Isolation and Characterization of a Cysteine Protease from the Latex of Araujia hortorum Fruits

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INTRODUCTION
Five catalytic types of proteases can now be recognized, in which serine, threonine, cysteine, aspartic, or metallo groups play primary roles in enzyme catalysis.The serine, threonine, and cysteine proteases are catalytically very different from the aspartic and metalloproteases in that the nucleophile of the catalytic site is part of an amino acid, whereas it is an activated water molecule in the other two groups.In cysteine proteases the nucleophile is the sulfhydril group of a Cys residue and the catalytic mechanism is similar to that of serinetype proteases in that the proton donor is a His residue.Although there is evidence in some cysteine peptidases that a third residue is required to orient the imidazolium ring of the His, there are a number of families in which only a catalytic dyad is necessary.Forty-one families of cysteine peptidases are recognized (a family is a group in which every member shows a statistically significant relationship of amino acid sequence to at least one other member of the family in the part of the molecule that is responsible for peptidase activity).Most plant cysteine peptidases belong to the papain family, including those of Asclepiadaceae, the milkweed family (Barrett et al., 1998).
tion of proenzymes, degradation of defective proteins, etc. (Rudenskaya et al., 1998), but the presence of a high concentration of proteolytic enzymes in some tissues is more difficult to explain.Many plants exude a latex containing a high amount of digestive enzymes, mainly cysteine and serine proteinases.This would suggest genetic resources involved in convergent evolutionary processes related to the overproduction and accumulation of proteases in different plant families (Boller, 1986).Proteolytic enzymes are tools which have been used in medicine as well as in industry for hundreds of years.However, in recent years, their utility has become of vital importance due to the availability of standard, potent, and active preparations with good solubility, stability and odor.Proteolytic enzymes of plant origin have received special attention in the field of medicine and industry due to their property of being active over a very wide range of temperature and pH.Proteases, which firmly maintain first place in the world market of enzymes, play an important role in biotechnology, given that proteolysis changes the chemical, physical, biological, and immunological properties of proteins.Enzymatic hydrolysis is strongly preferred over chemical methods because it yields hydrolyzates containing well-defined peptide mixtures and avoids the destruction of L-amino acids and the formation of toxic substances like lysino-alanine (Lahl and Brown, 1994;Mahmoud, 1994).
In spite of the fact that existing commercially available proteases have a high degree of proteolytic activity and are abundantly and cheaply available, there is a need for discovering new plant sources of potent, more active, and more specific proteolytic enzymes.Proteases are frequently present in the latex of Asclepiadaceae (milkweed family).Nevertheless, studies of these proteases are limited to a few species belonging to the genera Asclepias (Winnick et al., 1940;Greenberg and Winnick, 1940;Carpenter and Lovelace, 1943;Brockbank and Lynn, 1979;Lynn et al., 1980a;Barragán et al., 1985;Tablero et al., 1991) and Calotropis (Abraham andJoshi, 1979a, 1979b;Pal and Sinha, 1980;Sengupta et al., 1984).In a recent paper (Arribére et al., 1998) we reported the partial characterization of proteases isolated from the latex of five species of Asclepiadaceae grown in Argentina, as well as the purification and characterization of the proteases of one of them, Morrenia brachystephana Griseb.A further communication reported the presence of proteases in the latex of Morrenia odorata (Hook.et Arn.) Lindley (Arribére et al., 1999).
In this paper we report the characterization of a protease isolated from the latex of Araujia hortorum fruits.This species is a South American climbing plant that grows in the south of Brazil, Paraguay, Uruguay, and Argentina (Burkart, 1979).The latex has been used in folk medicine as a local application to warts (Watt and Breyer-Brandwijk, 1962).

Preparation of the Crude Extract
Latex obtained by superficial incisions of fruits, received on 0.1 M citrate-phosphate buffer (pH 6.5) containing 5 mM EDTA and cysteine, was first centrifuged at 16,000 × g for 30 min at 4°C.Gums and other insoluble materials were discarded, and the supernatant was ultracentrifuged at 100,000 × g for 60 min at 4°C.This new supernatant ("crude extract"), containing soluble proteins, was fractionated and conserved at −20°C for further studies.

Proteolytic (Caseinolytic) Activity Assays
Proteolytic assays were made using casein (Hammarsten type; Research Organics, Cleveland, OH) as substrate.The reaction mixture was prepared by mixing 0.1 ml of enzyme extract with 1.1 ml of 1% casein containing 12 mM cysteine, in a 0.1 M Tris-HCl buffer (pH 8.0).The reaction was carried out at 45°C and stopped 2 min later by the addition of 1.8 ml of 5% trichloroacetic acid (TCA).Each test tube was centrifuged at 3000 × g for 30 min and the absorbance of the supernatant measured at 280 nm.An arbitrary enzyme unit (caseinolytic unit, U cas ) was defined as the amount of protease which produces an increment of one absorbance unit per min in the assay conditions (Priolo et al., 1991).

Protein Determination
Proteins present in the crude extract were determined by Bradford's method (Bradford, 1976) using bovine albumin (Sigma Chemical Co., St Louis, MO) as standard.During chromatographic separation, the protein content of eluates was estimated by measuring the absorbance at 280 nm.Protein content of the active fractions was determined using Lowry's method (Lowry et al., 1951), with bovine albumin as standard.

pH Profile of the Proteolytic Activity
The effect of pH on enzyme activity of both the crude extract and the main purified proteolytic fraction was measured with casein (pH range 6.4-10.5)using 10 mM sodium salts of the following "Good" buffers (Good and Izawa, 1972): MES, MOPS, TAPS, AMPSO, and CAPS (Sigma Chemical Co., St Louis, MO).

Inhibitor Effect
The action of different inhibitors of cysteine proteases was evaluated by incubating the crude enzyme preparation for 10 and 30 min at 45°C with mercuric chloride and E-64.The residual caseinolytic activity after each incubation assay was measured as indicated above.

Purification of araujiain h I
The purification of the main proteolytic component (araujiain h I) was carried out by cation exchange chromatography (CM Sepharose CL-6B Fast Flow).Fourteen milliliters of the crude extract containing 75 mg of protein was loaded onto the column (Pharmacia K 15/30).The column was washed with 60 ml of 55 mM citrate-phosphate buffer (pH 6.4) and the bound material eluted with a linear gradient of sodium chloride (0 -0.6 M) in the same buffer.

Thermal Stability
Thermal behavior of the purified protease was evaluated by measuring the residual caseinolytic activity at 45°C (pH 8.5) during 2 min after incubation of samples for 2.5, 5, 10, 20, and 30 min at 37, 45, and 60°C.

Isoelectric Focusing (IEF)
IEF was developed on immobilized pH gradient gels of polyacrylamide (10%) in the pH range from 3 to 10 (Biolyte 3-10 carrier ampholytes, Bio-Rad, Hercules, CA) in a Mini IEF Cell (Model 111, Bio-Rad).Samples were concentrated by acetone precipitation and further centrifugation at 11,000 × g during 20 min.Deionization was performed by redissolving the precipitates in deionized water and repeating the whole treatment twice.Isoelectric focusing of proteases was carried out according to the conditions 100 V for 15 min, 200 V for the following 15 min, and 450 V for the last 60 min.Then, gels were fixed and stained with Coomassie brilliant blue R-250.

Native Electrophoresis
The crude extract as well as the active chromatography fractions were analyzed on a 12.5% polyacrylamide gel in a Miniprotean II Cell (Bio-Rad, Hercules, CA).Samples were precipitated with 5 volumes of acetone, redissolved in the sample buffer [62.5 mM Tris-HCl, pH 6.8, with 10% (v/v) glycerol and 0.25% (w/v) bromophenol blue], and centrifuged at 16,000 × g for 10 min.Samples were loaded on the gel, and the electrophoresis performed by changing the polarity of electrodes during 2 h at 35 mA.Protein bands were detected by Coomassie brilliant blue R-250.

SDS-Polyacrylamide Gel Electrophoresis
SDS-PAGE was carried out according to Laemmli (1970).Samples were precipitated with acetone as described above and redissolved in sample buffer containing 10 µM E-64 to prevent autodigestion and boiled for 5 min; then, β-mercaptoethanol was added and boiled again.Samples were then loaded on a polyacrylamide 14% separating gel overlaid by a 5% stacking gel.Electrophoresis was run in a vertical apparatus starting at 25 mA.When the samples left the stacking gel, the current was set at 50 mA until the bromophenol blue dye marker left the separating gel.The gels were stained with Coomassie brilliant blue R-250 and scanned for evaluation of the molecular masses using the Scion Image software.

Zymogram
To confirm the proteolytic activity of the bands, native electrophoresis unstained gels were contacted for 15 min at 56°C with an agarose gel imbibed in 1% casein solution (Westergaar et al., 1980) and then stained with Coomassie brilliant blue R-250.

Titration of Active Sites with E-64
A dose-response relationship was studied between the purified enzyme and increasing concentrations of E-64, a well-known cysteine proteinase inhibitor.The method was an adaptation of that developed by Barrett et al. (1982).Fractions (25 µl) of araujiain h I eluted from the ion exchange column were incubated with 25 µl of different concentrations (0-10 µM) of E-64 solutions for 30 min at room temperature.Then, the residual caseinolytic activity was assayed as follows: 55 µl of 1% casein solution with 12 mM cysteine (pH 8.35) was added to the mixture and kept for 8 min at 45°C; the reaction was stopped by the addition of 900 µl of 5% TCA and the absorbance of soluble peptides read at 280 nm.

Esterolytic Activity Determination
Measurement of endoesterolytic activity (Silverstein, 1974) was performed with N-α-carbobenzoxy-pnitrophenyl esters of some amino acids (Gln, Ala, Asp, Phe, Asn, Tyr, Trp, Gly, Leu, Val, and Pro).The synthetic substrates were obtained from Sigma Chemical Co.(St.Louis, MO).Assays were made at 40°C in 0.1 M Tris-HCl buffer (pH 8.0) containing 2 mM EDTA and 25 µM cysteine in the reaction mixture.Absorbance was measured at 405 nm every 10 sec for the first minute, and then every 15 sec.Both crude extract and araujiain h I were used for this assay.An arbitrary enzyme activity unit (U cbz ) was defined as the amount of protease that released 1.0 µM of p-nitrophenolate per min in the assay conditions.To determine the micromoles of p-nitrophenolate produced during the reaction, a standard curve (p-nitrophenol 15-70 µM) was carried out.

Protein Sequence Analysis
Alkylated araujiain h I was digested with Protease V8 (endopeptidase Glu-C) using a 1:25 enzyme protein ratio (w/w).This serine endopeptidase cleaves peptide bonds -COOH terminal to Glu residues at a rate that is about 3000-fold faster than that for Asp residues (Sprensen et al., 1991).Peptides were separated by reverse-phase HPLC using a Nova Pack C18 column (Waters) and a 60-min linear gradient of 0.1% trifluoroacetic acid (TFA) in water to 0.1% TFA in acetonitrile at 0.1 ml/min and were analyzed by mass spectrometry.A sample of the main peptide was adsorbed on a PVDF membrane (Millipore) and washed several times with deionized water.The N-terminal sequence was determined by Edman's automated degradation using a Beckman LF3000 protein sequencer equipped with a System Gold (Beckman) PTH-amino acid analyzer.Protein homology searches were performed using the BLAST network service (Altschul et al., 1997), indicating the specific residues which are identical ("identities"), as well as those which are nonidentical, but nevertheless have positive alignment scores ("positives").

Mass Spectrometry
Matrix-assisted laser desorption ionization/timeof-flight mass spectrometry (MALDI/TOF MS) was used for the determination of the molecular mass, as well as the degree of purity of active chromatographic fractions.MALDI/TOF mass spectra were acquired on a Bruker Biflex spectrometer equipped with a pulsed nitrogen laser (337 nm), in linear positive-ion mode, using a 19-kV acceleration voltage.Samples were prepared by mixing equal volumes of a saturated solution of the matrix (3,5-dimethoxy-4-hydroxycinnamic acid-sinapic acid) in 0.1% TFA in water/acetonitrile 2:1, and a 1-10 µM protein solution.From this mixture, 1 µl was spotted on the sample slide and allowed to evaporate to dryness.Proteins of known molecular mass were used as standards for mass calibration.ous recommendations (Barragán et al., 1985;Tablero et al., 1991).Specific activity was higher for the former, which was chosen for further analysis.When the crude extract was previously inactivated by treatment with HgCl 2 , the chromatographic pattern observed was identical to that of the untreated enzyme (data not shown).Table I shows the purification scheme: a sixfold purification degree (yield: 12%) was obtained for araujiain h I.
A slight shift in the optimum pH range (Fig. 2) was observed for araujiain h I (pH 8.0-9.5) in comparison with the crude preparation (pH 7.5-8.5).The enzyme exhibited a pI value higher than 9.3, as can be seen in Fig. 3; this basic nature was also observed in the Cysteine Protease from the Latex of A. hortorum Fruits 43

RESULTS AND DISCUSSION
Proteases contained in the latex of Araujia hortorum Fourn.fruits were studied.The crude enzyme extract (supernatant of 100,000 × g) showed higher caseinolytic activity in the presence of 12 mM cysteine.Highest activity (fourfold) was recovered when the crude extract was obtained with 5 mM EDTA and 5 mM cysteine.
Cation exchange chromatography of the crude extract yielded two main proteolytic peaks (Fig. 1): fraction I (araujiain h I), purified to mass spectroscopy homogeneity, and fraction II.The name araujiain h I is proposed for the new protease, according to previ-  ity increased with temperature: maximum proteolytic activity was obtained at 70°C for the crude extract (Fig. 5a) and at 60°C for araujiain h I (Fig. 5b).These results suggested a protective role played by the substrate and, in the case of the crude extract, probably by other proteins.Native electrophoresis of the crude extract showed two fractions, one of them with higher proteolytic activity (araujiain h I), as evidenced by the corresponding zymogram (Fig. 6).SDS-PAGE revealed that araujiain h I appeared as a unique band (Fig. 7), with a relative mass of about 25.5 kDa.In the titration of active sites with E-64 the residual enzyme activity gave a curve going downward with linearity at E-64 concentrations up to 5 µM.Extrapolation of the linear curve crossed the X axis at the E-64 concentration of 3.3 µM (Fig. 8), which matches with the protein concentration in the eluted fraction obtained by Lowry determination.Thus, the E-64 and the enzyme were found to react with each other on an equimolecular basis.By combining both methods, we calculated the molecular mass of araujiain h I to be about 24 kDa, in good agreement with the value obtained by mass spectroscopy (24,031), as shown in Fig. 9.As reported proteases isolated from the latex of Asclepias glaucescens (Barragán et al., 1985;Tablero et al. 1991), Morrenia brachystephana (Arribére et al., 1998), and Morrenia odorata (Arribére et al., 1999).Inactivation assays with HgCl 2 and E-64 suggested the possible cysteinic nature of the proteases present in the crude extract.When HgCl 2 was added, proteolytic activity almost completely reverted by adding cysteine to the incubation mixture.When E-64 was added, the inhibition obtained for crude extract was complete and irreversible (Table II).The activation, inhibition, and reactivating results would indicate dependence of the proteolytic activity upon the presence of active -SH groups in the enzymes present in the crude extract.This behavior was previously shown by proteases from other Asclepiadaceae species (Lynn et al., 1980a;Abraham and Joshi, 1979b;Tablero et al., 1991;Arribére et al., 1998Arribére et al., , 1999)).
As can be seen in Fig. 4, araujiain h I had a poor stability at high temperatures, but the caseinolytic activ- for other plant proteases (Harrach et al., 1998), molecular masses determined by SDS-PAGE are higher than those obtained by other methods.The results are almost coincident with those obtained for other proteases from Asclepiadaceae: M r values of Asclepias syriaca proteases are 21 and 23 kDa (Brockbank and Lynn, 1979;Lynn et al., 1980a), proteases from Asclepias glaucescens have M r of 23 kDa (Barragán et al., 1985;Tablero et al., 1991), the four calotropins isolated from Calotropis gigantea show molecular masses ranging from 23 to 27 kDa (Abraham andJoshi, 1979a, 1979b;Pal and Sinha, 1980), two proteases obtained from Morrenia brachystephana present molecular masses of 25.5 and 26 kDa (Arribére et al., 1998) and the proteases isolated from Morrenia odorata show M r of 24.2 and 25.8 (Arribére. et al., 1999).Endoesterolytic activity of the crude extract and araujiain h I, determined on N-α-CBZ-amino acid-pnitrophenyl esters, exhibited different patterns: both showed higher preference for the glutamine derivative, but in the crude extract it is followed by those of    aspartic acid, asparagine, glycine, leucine, tryptophan, tyrosine, phenylalanine, and alanine in decreasing order, while in araujiain h I the preference order is as follows: glutamine, alanine, aspartic acid, leucine, and tryptophan (Table III).
The N-terminus of araujiain h I was found to be blocked, as was previously reported for calotropins DI and DII isolated from Calotropis gigantea (Sengupta et al., 1984).Therefore, the sequence of an internal peptide obtained by hydrolysis with Protease V8 (endopeptidase Glu-C) was determined.Table IV shows the sequence of this fragment (41 residues) compared to those of 21 plant cysteine proteinases, none of which belongs to the Asclepiadaceae family, as only Nterminal sequences (Lynn et al., 1980b) and C-terminal sequences (Sengupta et al., 1984) of proteinases from species of this family have been reported.Despite this, a remarkable degree of homology was obtained (identities = 48-36%, positives = 70-55%) and several motifs are notably conserved (YPY in all cases; AF, NG, GXC, KVV or KVX or XVV, and VP, in most cases), suggesting that araujiain h I probably shares an ancestral gene with cysteine proteases obtained from taxonomically unrelated plant species.

Fig. 5 .Fig. 6 .
Fig. 5. (a) Enzyme activity of crude extract as a function of temperature; (b) enzyme activity of araujiain h I as a function of temperature.In both cases the determinations of activity were made on casein at pH 8.5.(a)(b)

Table I .
Purification of the Proteolytic Components Present in the Latex of Araujia hortorum Fig. 2. Effect of pH on proteolytic activity of crude extractsand araujiain h I.

Table III .
Kinetic Studies