Purification and properties of Bothrops protease A

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11 proteolytic enzyllle fro111the vctwt~i of Hotlwops jamzmcu, which hydrolyzes gelatin and t)e:lzoyl-r~~a~~ilIille amide, has heen previously drscrihed under the m111e of Kofhwpa proteasc .\ ( 1). This enzynle was stlowll to IF heat-w&ant ( 2) and to I)e different 1’i~oni the t)l,adykinin-l.eleasit~~ fact,or ( 3) and the tjlood-ctottillg etlzynw (4) of the venon~. It was purified by hcatitg followt~l J)y repeated precipitation with ~IIItllotliunl sutl’ate and zone etectmphoresis ( 1). III this work WC report, a nlethod which leads to t,tie sanie purification as the previous one ( 1) hut \vhich gives a higher yield. The activity of I~~thi~qx prot,easc .-I on se\-crat s;ltbstmt,es has twttl studied iii cotllparisoii to trypsiri. The dffxt of t-~ythogcii ion coiicctit.ixtiotl on t,tie stability of Rothrops protease .I, in tlie range of pII O-!).O, tins also twi1 studied.

I )tird venotn of 11. .j(tr(lrrccct (tttixtwe of vettonts collected befow 101i) was used as the startirtg trt:tteri:tl. The vettottt solution wts treated and ftxrt~ion:dly precipitated as pwviously dewribed (1). Ttw ftxrtiott obt:Crtrd at the level of 0.7-0.8 of s:ttrtr:ttiott with attttttottiuttl sulf:Ltr wits ditrlym~d

H!yd,o~~~sis of s!jnthelic .srtbsfrnfe,s. The hydrolysis of P-tc~lllrllcsulfon~l-L~arginin~ methyl rster (TAME) was measurrd ~)otentiometri~all~ by t.he technique of Schwrrt et trl. (13). For thr determination, 10 ml. of 0.005 III tris hufler CptI 8.0) containing 2.6 X lOV5 nroles of T$ME were us&. The hydrolysis was carried out at 25°C:. The amount trf libt~rated c:trbox~~l groups was titrated by adding 0.05 nrl. of 0.05 .V Sa0I-I :~rrd measuring the time required for the pH to rctrlrn to 8.0. With :ill sanrples, successive additions of the same amount of 0.05 h- SaOH were nxldr in order t,o observe the changes of hydrolysis rate occurring during flip reaction. These data. werr then plotted on :I gr:rph from which the initSial rate of hydrolysis was calculated. When n quantitative determination of hydrolytic potency Was ccjncentration WE so adjust(ld nxrde, the enzyrlre that the initial rate of Irydrolysis was srrudler t ban 3 u111oIes of substr:ttc per nlinutr. The hydrolysis

of 1,.lysino c%hyl cstclr (Lli:I’) was estimated photomet rirally by the hydrosam:~te technique of Hestrin (14) :ts modified by Roberts (15). For this determinatic~n the mol:w concentxltion of pH 8.0. the suhtrat~e was 0.08 in 0.25 N Iris I)nH’er, The hydrolysis of I)~nszl,~l~I,~:trg:illirle amide (ISAh) was measured bp the method of Schwert c1 a!. (13) :tdaptrd to the dilfusion apparatus of Tompkins and Kirk (l(i) :ts described by Henriques zt crl. (1). The substrate was tlissolvrd directly in 0.1 Jf tris I)utfcr, pH X.8. H,yrlro/ysis of pwfnraine. Prot,:inCnt: sulfat,e WVRSdissolved :rnd dialyzed :tgainst 0.1 JI phosphate buffer, pH 8.0. The reaction mixt,ures were prcparetl by mixing O.l25K~.O ml. of the prot,:imine solution with 0.2 ml. of the enzyme, :tlld 0.1 M phosphate bufl’er (pH 8.0) W:IS added to :t J-ml. end v111unw. ilftcr 20 minutes incubation at 3i”C., thr etlzynlic action was stopped by adding 1.0 ml. of gl:tciul xcetir wid, and the free amino groups \I-ere detcrnrined (17).

70 Tris HCI 0 GSM,pH 7.6

Tris. 01M

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I

Tris HCI 0.2M

80

100

90 Trls

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FIG. 1. Chromatography 011 DEA>J-cellulose of Hothrops protensr 11 preciI)it:ited at the level of 0.7-0.8 of saturation with ammonium sulfat?. The chromatogram was developed by steprise gradient with tris I)uf’fer, pH f.fi. The broken line A presents the extinction of fractions (vol. 20 ml,) at 280 mu (1 cm. light path); (01, relative specific hrllzovl-1..nrginirle relative specific p-tol~lt~~~rsulf~)~~~l-I.-:trgilli~rr: methyl rsteruse anridase activity; (0). activity; (@), relative specific blood-clotting :lctivit!-.

Figure 1 stttlttnai+zes the wsults ol~sttwd n-lie~i the tltaterial obtained froni lteab t.twtcd veiio~t~ ly precipitatioti with attltrtottiuttt salfat,e hetweett 0.i and 0.X of snt~ttmt~iotlwas suhnritted to chronlatograplty oti a I>E::ZEScellulose coluttiti, using tris hdfer, pII i.6, as clrrstit. It cat1 he sew that the proteins cotttaitted in the colun~~l n-et’c t~esolvcd into four distinct, peaks. The first two peaks, elrtt,ed with 0.0.5 Jl t’ris huf’fet,, cotttained 20 “; of the total protein added to the colttttltt attd did not slton- henzoyl-r,argitiirir niiiidase ( activity, p-tolttett~~sltlf’oti~l-I,-ai.~iriitie cstciase ( TAAI I<:asrj acti\+y, or blood-clotting activity. T11c third prot’titt peak, chtted wittt 0.2 A/ tt,is httfyet., rotttained iI0 5’; of the protein added t0 ttle COl~lllll~, 24 “; of the BAAse activity 11 ‘; of the 7’;\A\\l~Cascacti\-it,y, attd (j()“~; (11 tile t)lood-clott,itia activit,y of tile tota] c~lilottiat,o~t~aptled tiiatcrial. The fourth pl’oteitt peak, elutcd with 0~3 Al tris l,ltffer, cotitnirlrd 21 ( ; of t,lw proteitt added to the

rxhe)

coluintl, as ~11 as 47 “; of t,he J3A\,1se arid 65 5 of ThAIEase actirit,y of the chrotiiatographed material; it did not show any detcctahlc Mood-clot~ting activit?. Yractiott !)5, which corresponds t,o the fourth ptuteitt peak, had a ILL\sc or TIL\IKaw specific activity which was 13-4 t.ittles as high as the cht~otrlatographcd ntaterial (l’ig. 1) and 42 and 2:’ titttes as high as the crttdc vcttottl, cort*espottding therefore t,o ~othq~s protrasc A ( 1). ~~ltltongh the ft~act~iot~iswhich cotistitut,ed t,ltc third protciu peak wet’c also qttito active, their lLL\sc or T:2:\112nsc specific activities were atwut half as high as that of the ttraterial added to the coltmltl. Nofhwps prot,ease &\ (fraction 4, Tahlc I) u-as stttb niittfvl to t~rclii~ottiatogt~apti~ utttlet~ t,tte sat1ie coiiditiotis. So protein n-as elttted wit,li 0.0.; 01’0.1 :I/ t,ris hufi’cr, l)rtt 3i’; of t)lie protein, or 47 ‘; of IL1Asc activity, was eluted as :I \wy twoad hand when 0.2 ;I/ t,ris t)ttt’f(>r \\-a~ filtered through t)lte colrttrrtt. The wtmittittg 44 “J of the protein, or X ’ ; of the 13.\Lbx~a(‘tivity, was rrtttowd with 0.137/ t,t,is tmf’l’er as a very sharp proheitt peak. This wttt~aitted t,lic nrost, active fraction, ~vliirlt had a specific activity about 1,; “; higher tltatt of t,ltr starting satriplc (Tat+ I), while the prntciti cotit’aitwd itt the twoad hattd had :I specifics tllnI1 tllat of tllc ILLb3e act,ivity 20 ’:c ~lll:dlPI~ Li startjiitg tiiatct:ial.

Table I Lshow I’ that 0.07 “C of the original vcnon~ protein was recovered in the rechronlatographed material with yields of 3.4 aud 2.0’:; of BAAsc and ThIZII+:asr activities, respectively. Thus, after heating, 64 and 19% of the original HAAsc and TMIEase activities were respectively found in the supernatant; after precipitation with ammonium sulfate, 23 and 12% of the BAhsc and TA1IEasc activities, respectively, of the supernatmant~ were recovered in the fraction obtained bet,ween 0.7 and 0.8 of sabration with aunlonium sulfate; when this fraction was sub mitted to chronrato$raphy on DEAEcellulose, t’he highest BAAse and T.UIEase specific activit,irs were found in the fourth protein peak wit,11 yields of 1’7 and 65 “;, respectively.

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The hydrolvtic potency on TIZAITC of the preparation of Roth,*ops protease A obtained by a single chromatographic treatment8 (fraction 4, Table I) was conlpared with that of crystalline trypsin. I’or this purpose, the initia1 rates of hydrolysis, at several concelltration levels of TXL\IE, produced by cow stant aiiiount of trypsin or Rothrops protease The results (Icig. 2) 4 were measured. indicated that with both rnzynles maxinlu~ll rat,e of hydrolysis were not obtained even with substrate coiiccutratiou as high as 0.04 Al. From the esperinlental values, transformed according to Lineweaver and Uork (IS), it was concluded that the linlit rates of hydrolysis were 504 and 230 +uoles of TAME hydrolyzed per nliuut,e per n~illigram of protein enzynle for tlypsin and Bothwps proteasc .4, respcct,ively (Tab II). Therefore, when acting 011 TAAIE, the purified Botlwops proteasr A had about half the hydrolytic potency of crystalline trypsin.

It can be calculated front the figures presented in Table II that purified Roth.~~ps

protcase 21 had a hydrolytic activit,y 100 t~iiiics sliialler than that of wystalline trypsiu when hydrolyziu# 1X1:.

This suhstratc was used in different cow centrations k~etweeu 6.6 and 95 tug. per milliliter. A4t the limit rate of hydrolysis, ohserved at the higher levels of subst,ratc

at different pH levels (,Z>“C’.) attd sh\.ed 110 10~sof activity hetwett pH :I and !I C~‘lhl~l~~ III) up to “0 Ilollt~s. I)IscI-ssIos The

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for the main IL~Asc activity. In t,he present work it is shown that Roth.rops protease &\ contains also the bulk of T.L\IEase activity. The capacity of several of the chronlatographic fractions to liberate bradykillin was also studied. It was found that the fractiorl with highest TAI\IEase acti\:it,y had about t’he same Inradykillin-~eleasillg activit’y as the heated xwwn~. This confirms previous work of Hcnriqucs et al. (Sj. In t,hosc expernwnts (3) evidence was presented indicat,ing t,hat the blood-clottmiug PIU~IIW, the bradykillin-releasing enzyme, and Rolhwps protease A1 are distinct substances. If these t)hree enzymes are &erases, as proposed hy Habennann ( 20), the results of our chronlat,ographic cxperitnents can only lx rmderstood if thr Ti1AIEase which is contained in t,he peak of blood-clotting activit’y is slower than that which characterizes the Rothrops protease Ai peak. In fact, imp&ion of E’ig. 1 shows that the peak of blootlclotSting activity has a T~\.\lE:ase specific acti\-it,y half as high as that of the iilat,e:rial added to the colunm, while its blood-clotting specific activity is five times as high as the latter. Therefore the hypothesis of Habcrmann can only bc checked after the separation of the bradykinimreleasing enzyme and the blood-clotting enzyme of the \‘enotll in a purer state. 1~FFFlWS 1 2

1.

HENRIQCES, M.\s,

0. RI.,

H.,

L

K~NDELB.LI:JI,

yr~rds, 8. B., Hiochrnl. 2.

I~IJIBERG,

Intern.

ws \vR.\s,

4. k’.

It.,

A. ANI)

CJ.,

Frc’rr-

HEXRI-

J. 68, 5% (1058).

ROCH.\ E: SILVA, M., .lrch. I’hor~~~acodyn. 110, 22“ (1957). IT.,

LND