Trypanosoma cruzi: Isolation and characterization of a proteinase

EXPERIMENTAL

PARASITOLOGY

Trypanosoma H.A. Departamento

cruzi:

52, 199-209(1981)

Isolation

RANGEL, P.M.F.

and Characterization

of a Proteinase

ARA~~JO,D. REPKA, AND M.G.

de Microbiologia e Imunologia, lnstituto de Biologia (UNICAMP), C.P. 1170, Campinas, 13.100,

da Universidade Sdo Paula, Brazil

COSTA Estadual

de Campinas

(Accepted for publication 27 October 1980) ARA~JO, P.M.F., REPKA, D., AND COSTA, M. G. 1981. Trypanosoma and characterization of a proteinase. Experimental Parasitology 52, 199-209. Lysates of Trypanosoma cruzi epimastigotes were able to hydrolyze casein (K, = 2.5 mg/ml) as well as bovine and human hemoglobins (K, = 12.2 mg/ml); there was optimum activity was around pH 7.0. The proteinase activity detected with these substrates was enhanced by sodium diaminotetraacetate (EDTA) and reducing agents (SO:-, mercaptoethanol, cysteine) and was inhibited by sulthydryl reagents, thus suggesting an SH-dependent enzyme. Purification (60x) of the proteinase was carried out as follows: (1) precipitation at -20 C, pH 4.5, with 80% acetone, (2) gel fdtration on Sephadex G-200, (3) affinity chromatography on Sepharose 4B covalently linked to p-aminophenyl mercuric acetate. Only a single component (with an estimated molecular weight of 60,000) was detected in purified preparations by polyacrylamide gel electrophoresis. However, in addition to the major component identified as a proteinase, crossed immunoelectrophoresis experiments indicated the presence of at least three other antigens that apparently were devoid of proteinase activity. Optimum pH activity of the purified preparations was around pH 6.0 for casein and pH 3.0 for hemoglobins, but these activities probably are due to the one enzyme since they were altered identically by the same agents. INDEX DESCRIPTORS: Trypanosoma cruzi; Hemoflagellate; Protozoa, parasitic; Cultivation; Protease; Proteinase. RANGEL, H.A., cruzi: Isolation

The existence of proteases in Trypanosoma cruzi is suggested by the fact that this organism, which has a complex cycle, needs protein components for sustained growth and differentiation (O’Daly 1975, 1976). Moreover, proteolytic enzymes play important roles in the metabolism of microorganisms (Holzer et al. 1975) and it is likely that no organism is devoid of proteases. Our knowledge of T. cruzi proteases, however, is limited. Repka et al. (1972) showed that a crude extract of T. cruzi epimastigotes splits hemoglobin both at pH 3.0 and at pH 7.0. By using azocasein and some synthetic substrates, Itow and Camargo (1977) have shown that these extracts contain at least three different proteases. Data presented by Range1 et al.

(1976, 1977) indicated that the hydrolysis of the synthetic substrate tested could be inhibited by some esterase inhibitors which had no action on the hydrolysis of casein or of hemoglobin at neutral pH. Recently, Bongertz and Hungerer (1978) isolated and characterized a protease which showed esterase and transamidase activities. In this work, undertaken to study the enzyme which splits hemoglobin and casein at neutral pH, we show that isolation of the enzyme can be achieved by a three-step procedure and that the isolated enzyme presents different pH optima for casein and hemoglobin. MATERIAL

AND

METHODS

Soluble Fraction of the Parasite

The soluble

fraction

soma crud was obtained

(FS) of Trypanoas follows: para-

199 0014-4894/81/050199-11$02.00/O Copyright All rights

0 1981 by Academic Press, Inc. of reproduction in any form reserved.

200

RANGEL

sites of the Y strain (Silva and Nussenweig 1953) grown in liver infusion tryptose (LIT) medium (Fernandes and Castelani 1966) for 7 days at 28 C were washed three times with ice-cold 0.15 M NaCl. Washed parasites (90-95% epimastigote forms) were suspended in distilled water (5.6 x lo7 parasites/ml) and lyophilized. Portions of 0.5 g of the lyophilized material were suspended in 50 ml of ice-cold 0.15 M NaCl after it had been extracted twice with 50-ml aliquots of acetone, once with a 1.1 mixture of acetone and ether, and then three times with ether. Suspensions were kept at 0 C for 10 min and centrifuged at 302Og for 30 min at 5 C. The clear supernatant was kept at -20 C until used. Proteins

Casein, ribonuclease (RNase), and ovalbumin (OV) were obtained from Sigma Chemical Company; crystallized bovine serum albumin (BSA) was obtained from Pentex Biochemicals. Bovine and human hemoglobin, prepared from erythrocytes, were washed three times in 0.15 M NaCl. Packed erythrocytes (100 ml) were dialyzed for 24 hr against two changes, 10: 1 each, of distilled water. After centrifugation at 12,lOOg for 1 hr, the supernatant was lyophilized. Equine serum albumin (ESA), crystallized five times, was prepared according to the method of Adair and Robinson (1930). Human serum albumin (HSA) was obtained from pooled normal human sera by fractionation with ammonium sulfate followed by chromatography on CM-cellulose (0.7 meq/g) equilibrated with 0.05 @4 acetate buffer at pH 5.0. Human and rabbit IgG were prepared according to the directions of Kabat and Mayer (1961). Plasma proteins prepared in our laboratory were shown to behave as single precipitation systems when analyzed (with specific sheep or rabbit antisera to the

ET

AL.

whole serum) in crossed trophoresis tests. Immune

immunoelec-

Sera

Precipitating antibodies were obtained from rabbits immunized with either FS or its purified fractions by injecting a single dose of antigen (0.2 mg protein) incorporated in Freund’s complete adjuvant. Animals were bled 30 days after injection, and the sera were kept at -20 C after being heated at 56 C for 45 min. Protease

Assays

Protease assays were carried out by using a modification of Anson’s (1938) method and, unless otherwise stated, the proteolytic activity was tested by mixing O.l-ml aliquots of enzyme (properly diluted in 50 mM buffer) with 1.O ml of substrate (bovine hemoglobin 40 mg/ml or casein 6 mg/ml) dissolved in the same buffer. Controls for the assay contained only enzyme or substrate. After incubation of the mixtures at 37 C for 2 hr, 1 ml of 5% trichloroacetic acid (TCA) was added to every tube. Substrate or enzyme was then added to control tubes and a blank was prepared by mixing reagents in the following order: 1 ml TCA, 0.1 ml enzyme, and 1.0 ml substrate. Mixtures were then incubated at 45 C, for 15 min and centrifuged at 1085g for 30 min. Absorbance of supernatants was determined at 280 nm (l-cm square quartz cuvettes). Since the readings of substrate controls did not differ significantly from blanks, enzymatic activity was expressed as the difference in absorbance at 280 nm (AAZs,, .,) between control tubes of enzyme and the reaction mixture. To test effects on proteinase activity, equal-volume mixtures of the enzyme and effector solutions were incubated for 30 min at room temperature, then assayed ‘for proteinase activity either in the absence or presence of 15 mM 2-mercaptoethanol (2-ME) which previously had been added to

Trypanosoma

cruzi: PROTEINASE

the substrate solution. Relative activity was expressed as the ratio of proteolytic activity of the mixture to that of enzyme diluted to 50% with buffer (50 U/ml) assayed in the absence of 2-ME. Esterase activities in solution was tested by the method of Walsh and Wilcox (1970), and in gel plates by that of Uriel (1971). Enzyme Purification Precipitation with acetone. One liter of FS, kept at 0 C, was brought to pH 4.5 by the addition of 0.1 N HCl. The precipitate formed was discarded by centrifugation (302Og, 15 min) and the supernatant transferred to a refrigerated bath kept at -20 C. Four liters of acetone were then added slowly with constant stirring, care being taken that the temperature did not exceed -5 C. The mixture was kept at -20 C for 18 hr and centrifuged for 15 min at 302Og at - 15 C. The precipitate was suspended in 100 ml of cold 0.15 M NaCl and dialyzed against 5 liters of this diluent at 0 C for 15 hr. The remaining precipitate was removed by centrifugation and the supernatant containing 66% of the original proteinase activity was either lypophilized or kept at -20 C until used. Gel filtration chromatography. Proteinase samples (100 mg protein in 5-ml aliquots) were analyzed by gel exclusion chromatography on Sephadex G-200 columns (95 x 2 cm) equilibrated with 50 mM acetate buffer, pH 5.0, as described by Flodin (1962). Elution was carried out at 0 C with the same buffer at a rate of 10 ml/hr and by collecting 2-ml fractions. Reference standards of Dextran Blue, rabbit IgG, BSA, and OV were filtered under similar conditions on the same column before and after filtering the enzyme samples, in order to determine elution volumes. Affinity chromatography. Sepharose 4B, covalently linked to p-aminophenyl mercuric acetate (p-APMA-Sepharose), prepared according to the method of Sluyter-

201

man and Wijdenes (1970), reacted with 1.1 mM DTNB/ml matrix. The enzyme fraction obtained from the Sephadex filtration previously had been dialyzed overnight (against 50 mM acetate buffer, pH 5.0, containing 10 mM sodium sulfite, 0.1 A4 KCl, 1 mM EDTA, and 10% dimethylsulfoxide (DMSO)) and was applied to columns (5 x 1 cm) of the absorbent previously equilibrated with the same buffer. Elution was accomplished by using the same buffer at 20 ml/hr and by collecting I-ml fractions until the Azso nm reading was 50.010. The absorbed material was eluted by using 50 mM acetate buffer, pH 5.0, containing 15 mM 2-mercaptoethanol. Disc gel electrophoresis. Polyacrylamide (7%) gel columns (10 X 0.5 cm) were prepared in 50 mM TRIS-HCI buffer, pH 7.5, as described by Fairbanks et al. (1971). Electrophoresis was done on duplicate sets of columns (3 MA/tube) until the pyronin marker reached 1 cm from the tube bottom. After electrophoresis, one of the duplicate columns was stained for proteins with Coomassie brilliant blue and the other was used for localization of the enzymatic activity. This was achieved by immersing columns for 2 hr at 37 C in 0.15% casein dissolved in phosphate buffer, pH 7.0, containing 5mM EDTA and 15 mM 2-ME. Gels were then suspended by pins on corks and incubated at 37 C for 4 hr in a moist atmosphere, after which they were fixed and stained with Coomassie brilliant blue in the usual way. Molecular Weight Estimation ESA (69,000), OV (45,000) and RNase (13,700) were used as standards for the estimation of the molecular weight of the purified proteinase on SDS-5.6% polyacrylamide gels by the procedure of Fairbanks et al. (1971). Immunodiffusion Tests Double-diffusion tests were performed by the method of Ouchterlony (1958). Crossed

202

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immunoelectrophoresis experiments (Week 1973) were conducted at 5 C under the following conditions. Plates (12 x 9 cm) contained 1% agarose gel in 50 mM Verona1 buffer pH 8.6; the applied current was 6 V/cm for 1 hr for the first dimension and 2 V/cm for 18 hr for the second dimension; immune serum was diluted 1: 10 into gels. The detection of proteolytic activity after crossed immunoelectrophoresis was performed similarly to the procedures of Uriel (1971) for disc gel electrophoresis. Protein arid Polysaccharide Determinations

The methods of Lowry et al. (1951) and of Dubois et al. (1956) were used for protein and polysaccharide determinations, respectively . Reagents and Other Materials

The following reagents were obtained from commercial sources: 2-mercaptoethanal, cysteine, dithiotreitol, sodium ethylenediaminotetraacetate (EDTA), diisopropyl fluorophosphate (DFP), p-tosyllysine chloromethyl ketone (TLCK), p-tosylphenylalanine chloromethyl ketone (TPCK), p-aminophenyl mercuric chloride (p-APMC), p-toluenesufonyl-L-arginine methyl ester (TAME), N-benzoyl-DL-arginine-2-naphthylamide (HCl (BANA), N-acetyl-DLphenylalanine P-naphthyl ester (AFNE), N-carbobenzoxyltyrosine p-nitrophenyl ester (CNT), N-benzoyl-L-arginine ethyl ester HCl (BAEE), 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), N-ethylmaleimide (N-EMI), fast garnet GBC salt, CM-cellulose, and DEAE-cellulose from the Sigma Chemical Company; acrylamide; bis-acrylamide, and TEMED from Canalco, Rockville, Maryland, U.S.A., sodium dodecyl sulfate (SDS), tris(hydroxymethyl)aminomethane (TRIS), and dimethylsulfoxide (DMSO), from Fisher Scientific Laboratories, Springfield, New Jersey, U.S.A.; ammonium persulfate, agarose, and Coomassie brilliant blue from Bio-Rad Laborato-

ETAL.

ries, Richmond, California, U.S.A.; Sephadex G-200, Sepharose 4B, and Blue Dextran from Pharmacia, Uppsala, Sweden. The p-aminophenyl mercuric acetate (pAPMA) (mp 166-167 C) was prepared in our laboratory (according to the method of Dimroth (1902). RESULTS

As reported earlier (Range1 et al. 1977) the soluble fraction (FS) of the Trypanosoma cruzi epimastigote was shown at pH 7.0 to hydrolyze the following compounds: N-benzoyl-DL-arginine 2-naphthylamide (BANA), N-carbobenzoyltyrosine p-nitrophenyl ester (CTN), and N-acetyl-DL-phenylalanine P-naphthyl ester (AFNE). The FS showed a zone of optimum proteolitic activity in the range of pH 6.6-7.4 when tested with either casein (6 mg/ml) or hemoglobin (40 mg/ml) dissolved in 50 mM phosphate solution, with pH varying between 6.0 and 9.5 (Fig. 1). Tests in the acidic pH range performed with hemoglobin (dissolved in 50 mM citrate-phosphate buffer) showed a second zone of optimum activity around pH 3.0. This second zone was not investigated because casein is insoluble in that pH range. The proteinase activity tested at pH 7.0 could also be detected by using the different proteins indicated in Table I. As can be seen, the highest activities were observed with casein and bovine hemoglobin. Kinetic determinations, using either casein or hemoglobin indicated that no further hydrolysis was observed after 90 min incubation at 37 to 45 C (Fig. 2). A Lineweaver-Burke plot of the results obtained at pH 7.0, 37 C with varying concentrations of substrate indicated a K, of 2.5 mg/ ml for casein (Fig. 3) and a K, of 12.2 mg/ ml for both human and bovine hemoglobins. The proteinase activity, tested with either casein or bovine hemoglobin, was enhanced by EDTA as well as by SH compounds and was inhibited by iodoacetamide, iodoacetate, N-ethylmaleimide, so-

Trypanosoma Hemoglobin

cruzi: PROTEINASE

203

-O-~--O-

Casein

$1,;

I. 5.0

.,.I.,,7 10.0 PH

FIG. 1. F’roteolitic activity of FS (the cruzi soluble fraction; 0.5 mg protein/ml) ying pH with either casein (6 mg/ml) in phate buffer or with bovine hemoglobin 50 rnM citrate-phosphate buffer.

Trvpanosoma tested at var50 mM phos(40 mg/ml) in

Y. 0

100 TIME

dium tetrathionate, and mercuric ions (Table II). However, except when iodoacetate was used, the inhibition could be reversed by adding 15 n-N 2-ME. Pepstatin, esterase inhibitors (DFP, TLCK, TPCK), and heavy metal ions (Ca2+, Mg2+, Pb*+) had no apparent action. A linear relationship was observed between proteolytic activity and the amount of FS using the following conditions: casein

(min.)

FIG. 2. Influence of time and temperature on the proteolytic activity of the Trypan~~~ma cruzi soluble fraction (FS) (0.5 mg protein/ml). Casein (6 mgiml) was used as substrate.

(6 mg/ml) dissolved in 50 mM phosphate buffer, pH 7.0, containing 5 mM EDTA and 15 rrJ4 2-ME, and incubated at 37 C for 2 hr. For routine assays a provisional unit was defined as the amount of proteinase able to give a AA = O.OOl/min, under the above conditions.

TABLE1 Eroteolytic Activity (AZso .,) of the Soluble Fraction (FS) of Trypanosoma cruzi, Tested on Different Protein Substrates

-

Substrate Casein Bovine Bovine Human Equine

from milk hemoglobin albumin albumin albumin

Concentration bxdml) 20 40 30 30 30

FS” (mg protein/ml) 0.8

0.4

0.2

0.1

1.07 0.81 0.15 0.12 0.08

0.49 0.48 0.08 0.07 0.02

0.16 0.34 0.03 0.04 0.00

0.07 0.22 0.02 0.02 0.00

n Results expressed as the average AA,,, “,,, of three determinations. minations was not greater than 10% of the average.

The standard deviation of these deter-

204

RANGEL

ET

AL.

Isolation of the Proteinase

Proteinase was purified by the following steps. Some data obtained during each step is summarized in Table III. (a) Precipitation

3 (mg/ml)-’

FIG. 3. Lineweaver-Burke plot of reaction velocity @A Zlo,,m/min)of Trypan~~~ma crazi proteinase. Tests performed with casein in 50 mh4 phosphate buffer, pH 7.0, at 37 C for 2 hr.

with 80% acetone at

-20 C. The fraction obtained after this purification step, as well as the crude extract, when tested with 50 mM phosphate buffer, pH 7.0, was able to hydrolyze casein (200 U/mg protein) or hemoglobin, BANA, AFNE, and CTN. However, the action on these synthetic substrates could be inhibited by 1 mM concentrations of esterase inhibitors (DFP, TPCK, and TLCK) which had no action on proteinase activity. (b) Sephadex G-200 chromatography. A

TABLE II Effect of Activators and Inhibitors on the Trypanosoma cruzi Proteinase Activity of the Soluble Fraction (FS)

Effector None Ca*+ Mg2+

PbZ+ EDTA

2-Mercaptoethanol

Sodium sulfite Cystein Hg2+ p-Chloromercuribenzoate p-Aminophenyl mercuric acetate N-Ethylmaleimide Sodium tetrathionate Iodoacetamide Iodoacetic acid Diisopropyl fluorophosphate Tosyllysil chloramethyl ketone (TLCK) Tosylphenyl chloromethyl ketone (TPCK) Pepstatin

Final concentration wf) 1 I

1 0.2 1 5 1 5 20 50 20 15 1 1 1 1 1 IO I I 1 1 1 0.2 1 5

Relative activity Absence of 2-ME

Presence of 15 mM 2-ME

1.0 1.0 1.0 1.0 1.6 2.4 3.0 2.3 3.0 3.0 3.0 3.0 3.0 0.0 0.0 0.0 0.0 0.8 0.2 0.2 0.0 1.0 0.7 1.0 1.0 1.0 1.0

3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 1.5 3.0 3.0 3.0 3.0 3.0 2.5 0.0 3.0 3.0 3.0 3.0 3.0 3.0

Trypanosoma

cruzi:

PROTEINASE

TABLE III Methods for the Purification of the Trypanosoma

Fraction Initial FS 80% Acetone precipitate F3 of Sephadex chromatography F3-2 of affinity chromatography

Volume (ml)

Total units

Specific activity (units/mg)

cruzi

205 F’roteinase Recovery (%I

Fkification (-fold)

1000 50

30,000 20,000

40 200

100 66

1 5

50

12,000

800

20

40

20

4,800

2400

16

60

pattern similar to that presented in Fig. 4 resulted when the fraction obtained in the previous step was filtered in Sephadex G-200 columns. As can be seen in that figure, separate assay of collected fractions with BANA, casein, and AFNE, showed that the peaks of these enzymic activities were eluted at different volumes. The fractions (pooled as indicated in that figure) displayed different properties. Fractions 1, 4, and 5 were devoid of proteinase activity. Fraction 2 was able to hydrolyze casein (350 U/mg protein) and BANA. Fraction 3 hydrolyzed casein (800 U/mg protein) or CTN and AFNE but was unable to hydrolyze BANA. (c) Affinity chromatography. Filtration of F3 through p-APMA-Sepharose columns gave two fractions. The first (F3-I), not retained by the column, hydrolyzed

FIG. 4. Sephadex G-200 chromatographic pattern of the Ttypanosoma cruzi soluble fraction (FS) purified by acetone precipitation.

both casein (500 U/mg protein) and AFNE. The second (F3-2), obtained by eluting the absorbed material with 15 n-&f 2-mercaptoethanol, was able to hydrolyze casein (2.400 U/mg protein) but was unable to hydrolyze AFNE, CTN, or BANA. Properties of Puri’ed

Enzyme

The purified enzyme preparations (5 mg protein/ml) contained less than 10 mg carbohydrate per gram of protein. Such preparations (F3-2), analyzed by disc gel electrophoresis, exhibited a single protein band which had proteinase activity (Figs. 5C and D). Double-diffusion tests, with varying concentrations of F3-2 and polyspecific anti-FS sera, showed only a single precipitation line that had proteinase activity. Crossed immunoelectrophoresis experiments using these sera indicated the presence of a major precipitation system which exhibited proteinase activity, and three other faint precipitation bands in which no proteolytic activity was detected (Figs. 5A and B). No hydrolytic activity toward BANA or AFNE could be detected on these plates. Similar experiments, performed by using immune sera specific for purified F3-2, detected three different precipitating systems in both crude FS and purified enzyme preparations. Also, in this case, no BANA or AFNE hydrolytic activities were detected. SDS-polyacrylamide gel electrophore-

206

RANGEL

ET AL.

by the intensity of staining, increased while the concentration of the first one decreased with increasing time of incubation with dithiotreitol. The pH optimum activity of purified proteinase was found to be around 6.0 when casein was tested. When bovine hemoglobin was used as substrate the optimum was situated around pH 3.0 (Fig. 6). The results presented in Table II were obtained when the action of inhibitors and activators on purified enzyme was tested by using both casein (6 mg/ml) in 50 rnM phosphate, pH 6.6., and hemoglobin (40 mg/ml) dissolved either in pH 3.0 or in pH 6.0, 50 mA4 citrate-phosphate buffer as substrate. DISCUSSION

Our data show that crude extracts of Trypanosoma cruzi culture forms contain an endopeptidase. This endopeptidase probably is SH-dependent. Indeed, the activity of this enzyme, whether in crude extracts or in

FIG. 5. (A) Crossed immunoelectrophoresis of the Trypanosoma cruzi soluble fraction (FS) and of the purified enzyme (F3-2); (B) detection of caseinolytic activity of FS and of F3-2 after crossed immunoelectrophoresis (anti-FS in dilution 1: 10 was used in both plates A and B); (C) polyacrylamide gel electrophoresis of the purified enzyme (F3-2) (0.5 mg protein/ ml); (D) localization of caseinolytic activity of the purified enzyme (F3-2) after polyacrylamide gel electrophoresis.

sis, performed in the absence of dithiotreitol, detected only a single band corresponding to an estimated molecular weight of 60,000. When the sample of F3-2 was previously incubated at 37 C for 30 min with dithiotreitol, two bands were apparent: a strongly stained one (corresponding to 60,000) which had proteinase activity and a faintly stained one (corresponding to 6000) which was devoid of proteinase activity. The concentration of the latter, estimated

FIG. 6. Proteolytic activity of Trypanosoma cruzi purified enzyme (0.2 mg protein/ml) tested at varying pH with either casein (6 mg/ml) in 50 mM phosphate buffer or bovine hemoglobin (40 mg/ml) in 50 mM citrate-phosphate buffer. Substrates solutions contained EDTA (5 mM) and 2-mercaptoethanol(15 m&f).

Trypanosoma

cruzi: PROTEINASE

purified preparations, was enhanced by thiol compounds and was inhibited by reagents that attack SH groups. This inhibition (except for that induced by iodoacetate, which usually binds covalently to sulfhydryl groups (Means and Feeney 1971)) could be reversed by the action of 2-ME; this was expected for an SH-dependent enzyme. Moreover, the enzyme could be isolated by using a mercurial Sepharose adsorbent which reacts preferentially with SH groups from enzyme active sites (Sluyterman and Wijdenes 1970). The isolated enzyme was nearly homogenous by several criteria. Disc gel electrophoresis experiments, which are able to detect proteins at concentrations of less than 50 pg (Fairbanks et al. 1971), detected only a single component in purified enzyme preparations (5 mg protein/ml). This permitted an estimate of less than 5% for the concentration of probable contaminants. In agreement with this interpretation, Ouchterlony tests with plurispecific a-FS sera detected only a single precipitation line and that line had proteinase activity. Crossed immunoelectrophoresis experiments, employing the same plurispecific sera, and purified enzyme preparations, always detected a major precipitating system which had proteinase activity. In addition to this system, three faint precipitation lines could be observed but these lines were devoid of proteinase activity. Thus, there were indications of the presence of at least three contaminants. Similar observations were made by using the sera of rabbits immunized with purified preparations. However, since this method can detect very low antigen concentrations (1 pg/ml) and these contaminants were not detected by other sensitive methods, we conclude that the isolated enzyme is at least 95% pure. Additional evidence supporting this view was adduced by SDS-polyacrylamide gel electrophoresis. When these experiments were performed in the absence of dithiotreitol only a single component with a mo-

207

lecular weight 60,000 was detected. In spite of the fact that molecular weight determinations by this method are not reliable unless the molecule is fully reduced (Fairbanks et al. 1971), it can be assumed that the molecular weight of native proteinase is really close to 60,000. This estimate has been confirmed by Sephadex G-200 gel filtration of partially purified enzyme preparations and by SDS-polyacrylamide gel electrophoresis of purified enzyme, previously reduced with dithiotreitol. In the latter instance, a low-molecular-weight component was also evident. This component apparently results from autodigestion of the proteinase molecule since its concentrations in purified preparations increased while that of proteinase decreased during incubation under conditions that favored proteinase action. Our overall data are consistent with the view that, at minimum, purified enzyme preparations consist 95% of proteinase molecules. However, the data on optimum pH activity introduce some element of doubt. When casein was used as substrate, the optimum activity was found to be around pH 7.0 when crude preparations were tested and around pH 6.0 when purified preparations were tested. This difference can probably be ascribed to the action of other proteases in T. cruzi extracts (Itow and Camargo 1977). However, the data obtained by using hemoglobin as substrate hardly fit this explanation. In this case, although high levels of proteolytic activity could be observed in the vicinity of pH 7.0, the optimum activity was found around pH 3.0. Experiments performed using T. crud crude extracts and hemoglobin could be interpreted by assuming the existence of at least two proteinases: one neutral and one acid. However, such an interpretation for data on purified proteinase can hardly be sustained; a single enzyme only was detected by different criteria. Moreover, the same inhibitors and activators that were effective at pH 7.0

RANGEL

208

when crude preparations were tested with either casein or hemoglobin were equally effective vis-a-vis purified enzyme (either at pH 7.0 with either substrate or at pH 3.0 with hemoglobin). To maintain the assumption that there are two Trypanmmna cruzi proteinases would also require the assumption that both enzymes have almost identical physicochemical and immunologic properties. A more consistent explanation for the present findings is the assumption that the optimum pH activity of the proteinase is strictly dependent upon the physicochemical state of the substrate. This has already been shown to occur with other enzymes (Walsh and Wilcox 1970). As a matter of fact, casein is decreasingly less soluble below pH 6.0, rendering activity determinations in acid pH range difficult. ACKNOWLEDGMENTS This work was supported by funds from the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), Coordenadoria de Aperfeicoamento do Pessoal de Ensino Superior (CAPES), and Conselho National de Pesquisa (CNPq). We are indebted to Professors R. E. Bruns and J. Miller, Instituto de Quimica, UNICAMP, for grammatical revision. REFERENCES ADAIR, G. S., AND ROBINSON, M. E. 1930. The specific refraction increments of serum albumin and serum globin. Bioch~mica/ Journal 24, 933- 1011. ANSON, M. L. 1938. The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. J. Gen. Physiol.

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195-202. FLODIN, P. 1962. “Dextran Gels and Their Applications in Gel Filtration,” 3rd ed. Phamacia, Uppsala. HOLZER, H., BETZ, H., AND EBNER, E. 1975. Intracellular proteinase in microorganisms. In (B. L. Horecker and E. R. Stadtman, eds.), “Current Topics in Cellular Regulation” Vol. 9, pp. 103- 156. Adacemic Press, New York. ITOW, S., AND CAMARGO, E. P. 1977. Proteolytic activities in cell extracts of Trypanosoma cruzi. Journal of Protozoology, 24, 591-595. KABAT, E. A., AND MAYER, M. M. 1961. “Experimental Immunochemistry,” 2nd ed. Charles C. Thomas, Springfield, Ill. LINEWEAVER, H., AND BURKE, D. 1934. The determination of the enzyme dissociation constants. Journal of the American

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MEANS, G. E., AND FEENEY, R. E. 1971. “Chemical Modification of Proteins.” Holden-Day, San Francisco. O’DALY, J. 1975. A new liquid medium for Trypanosoma

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O’DALY, J. 1976. Effect of fetal calf serum fractions and proteins on division and transformation of Trypanosoma

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