Tadsa Vd. 30, No.3. pi. Ul-l.W.1W2 Frinldbofatmimin.

PURIFICATION AND PROPERTIES OF A KININOGENIN FROM THE VENOM OF LACHESIS MU?-” (BUSHMASTER) MARCELOR. V. DIN& and EDUARDOB. OLIVBIRA*+ ‘Funda& EzcquiclDiaq Bdo Horimntc,MinaaGerais;and 2Dspartment of Bi-,

Mcdkine, University of !%o Paulo, 14049 Rib&30 Prcto, Sib Paula, Brad

Facultyof

(Rccciwd1 July 1991; accepted3 Decem&r 1991)

and properties of a hi. R. V. I)INn. and E. B. OLIWIU. PuriCxtion kininogenin from the venom of Luchesir muta (bushmaster). Toxicon 30, 247-258, 1!992.-An acidic kininogedn from Luchesis muta snake venom was pur&d to apparent homogeneity by a combination of gel filtration, isoelectric focusing and preparative gel electrophoresis. It was shown to be a highly stable serine protease (mol. wt 27,900; pI 5.4) capable of releasing bradykinin from low mol. wt bovine kininogen and of cleaving some synthetic chromogenic peptides with the following catalytic efficiencies (K&/Km, M-‘.sec-I): (1.92 x lo’); H-D-Val-Leu-Arg-p N-benzoyl-Phe-Val-Arg-pnitroanilide nitroanilide (1.55 x 10’); N-acetyl-Phe-Arg-pnitroanilide (3.98 x 10r); no hydrolysis was observed with N-benzoyl-Arg-pnitroanilide. A marked and sustained hypotensive effect was recorded following iv. injection of puri6ed kininogenin into rats. Tachyphylaxis was observed after repeated i.v. injection of the enzyme, a phenomenon accompanied by a decmase of only 15% in the total circulating rat kininogen. Both the in viva action and the enzymatic properties of the L. muta kininogcnin indicate that this enzyme might be helpful for understanding the kinin-kininogen system.

MTRODUCI’ION

Swrcn venoms from species belonging to the family Viperidae possess high kinin-releasing activity. The corresponding enzymes, named kininogenases, kininogenins, or kallikreinlike enzymes, have been isolated and characterized from venoms of several species, such as Agkistrodon halys biomhofi (IWANAGA et al., 1%5), Echis coloratus (COHENet al., 1970), Bitis gabonica (MHBS, 1970), Vipera ammoa’ytes ammoa’ytes (BAILBYand SHJKKINI, 1976), Crotalus atrox (BJARNASON et al., 1983), Crotafus scutulatw scutulatus (SCHWARTZ and Btnnua, 1985), Bitir a&tans (SFXOCWCHIet al., 1986), Vipera berus berus (SAMELet al., 1987), Vi@era aspis aspis (KOMORI and SUGII-UIU, 1988), Crotalw viri& virtiis (KOMORI et al., 1988) and Agkistrodon caliginosus (YABUKI et al., 1991). In comparative studies (DEUTXH and DINIZ, 1955; Osru~ et al., 1969; MEBS, 1970), L.uchesis muta venom was described as containing high levels of kininogenin activity and insignificant amounts of kininases, thus rendering such venom a suitable source of the former enzyme. The present *Authorto whom correspondence should be ad-.

248

M. R. V. DINJZ and E. B. OLJVEIRA

work reports the purification and characterization of an acidic kininogenin from L.mutu venom; it was also aimed at defining the usefulness of the purified enzyme as a biological tool for studying the kininogen-kinin system.

MATERIALS AND METHODS Luchesis mum venom was obtained by manual milking of a single specimen kept at the Funda+o Exequiel Dias serpentarium; the snake had been collected in Para State, Northern Brazil. Sephacryl S-200, Sephadex G-75. DEAESeuhacel and mol. wt marker moteins were ~u~~hasxl from Pharm&a Fine Chemicals. Sweden. Brakykinin was obtained from Sandoz Co., U.S.A. L-BApNA,* phenyhnethylsulfonyl fluoride and buffers used in the preparative isoelectric focusing experiment were purchased from Sigma Chemical Co., U.S.A. The chromogenic substrates N-Bz-L-Phe+Val-L-Arg-pNA and H-D-Val-L-Leu-L-Arg-pNA were from Kabi Diagnostics. Sweden, and N-acetyl-L-Phe-L-Arg-pNA was kindly supplied by Dr L. Juliano, EPM, S8o Paulo. Captopril was a girt from Squibb Institute, NJ, U.S.A. Partiahy purified kininogen was obtained from the soluble fraction of heat-treated &rated bovine plasma (61°C. 60 min) by a combination of salt fractionation, DEAESephacel and Sephacryl S-200 column chromatographies. Only the kininogen form with chromatographic and physicochemical properties similar to those of the monomeric low mol. wt bovine kininogen described by KOMNA et cl. (1974) was used as a substrate for identifying the kininogenin from the L. mute venom.

Assay of kininogenin activity

The kininogenin activity was determined at 37°C by measuring the amount of kinin released after a 5 to 15 min incubation of 0.48 ml of substrate solution (20 PM low mol. wt kininogen in 0.1 M Tris-HCl buffer, pH 7.5) with 0.02ml of a diluted enxyme preparation. Under these conditions the amount of kinin released was proportional to the incubation time, care t&g taken not to exceed 15% substrate consumption. After stopping the reaction by lowering the tem~rature of the incubation mixture in an ice bath, the kinin formed was estimated by the isolated guinea-pig ileum bioassay (Rm E. S~LVAet cl., 1949). For scmening purposta throughout the enxyme purification the heights of the recorded contractions were compared with those produced by increasing amounts of synthetic bradykinin within the linear portion of the doscresponse curve (log dose against the corresponding contraction heights). For a more quantitative estimation of the kinin released by the pools of kininogenin obtained at the end of each purification step, the analysis of variance of data obtained by the four-point assay (2 x 2 Latin square design) as described by FINNEY(1978) was Performed. One unit of kininogenin activity was detined as the amount of enxyme which releases I.O~nmoieof bmdykinin per min. under the described conditions. For assaying the kininogenin activity at different pH values, we used a bunkered saline solution containing 0.02 M of each acetic acid, Bis-Tris. Mops and Tris, the pH adjusted with 1 M HCI or 1 M NaOH.

Enzyme p@icalion Dried L. muta venom (400 mg) was suspended in 10 ml of 0.05 M NaCl, and the insoluble material removed by centrifugation (3500 xg. l&in) atI& I5 hr standing at 4°C. The clear supcrnatant was applied to a Seuhacrvl S208 column (1.8 x 190 cm) and eluted as &bed in the leaend to Fia. 1. The lyo~hilized material thus obtained was dissolved in 1.0 ml- of distilled water and applied to-a Sephad& G-75 column (1.0 x 95 cm) eluted with distilled water at mm temperature, at a flow rate of 3.1 ml/hr. Fractions of I.0 ml were colleoted and their protein contents and kininogenin activities determined as described in Fig. 1. The preparative llatbed (1979). using the electrofocusing step (Fig 2) was performed according to the method of Pamm~~ and ti amphoteric and non-amphoteric buffer mixture described as Standard plus Basic (0.02 M each) to generate a pH gradient. Focusing was carried out at 4°C on a gel tray measuring 25 x 2 x 0.5 cm (20 ml sample), with the voltage gradually &teased from 200 to loo0 V to limit the power dissipated in the gel to approxiaately 2 W. The preparative electrophoresis puritlcation step was carried out on a 7 to 13% gradient polyacrylamide gel slab (12 x 10 x 0.16 cm) using a simplified discontinuous but& systen in which DAM& (1964) stacking and sample but&m, pH 6.7, were omitted. After the run, a narrow strip of gel was cut from each side of the slab and stained with Coomassie blue for locating the protein bands. The mnsinder of the gel was transversely cut into 40 slices from which protein was extracted twice with 0.5 ml of 0.05 M Tris-HCl buffer, pH 7.5. for 24 hr. The

+A&evhrjons: L-BApNA, N-a-benxoyl-r.-arginine-p-nitroanilidt; Bis-Tris, bis [Zhydroxyethyl] iminotris [hydroxymethyl~~ HPLC, high performance liquid chromatograp~ MOPS, 3-B+motpholinol propane_ sulfonic acid; PAGE, polyacryhunide gel electrophoresia; pNA, pnitroanih~ SDS, sodium dodecyl sulfate.

L47cksis muta Venom Kininogenin

300 EFFLUENT FIG. 1. f&L

249

400 VOLUME (ml1

FlLTRATlON CHROMKTOGMPHY OF ‘toUlg OF CRUDE LrrchcSiStflUt0 VENOM OVER #~HMXWL S-200 COLUMN (1.8 x 180 cm).

Elution was performed with 50 mM NaCl at room temperature at a flow rate of 20 ml/hr. The collected fractions (6.1 ml each) were assayed for kininogenin activity using bovine low mol. wt kininogen as substrate. and their protein contents wxxe estimated by A,, measurements. The horizontal bar indicates the pooled active fractions which were dialy-scd against water and lyophilized.

6

Ro. 2. FLATBELI -C3

24

PAllRUN OF THE FTIAClTONSDISPIAYINO G-75 COLUYN.

30

KININWENIN

ACIIVITY

REalvEuEDFRDMTHEsEPHADa

run was carried out at 4°C for 18 hr. atIer which the supporting gel was transversely sectioned into 31 fractions whose contents were transferred to plastic tutxa and suspended in 2.0 ml of distilkd water. Each individual fraction was characterized by its protein content (Am_,) and its kininogenin activity using bovine low mol. wt kininogen as substrate, as indicated by the vertical bars. The pH gradient was plotted based on pH measurements taken from the isolated fractions.

The

M. R. V. DING! and E. B. OLIVEIRA

250

kininogenin activity of each fraction was determined using low mol. wt kininogen as substrate, as described previously.

Prorein

&reminarioa

Rotein concentrations were determined from the absorbance at 28Onm measured in a Beckman DU-2 spcctrophotometer, using bovine serum albumin as a standard (a solution of 1 mgJml in Tris-bulked saline, pH 7.5, has an absorbance of 0.66).

SDS-palyacrykami& gel ekc~rophoresis

Electrophoresis under denaturing conditions was performed on an 8 to 18% linear gradient polyacrylamide gel slab using the discontinuous bulk system described by LAEUMLJ(1970). Mol. wt marker proteins WQC:bovine serum albumin (69,ooO), ovalblrmin (4S,ooO), aldolase monomer (39$X0), chymotrypsinogen A (25,OOO)and ribonuclease A (13,700).

Aminopep&se asray Aminopeptidase activity was assayui by incubating 2Onmoles of Lys-bradykinin with diluted enyme preparations in 200 4 of 0.02 M Tris-HCl buffer, pH 7.5. for 30-60 min at 37°C. After stopping the reaction by addition of 200 d of 0.2 N scdium citrate, pH 2.2, containing 15% glycerol, the released amino acids were measured by automated amino acid analysis.

De~errninatlon of

kineticpmmwers The steady-state pammews Km and Kcat were determined for the hydrolyses of the chromogenic suhotrates listed in Table 1, as described by SIL~EIRAet al. (1989). using a computer program called VMKM ~UIA, 1972).

Kinin identijication The identification of kinins was made by comparison of their retention timea obtained by HPLC analyses with

those of synthetic bradykinin, Lys-bradykinin and Met-Lys-bradykinin (10 nmole each). Digestion of bovine low mol. wt kininogen (40 a prepared in 0.24 ml of 0.01 M Tris-HCl buffer, pH 7.5) was performed with 0.1 units of pmi5ed kininogenin for 10 min at 37°C. After addition of 1.0 jd of formic acid, the digestion mixture was chromatographed on a @ondapak Cl8 column (Waters Asso~Gtes, 3.9 x 300 mm) developed under isccratic conditions (16% acetonitrik in 0.04 M formic acid, pH ajusted to 3.15 with triethylsmine) at a 500~ rate of 2.0 ml/min. Peptides were monitored by absorbance at 220 mn.

Blwd pressure measuremms

The effect of kininoganin injections in the rat blood pressure was meawedinanimalawithchronically implanted cannulas, so that experiments extending over a period of 2&36hr could be carried out without provoking any response common to painfid stimulation such as 5ight reaction, vocalization or autonomic ~.MaleW~tarrats.n=8(~250g)wertused.Thcdaybcfortthccxperimenttheratswcre an&be&d with ether. and polyethylene cannulas (PE 10. Clay-Adams, NJ, U.S.A.) were w into the femoral artery for direct blood pressure measurements and blood collection, and into the femoral vein for i.v. injection. During the experiments puri5ed enzyme (up to 100 units), synthetic bradykinin (l-5 jig) or captopril (0.20-0.25 mg) were dissolved in saline (0.1 to 0.2 ml) and iqjeckd i.v. as a sin* bolus through the implanted in rats with chronically implanted cammlas cannula. The detailed procedures for blood pmssure wts were demibed previously (SALMW eI al.. 1986). The experiments in conscious rats wxe performed in and guidelinal of the Anwrkan Physiological So&y. aaxmkuxwithtkethiwJstanda&

RESULTS

Enzyme purification The purifkation of an acidic kininogenin from L. muta venom was achieved by a combination of gel filtration, isoelectric focwing and preparative PAGE (Table 2). The crude venom was fractionated on a Sephacryl S-200 column (Fig. l), leading to a

Loch&

mufa Venom Kininogcnin

TABLE1. KINJ?IX pmumms

RX

OF pEpTIDE

THE KININODENlN-CATALYSED

CHllOMmC

N-Bz-PbVal-Aq-pNA H-D-Val-Leu-WNA N-AC-F%e+kgqNA N-Bz--NA

?IYDIlOLYXCi

!WBTlXAlES

Km’ (M)

Substrate

251

Kcat+ (=-‘)

Kcat/Km (M-l.sec-l)

(1.70f0.17)~ lo-’ 3.26kO.28 1.92x l(r 1.55 x 104 (3.00f0.31) x IO-’ 4.64kO.46 (1.81f0.18)~ lo-’ 0.72kO.04 3.98& 102 No hydrolyxis detected

*Data are presented as means *S.E. (n = 4-5).

kininogenin preparation almost free of other previously described proteolytic enzymes of L. mufu venom (SILVAet ol., 1985). Further purification was carried out by gel filtration on Sephadex G-75 column, which resulted in the elution of a single protein peak (data not shown). The enzyme activity was recovered in a high yield from fractions corresponding to the first half of the peak. The flatbed electrofocusing purification step (Fig. 2) separated multiple forms of kininogenins, whose pls ranged from 4.5 to 8.0. This charge heterogeneity precluded pooling of all active fractions, since SDS-PAGE analysis of each individual active fraction revealed a variety of contaminant proteins. Therefore, an acidic kininogenin, pl 5.4, was recovered from fraction 6 (Fig. 2) and subjected to further purification. As a result of the exclusion of several forms of kininogenins from the purification schedule presented in Table 2, the recovery of enzyme activity in the electrofocusing purification step was only 12%. The final purification procedure took advantage of the high resolving power of preparative PAGE. Five protein bands were detected by Coomassie blue staining, and kininogenin activity was associated only with the two fastest migrating bands. The protein recovered from the second fastest migrating band (specific activity 600 units/mg) was used for subsequent biochemical characterization of the L. muta venom kininogenin. SDS-PAGE analysis and antigenicity Figure 3 shows the pattern of the SDS-PAGE analysis of the whole L. muta venom and of the active fractions obtained after each puriftcation step. It was observed that the purification scheme led to a progressive enrichment of a protein of mol. wt 29,700, which TNIIJ? 2. SUMbIUY

OF ~UIUFICATION

Proccdul-es Unfractionatcd venom Gel 6ltration on stphacryl~~ Gel 6ltration on Sephadex G-75 Prepafa~ve ektfoplgz&el clectrophorenis

OF AN ACIDIC

KININOGEMN

FROM THE VWOhi

Specific activity (mWW)

OF

L. m&Z

Total protein (W)

Total kininogenin activity (units)+

394.0

16.510

42

53.0

9900

187

59.9

28.4

9380

330

56.7

2.60

1080

418

6.5

0.58

348

600

2.1

+Mcasurcd wing bovine low mol. wt kininogenin as the substrate. tBaszd on the owxall recoycry of enzyme activity.

Yield? W) 100

252

M. R V. DINIZ and E. B. OLIVEIRA

Ro. 3. SDS-PAGE ANALYSIS op nim L. muta VEIWMAND FRACCIOX? DBPLAYlNC3

I;MINOOEMN

AcnvrrY.

Ekctrophorcsis was performed on an 848% linear gradknt polyacrylamide gel slab. In lane A wasrunamixtureofmol.wtprottinmarkcrs(SIrgeach)containing:(a)bovintsenunalb~ @) ovalbum& (c) chymotrypsinogen; (d) ribonuckaw A. The aampks loaded in lanes B to F correspond to kininogenin preparations of daream@ qxcitic activities, an deaxibcd in Table 2: lane (B) pqarative PAGE (12 &; (C) preparative isoekctric focusing (5 jig); (D) Sepbadex G-75 (25 crp); (E) Sephacryl !MtMJ(35 pg); (F) unfractionated venom (1SOpg). Protein bands were stained with Coomassie blue.

migrated as a homogeneous band after the preparative PAGE step. The homogenous kininogenin (pZ 5.4) showed only one precipitin line when assayed by double immunodiffusion with antiserum against the whole venom. Lines of identity were observed when the

253

L.achesis muta Venom Kininomn

5

IO

I5

20

TIME (mid mt0u L. mta

~.4.hNlTFXXKINOFlHEKINlN~BY~-

vmoM.

The

retention timen of synthetic bradykinin (BK). Lys-bradykinin (LBK) and Met-Lys-BK (M-LBK) obtained by HPLC analysis (upper chromatogram) were compared with that of the pzptide released by enzymatic digestion of bovine low mol. wt kininogen (lower chromatogram). The analyxa were performed on a +mdapak column (3.9 x 300 mm) developed under isocratic conditions (I 6% acetonitrile in 0.04 M formic acid, with pH adjusted to 3.15 with tricthylamine) at flow rate of 2.0 ml/min. Peptides were detected by absorbana at 220 nm.

purified enzyme was assayed beside the partially puSed obtained in Fig. 2 (data not shown).

kininogenins of various pls

mm Hg :EE

50 I

0

1

3 TIME

3.0

33

I 36

TIME(mnV

46

9’

i(rmnI

51 TIMEhnl

54

FlG.5.EFFEcrGF~mKINlNoGENlN UUECll0l-U ON RAT AlllERlAL BLOOD -. The purikd enzyme (70 units, specitk activity 330 units/n& was administered into the vein of a normotensive rat at the times indicated by E above the physiograph tracing. The responsiveness of the prepamtion towards bradykinin injection remakd essentially uochangal throughout the experiment: 5.OH of the synthetic peptide cad a short-lasting (g-15 set) pressure fall of approximately 50 mmHg. Blood samples (0.4 ml) were wit+awn prior to ir+ction of kininogenin and at the end of tk experiment for total khkogcn &termmation. Data shown in this figure are IeprcRntative of two CqMiments.

254

M. R. V. DINIZ and E. B. OLIVEIRA

Enzyme specificity The purifted kininogenin displayed amidase activity on peptide chromogenic substrates and bovine kininogen. Table 1 shows the catalytic efficiency of the enxyme for four N-terminal derivatives of the Arg-pNA moiety, indicating a marked effect of neighboring structures on the hydrolysis of the Arg-pNA amide linkage. Figure 4 shows a comparison of the HPLC elution pattern of three synthetic kinins with that observed when the product formed by the action of the kininogenin on the bovine low mol. wt kininogen was analysed under identical conditions, allowing for the identification of the kinin released by this enzyme. The kininogenin was devoid of aminopeptidase activity when assayed with Lys-bradykinin as substrate (data not shown).

Activity and stability under various conditions The kininogenin activity was measured at different pH values or in the presence of certain inhibitors. This enzyme was inactive at pH values below 4.5, while raising the pH from 5.0 to 7.0 gradually increased the activity (8% at pH 5.0,45% at pH 6.0 and 100% at pH 7.0). Further increase of the pH up to 9.0 had an opposite effect (85% at pH 8.0 and 70% at pH 9.0). Well-known competitive inhibitors for kallikreins, such as soy bean trypsin inhibitor (0.3%), aprotinin (200 units/ml) and benzamidine (3 x lo-’ M), had no effect upon the kininogenin activity when assayed at pH 7.0 at the concentrations indicated. The stability of the kininogenin (specific activity 330 units/mg, 1.0 mg/ml in 0.05 M Tris-HCl buffer, pH 7.0) was tested under various experimental conditions. Less than 2% of the activity was found tier phenylmethylsulfonyl fluoride treatment (2.0 mM, 60 min at 25°C). Incubation in the presence of guanidinium hydrochloride (6.0 M, 24 hr at room temperature) caused only 15% loss of kininogenin activity. Thermal stability measurements indicated that the enzyme is fully stable at 25”C, even after a 24hr incubation period, while it lost 50% of its activity following incubation periods of 70 min at 60°C and 17 min at 100°C.

In vivo action of the L. mum kininogenin The hypotensive effect of repeated i.v. injections of kininogenin on rat blood pressure is shown in Fig. 5. The amount of enxyme injected each time (70 units) was chosen as a compromise between those causing negligible effect (n = 2,30 units) and shock (n = 3,100 units). The first injection elicited an abrupt fall in blood pressure of about 75 mm Hg, followed by a period of blood pressure recovery lasting over 15 min. The second dose had a less pronounced hypotensive effect, while the third dose caused no noticeable change in arterial blood pressure. Captopril injection (i.v., 1.0 mg/kg), potentiated the hypotensive effect of the enzyme (data not shown), strongly suggesting the participation of bradykinin as the hypotensive factor. The kininogen contents of blood samples withdrawn prior to kininogenin injection and at the end of the experiment shown in Fig. 5 were determined according to the method of DINIZ and CARVALHO(1963). It was observed that repeated doses of enzyme depleted the rat of only 16% of its total circulating kininogen. Since the responsiveness of the rat towards bradykinin injection (5 pg) remained unchanged throughout the monitoring period, the induction of tachyphylaxis following repeated injections of enzyme was attributed to the depletion of a pool of kininogenin-sensitive kininogen from the rat circulation.

L.achesisntutoVenomKinino@n

255

DI!XXSSION

A homogeneous kininogenin preparation was obtained from the venom of L. muta by a combination of gel filtration, isoelectric focusing and preparative gel electrophoresis. This enzyme was a remarkably stable serine proteinase of mol. wt 29,700 and pZ 5.4, corresponding to one of the separable forms of kininogenin generated by preparative isoelectric focusing. Tbe observed charge heterogeneity of the kininogenin activity in the L. mura venom (Fig. 2) resembled that described for the homologous thrombin-like activity, whose polymorphism arose from a non-uniform distribution of sialic acid residues among the distinct molecular forms (Snvnrt~ et al., 1989). The spreading of the kininogenin activity over the pZ range of 4.5 to 8.0 (Fig. 2) greatly impaired the yield of purified enzyme (Table 2). The kininogenin from L. muta venom released bradykinin directly from bovine low mol. wt kininogen (Fig. 4), a feature shared with kininogenases found in venoms of other species (IWANAGA and SUZUKI, 1979), although both bradykinin and kallidin may be released from natural kinin precursors or synthetic kininogen analog (WEBSIER and PIERCE,1963; KOMORIand SUGIHARA, 1988). The kinetic parameters for the kininogenin-catalysed hydrolysis of peptide chromogenic substrates revealed that a productive enzyme-substrate interaction was markedly dependent on the peptide structure adjacent to the scissile bond of the substrate. No hydrolysis of the Arg-pNA bond was detected on the substrate Bz-Arg-pNA, whereas the values of catalytic efficiency (Kcat/Km) observed on the cleavage of the Arg-pNA bond of other substrates varied as much as 48-fold (Table 1). The kallikrein-like enzymes from Crorahs aahantew (MARKLAND et al., 1982) and from C. atrox (BJARNASON et al., 1983) were active upon the plasma kallikrein substrate H-D-Pro-Phe-Arg-pNA and upon the glandular kallikrein substrate H-oval-L.eu-Arg-pNA. These findings are additional evidence for the similarities between these enzymes and kallikreins, with which they were shown to share sequence homologies. However, the preferred substrate for the L. muta kininogenin, an enzyme devoid of thrombin-like activity (S~~EIRA et al., 1989), was Bz-Phe-Val-ArgpNA, a substrate designed for thrombin assay in human plasma. Indeed, some reports have indicated that specificities of proteolytic enzymes from different snake venoms toward synthetic chromogenic peptides do not necessarily reflect their action upon natural substrates (SILEIRA et al., 1989; TJZNGet al., 1989; OHTANIet al., 1988). The purified kininogenin from L. muta venom displayed a specific activity of 600 units/mg (Table 2). A survey of the specific activities ascribed to purified kinin-releasing enzymes from various snake venoms demonstrated values ranging from 0.42 units/mg for that from Vipera asper asper (KOMORIand SUGIHARA,1988) to 58 units/mg for that from Agkisfroaim cafiginosus (OHTANIet al., 1988). Factors of various kinds may contribute to such a wide range of values, namely, lack of standard method of assay for these enzymes, evolutionary adaptation generating functionally related proteins of different specificities and intrinsic activities, and strategies employed in enzyme purification. With respect to this latter factor it should be stressed that we monitored the enzyme activity using kininogen as a substrate throughout the purification steps (Table 2), a procedure seldom used (Saro et al., 1965; COHENet al., 1970). The majority of the kinin-releasing enzymes have been purified as arginyl ester hydrolases and subsequently shown to form kinin on incubation with kininogen. In the case of the L. muta venom its two major arginyl ester hydrolases are devoid of kinin-releasing activity (SILVAet al., 1985). Notwithstanding, the purified L. muta kininogenin was active towards N-a-benzoyl-t_-arginine ethyl ester and

256

M. R. V. DINIZ

and E. B. OLIVEIRA

N-a-tosyl+arginine methyl ester (data not shown), a feature common to other purified snake venom kininogenases (ROTHSCHILDand ROTIISCHILD,1979). The observed hypotensive effect of i.v. administration of purified kininogenin in rats (Fig. 5) was consistent with the in vitro bradykinin-releasing activity of the enzyme using bovine kininogen as the substrate (Fig. 4). However, a rather high dose (70 units) was required to provoke an intense, long-lasting hypotension. A similar result was obtained with as little as 0.042 units of purified kallikrein-like enzyme from Vipera arpb aspis venom (KOMORIand SUGIHARA,1988), an enzyme reportedly insensitive to rat plasma inhibitors. To account for the relatively low in viva activity of the L. mutu venom kininogenin, we suggest that it might be strongly inhibited by rat plasma substances such as a,-macroglobulin, a,-antitrypsin or other inhibitors. The mechanism of the hypotensive effect of i.v. administration of purified L. mutu kininogenin in rats was investigated (Fig. 5). We observed that repeated doses of enzyme induced tachyphylaxis, a result resembling the temporary desensitization of the hypotensive effect of crude rattlesnake venom injection in experimental animals (E&SEXand MARKOWITZ, 1930). The tachyphylactic effect induced by purified L. muta kininogenin was accompanied by a decrease of about 15% in the total circulating kininogen concentration, suggesting that the action of the enzyme was restricted to some particular type of rat kininogen whose consumption caused tachyphylaxis (Fig. 5). There have been no previous reports of in vivo action of purified snake venom hypotensive factors regarding their abilities in affecting kininogen stores. It has been shown, however, that a complete kinin precursor deficiency can be induced by administration of some crude crotalid venoms in experimental animals (MARGoLrs et al., 1965; ROTHSCHILD and ALMEIDA, 1972). On account of this property, crude snake venoms have been suggested as useful experimental tools for demonstrating possible roles of kinins as mediators of various physiopathological reactions (MARGOLIS et al., 1965). However, the description of other venom factors capable of altering the cardiovascular system by different mechanisms (B~NILLA and RAMMEL,1976; HUANG and LEE, 1984; CEVESEet al., 1984; KOMORIand SUGIHARA,1990) may impose some difficulties in interpreting results obtained with crude venoms used as experimental tools. purified venom factors, such as the L. mutu venom kininogenin described in this work, can be used to advantage as an in vivo biological tool owing to their specificities. Although potent bradykinin antagonists are now available for studying the physiological role of the kinin-kininogen system (WIRTH et al., 1991), specific enzymes may still be useful in the investigation of how different kinin precursors participate in blood pressure regulation and other biological processes. Acknow/&~t+We thank Drs M. -GUM and M. C. 0. SALGADOfor theirhelp on the kinetic analynis and pharmacological assays, rcqxxtively. This work was supported in part by Consclho National de Desenvolvimcnta Ciitiiico c Tamol6gico (CNFQ). Taken in part from the thesis submitted by M.R.V.D. to the Uniwxsidadc Federal de Minaa Geraie in partial fu&lment of tlx rcqdrcmcnts for the M.Sc. degree.

REFERENCES BAIUW. G. S. and SHIPOLINI, R. A. (1976) F’urifbtion and properticsof a kininogcnin from the venom of Qoera amnu+tes ammodytes. B&hem. J. 153,409+14. enqmes ~ARNASON. J. B., BARBH, A., DIRENZQ G. S., CAYPBEU, R. and Fox, J. W. (1983) --like from Crolalns atrox venom. J. biol. Chem. 238, 12,%6-12.573. BONILU, C. A. and REPEL, 0. J. (1976) Comparative biochemistry and phmacology of salivary ghnd

Mti

muI0 Venom Kininogcnin

257

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