN* J. F. BAUG...
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