Inactivation and cleavage of radish peroxidase by various reducing agents

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Phytochemistry\ Vol[ 38\ No[ 0\ pp[ 12Ð16\ 0887 Þ 0887 Elsevier Science Ltd[ All rights reserved Printed in Great Britain 9920Ð8311:87:,Ðsee front matter

Pergamon PII] S9920Ð8311"86#90022Ð2

INACTIVATION AND CLEAVAGE OF RADISH PEROXIDASE BY VARIOUS REDUCING AGENTS LEE MI YOUNG and KIM SOUNG SOO$ Department of Life Science\ College of Science\ Soonchunhyang University\ Shinchang\ Asan\ Choongnam\ 226!634\ Korea^ $ Department of Biochemistry\ College of Science and Bioproducts Research Center\ Yonsei University\ Seoul\ 019!638\ Korea "Received in revised form 00 November 0886#

Key Word Index*Raphanus sativus L[^ Cruciferae^ radish^ inactivation and cleavage^ per! oxidase^ reducing agents[

Abstract*The inactivation and cleavage of Korean radish "Raphanus sativus L[# peroxidase "EC[ 0[00[0[6# by three reducing agents\ dithiothreitol "DTT#\ ascorbate and 1!mercaptoethanol "1ME# was examined[ Upon incubation of the enzyme with DTT\ inactivation followed by degradation and cleavage of the anionic isoperoxidase A0 "Mr] 32 k# was observed\ whereas\ inactivation and degradation took place almost sim! ultaneously in the presence of ascorbate[ Nevertheless\ the cleaved products generated from the inactivated enzyme showed similar Mr values of about 29 k\ 12 k and 07 k in both cases[ When 1ME was used\ the formation of larger Mr aggregates of 55 k and 58 k was observed without cleavage of smaller peptides[ When the enzyme was inactivated by DTT and ascorbate\ dimethyl sulfoxide and ethanol protected the enzyme inactivation almost completely in both cases\ but these scavengers did not reverse the inactivation signi_cantly when 1ME was used as a reducing agent[ The singlet oxygen scavenger histidine prevented the enzyme from inactivation by ascorbate and 1ME to some degree\ but it failed to inhibit enzyme inactivation by DTT[ These results suggest that there might be di}erent inactivation and cleavage mechanisms of radish peroxidases\ depending upon the reducing agents used[ Þ 0887 Published by Elsevier Science Ltd[ All rights reserved

The present study was undertaken to investigate the mechanism of inactivation and degradation of Korean radish peroxidase by reducing agents such as DTT\ ascorbate and 1ME[ The characteristics of inac! tivation and cleavage patterns of isoperoxidase A0 by these reducing agents were compared in detail[

INTRODUCTION

Recently it has been shown that several enzymes are inactivated and degraded by the oxidative modi! _cation system comprised of a reducing agent\ oxygen and iron "or copper# ð0Ł[ The proteins cleaved by the oxidative modi_cation system include Saccharomyces cerevisiae glutamine synthetase ð1Ł\ Escherichia coli glutamine synthetase ð2Ł\ erythrocyte superoxide dis! mutase ð3Ł and low density lipoprotein ð4Ł[ This speci_c inactivation event is thought to be the result of the generation and reaction of active oxygen species at speci_c iron or copper binding site on these proteins ð1Ł[ However\ Davison et al[ observed that active oxy! gen species were not directly involved in the ascorbate mediated catalase inactivation with copper ð5Ł[ In this case\ semidehydroascorbate formed from the oxi! dation of ascorbate was found to be the most likely damaging agent[ Therefore\ there might be the possi! bility of di}erent inactivation and degradation mech! anisms depending upon the proteins and reducing agents used[

RESULTS AND DISCUSSION

Inactivation of Korean radish peroxidase by reducin` a`ents In the study presented here\ we found that Korean radish isoperoxidase A0 was inactivated in the pres! ence of DTT\ ascorbate and 1ME in a concentration and time!dependent manner[ Figure 0"a# shows the e}ect of various concentrations of these reducing agents on the activity of isoperoxidase A0 "_nal con! centration 9[0 mM#\ suggesting that DTT and 1ME inhibited the enzyme with similar e}ectiveness\ and that they were much more potent than ascorbate in inhibiting peroxidase activity[ Figure 0"b# shows time!dependent inactivation of isoperoxidase A0 by DTT in the presence of EDTA and FeCl2[ Isoperoxidase A0 "_nal concentration

 Author to whom correspondence should be addressed[ 12

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L[ M[ YOUNG and K[ S[ SOO

Fig[ 0[ "a# Inactivation of isoperoxidase A0 with several reducing agents[ The enzyme "9[0 mM# was incubated at 14> for 0 h with various concentrations of dithiothreitol "R*R#\ ascorbate "ž*ž# or 1!mercaptoethanol "Ž*Ž#[ "b# E}ect of EDTA and FeCl2 on the inactivation rate of isoperoxidase A0[ The enzyme "9[4 mM# was preincubated with 4 mM EDTA "ž*ž# or 49 mM FeCl2 "Ž*Ž# in 09 mM sodium phosphate bu}er "pH 5# for 04 min\ then treated with 0 mM dithiothreitol[ Remaining enzyme activity was compared with the enzyme treated with dithiothreitol "R*R# at various time intervals[

9[4 mM# lost about 29) of its activity when the enzyme was incubated with 0 mM DTT for 19 min[ The addition of 4 mM EDTA almost completely pro! tected the enzyme activity under the same experi! mental condition\ while the enzyme inactivation was much more stimulated by 49 mM FeCl2[ However\ the protective e}ect of EDTA in chelating the adventi! tiousiron was not as e}ective when the incubation time was over 19 min at pH 5[ The protective e}ects of EDTA were reported in glutamine synthetase\ alcohol dehydrogenase and glyceraldehyde!2!phosphate dehydrogenase ð6Ł in the presence of exogenous iron and reducing agents although they are not iron!con! taining metalloenzymes[ SDS!PAGE analysis of the cleaved peroxidases by reducin` a`ents When isoperoxidase A0 "_nal concentration 16[5 mM^ 25 mg# was treated with 19 mM DTT for 49 h\ we could not detect cleaved products at all\ although the enzyme lost about 89) of its activity "lane 3 of Fig[ 1#[ On the other hand\ the enzyme began to be degraded and cleaved into several discrete smaller peptides of 29 k\ 12 k and 07 k as soon as the enzyme activity disappeared completely at 9[0 M DTT "lanes 4\ 5\ 6 of Fig[ 1#[ Therefore\ it is likely that the cleavage event does not occur until the enzyme loses its activity completely[ On the other hand\ 9[0 M ascorbate treatment for 49 h did not induce enzyme inactivation and cleavage as shown in lane 2 of Fig[ 2[ The cleaved peptide fragments were observed when the enzyme lost only 09) of its activity at 9[1 M ascorbate "lane 3 of Fig[ 2#[ The measured Mrs of cleaved peptides were about 29 k\ 12 k and 07 k\ which were similar to the Mr values of the fragments generated by the DTT inactivation system[ These results suggest that enzyme inactivation and degradation took place almost simul!

taneously\ and that these two steps might not be strictly separate phenomena when ascorbate was used ð7Ł[ When the concentration of ascorbate was over 9[2 M\ the enzyme was degraded thoroughly and the 32 k main band disappeared completely "lanes 4 and 5 of Fig[ 2#[ In Fig[ 3\ the e}ect of 1ME concentration on the inactivation and cleavage is shown[ At 49 mM 1ME\ the enzyme lost all of its activity[ In this case\ a notable observation was the formation of higher Mr aggre! gates of 55 k and 58 k which could not be found in DTT and ascorbate mediated cleavage system "lanes 2 and 3 of Fig[ 3#[ Furthermore\ we could not observe any smaller cleaved products derived from the enzyme by 1ME\ although there remains the possibility that thoroughly degraded very small peptide fragments could not be detected as bands on the gel by Coom! assie brilliant blue R!149[ This suggestion is well sup! ported by the observation that the more 1ME con! centration was increased\ the thinner the intact band of 32 k became\ without the increase of cross!linked larger peptide fragments[ The generation of cross! linked higher Mr adducts through radical mediated inactivation of yeast glutamine synthetase by DTT was previously reported ð1\ 8Ł[ In our experimental condition\ however\ only 1ME caused peroxidase to make higher Mr aggregates[ Effect of radical scaven`ers on the isoperoxidase A0 inactivation Various radical scavengers were used to examine whether free radicals might be involved in the inac! tivation of peroxidase by reducing agents[ In Table 0\ the e}ect of several radical scavengers on the inac! tivation of isoperoxidase A0 by reducing agents was shown[ Upon incubation of 9[0 mM isoperoxidase A0 with three reducing agents "DTT\ ascorbate and 1ME#\ di}erent protective e}ects were obtained

Cleavage of peroxidase by reducing agents

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Fig[ 1[ E}ect of dithiothreitol concentration on the inactivation and cleavage of iosperoxidase A0[ The enzyme "16[5 mM^ 25 mg# containing various concentrations of dithiothreitol was incubated at 14> for 49 h[ Lane 0\ Mr markers "85 k\ 34 k\ 20 k\ 19 k and 03 k#^ lane 1\ no DTT^ lane 2\ 09 mM DTT^ lane 3\ 19 mM DTT^ lane 4\ 099 mM DTT^ lane 5\ 199 mM DTT^ lane 6\ 299 mM DTT[

Fig[ 2[ E}ect of ascorbate concentration on the inactivation and cleavage of iosperoxidase A0[ The enzyme "16[5 mM^ 25 mg# containing various concentrations of ascorbate was incubated at 14> for 49 h[ Lane 0\ Mr markers "85 k\ 34 k\ 20 k\ 19 k and 03 k^ lane 1\ no ascorbate^ lane 2\ 9[0 M ascorbate^ lane 3\ 9[1 M ascorbate^ lane 4\ 9[2 M ascorbate^ lane 5\ 9[4 M ascorbate[

depending upon the reducing agents used[ When the reaction condition was adjusted to obtain 49) enzyme activity inhibition by DTT\ dimethyl sulfoxide protected the enzyme almost completely[ Ethanol and mannitol showed some protective e}ect against DTT\ whereas thiourea and histidine had no e}ect[ Ethanol and dimethyl sulfoxide also protected the enzyme inactivation due to ascorbate\ although they did not reverse the inactivation by 1ME signi_cantly[ Con! sidering the above results\ reactive oxygen species are thought to be involved in the inactivation\ since rad! ical scavengers such as ethanol and dimethyl sulfoxide prevented the enzyme inactivation[ Curiously\ his! tidine reduced peroxidase inactivation by ascorbate and 1ME to some degree\ indicating presumptive evi!

dence for a role of singlet oxygen ð5Ł[ However\ the possibility that it scavenges hydroxyl radicals or serves merely to chelate metal ions must be considered\ as suggested in the NADH oxidase system ð6Ł[ Effect of substrate and several compounds on the cleav! a`e of isoperoxidase A0 by DTT The e}ects of substrate and several compounds on the inactivation and cleavage of isoperoxidase A0 are shown in Table 1[ The enzyme "_nal concentrations 16[5 mM\ 25 mg# preincubated with 9[4 M guaiacol retained 84) of the activity\ suggesting that guaiacol might prevent accessibility of DTT to the enzyme[ Recent reports indicate that a site!speci_c oxidative

15

L[ M[ YOUNG and K[ S[ SOO

Fig[ 3[ E}ect of 1!mercaptoethanol concentration on the inactivation and cleavage of isoperoxidase A0[ The enzyme "16[5 mM^ 25 mg# containing various concentrations of 1!mercaptoethanol was incubated at 14> for 49 h[ Lane 0\ Mr markers "85 k\ 34 k\ 20 k and 19 k^ lane 1\ no 1!mercaptoethanol^ lane 2\ 49 mM 1!mercaptoethanol^ lane 3\ 9[0 M 1!mercaptoethanol^ lane 4\ 9[1 M 1!mercaptoethanol^ lane 5\ 9[2 M 1!mercaptoethanol^ lane 6\ 9[4 M 1!mercaptoethanol[

Table 0[ E}ect of various scavengers on the inactivation of isoperoxidase A0 by reducing agents Loss of activity$

Scavengers%

Ascorbate "4 mM\ 09 min#

Dithiothreitol "0 mM\ 09 min#

1!Mercaptoethanol "0 mM\ 09 min#

None Ethanol Dimethyl sulfoxide Mannitol Thiourea Histidine

099 4 02 84 027 53

099 34 9 58 033 093

099 71 62 007 83 69

 Concentration of iosperoxidase A0 is 9[0 mM[ $ Results are expressed as a percentage of the activity loss without scavenger additions[ Thus\ values less than 099) indicate protection from inactivation[ In the absence of scavenger additions\ about 49) of the peroxidase activity was lost during the inactivation[ % Concentration of scavengers is 149 mM except ethanol "799 mM#[

modi_cation system generates activated oxygen derivatives that attack His or other oxidizable amino acids "e[g[ Cys\ Met\ Trp and Tyr# in the catalytic site of the enzyme ð09Ł[ In this regard\ it is noteworthy that the guaiacol!protected enzyme did not generate 12 k cleaved peptide which had been apparently observed in the DTT treated sample "Table 1#[ The same cleavage patterns without 12 k peptide were found in the EDTA treated enzyme having 19) of the activity\ although complete protection of EDTA against glutamine synthetase cleavage were reported ð1Ł[ Dimethyl sulfoxide treated enzyme showed three discrete peptide fragments with identical Mr to DTT treatment sample\ suggesting that dimethyl sulfoxide did not protect against enzyme cleavage by DTT at

Table 1[ E}ects of substrate "9[4 M# and various compounds "9[4 M# on the cleavage and inactivation of isoperoxidase A0 by dithiothreitol[ The enzyme "16[5 mM^ 25 mg# was pre! incubated with several compounds for 2 min and sub! sequently reacted with dithiothreitol "9[4 M# for 2 h at 24> before SDS!PAGE

Addition

Enzyme activity ")#

Enzyme only 099 DTT 9 DTT¦guaiacol 84 DTT¦dimethyl sulfoxide 19 DTT¦EDTA 19

Formation of 12 k peptide

Yes No Yes No

Cleavage of peroxidase by reducing agents

all "data not shown#\ although it was an e}ective pro! tector against enzyme activity inhibition as shown in Table 0[ Therefore\ the inactivation and degradation phenomena observed in Korean radish peroxidase seem to be ascribed to thiol:metal ion:O1 oxidative modi_cation system\ and the existence of heme iron in the peroxidase structure might make the enzyme more susceptible to easy attack by reducing agent in the presence of O1 ð1\ 06Ł[ EXPERIMENTAL

Experimental plant and enzyme puri_cation The fresh Korean radish "Raphanus sativus L[# root was obtained from a local market in Seoul and used for the puri_cation of isoperoxidase A0[ The enzyme puri_cation was performed as previously reported in our laboratory ð00Ð02Ł[ Peroxidase assays and protein determination The peroxidase activity with guaiacol as a substrate was assayed by a modi_ed method of Ref[ ð03Ł[ Protein concn was determined by the method of Ref[ ð04Ł[ Enzyme cleava`e reaction The cleavage reaction mixture was composed of Korean radish isoperoxidase A0 and 09 mM Na!Pi bu}er "pH 5# containing various concns of reducing agents such as DTT\ ascorbate and 1ME[ Iso! peroxidase A0 "_nal concentration 16[5 mM^ 25 mg# was incubated with 09 mM Na!Pi bu}er "pH 5# con! taining 9[4 M DTT for 49 h at 14> in a total vol[ of 29 ml[ In order to investigate the e}ect of concns of vari! ous reducing agents such as DTT\ ascorbate and 1ME on the inactivation and cleavage of isoperoxidases\ the reducing agents were added at di}erent _nal con! cns "09 mMÐ9[4 M# in 09 mM Na!Pi bu}er "pH 5# at 14> for 49 h\ and then the residual enzyme activity was measured[ Effect of substrate and several compounds on the cleav! a`e of isoperoxidase A0 by DTT To investigate the e}ect of substrate and several compounds on the inactivation and cleavage process of iosperoxidase A0 by DTT\ the enzyme "16[5 mM^ 25 mg# was incubated with 9[4 M DTT for 2 h at 14> in the presence of substrate or other compounds in a

16

total vol[ of 29 ml[ These additions include substrate "guaiacol# of 9[4 M\ 9[4 M dimethyl sulfoxide and 9[0 M EDTA[ SDS!polyacrylamide `el electrophoresis After the cleavage reaction was performed\ the reac! tion mixtures were analysed by SDS!PAGE according to methods of Ref[ ð05Ł[ Acknowled`ements*This work was supported by a grant from the Korea Ministry of Education "BSRI! 85!3319#[ REFERENCES

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