Properties of a proteolytic enzyme from Schistosoma mansoni

Comp. Biochem. Physiol., 1972, VoL 42B, pp. 205 to 220. Pergamon Press. Printed in Great Britain

PROPERTIES OF A PROTEOLYTIC ENZYME FROM SCHISTOSOMA MANSONI* M. C. V. S A U E R and A. W. S E N F T t Division of Biological and Medical Sciences, Brown University, Providence, Rhode Island 02912

(Received 13 November 1971) Almtract--1. A globinolytic protease from adult Schistosoma mansoni worms has been purified seventy-one-fold with a 52 per cent recovery yield. The isoelectric point of the enzyme was found to be 3"55 by an eleetrofocusing technique. 2. The molecular weight is estimated at about 27,000, as determined by Sephadex gel filtration and polyacrylamide gel electrophoresis. 3. Analysis of the hydrolyzate of this enzyme shows that the proportion of the aspartate plus glutamate in the protein is about 20 per cent. 4. The, enmyrne shows an optimum pH range from 3"9 to 4"5 for the digestion of globin, and a K,~ of 0.137 mM for this substrate. 5. Phenylalanine was found to be a competitive inhibitor of the enzyme with an estimated K~ of 3.5 mM. 6. A study of the reaction products indicates that the principal end-products are small peptides; essentially no free amino acids are formed. 7. Phenylalanine has been found to be the most common N-terminal residue of these peptides. INTRODUCTION FOR MANY years it has been axiomatic that schistosomes ingest red cells as a source of food. Worms can be shown by microscopic observation to swallow erythrocytes, and the adult parasite ceca are characteristically filled with a black slurry of hemolyzed red cells. Hematin crystals have been described in the gut of the worms (Rogers, 1940), while deposits of schistosome "pigment", that is, the residue of the red cell meal, are a constant finding in the liver of infected mammals. T h e role of the digested hemoglobin in the nutrition of these worms is not yet fully understood. It is known that the addition of erythrocytes to serum-based cultures of 5-day-old schistosomulae stimulates growth and maturation (Clegg et aL, 1971); and it has been shown that the addition of red cells, hemoglobin or globin to defined culture media increases growth and survival of adult Schistosoma mansoni in vitro (Cheever & Weller, 1958). Labelled red cells, containing 14C-leu-hemoglobin have been intravenously administered to schistosome-infected mice. T h e r e * Supported by NSF Grant GB-30646. To whom reprint requests should be addressed. 8

2O5

206

M. C. V. SAUERAND A. W. SENFT

appeared to be generalized incorporation of the label throughout the worm's body (Zussman et al., 1970). In other studies, the label from 14C-leucine red cells has been localized within the eggs deposited in the liver of infected mice (Wichser, 1970). The presence of a proteolytic enzyme, with marked specificity for hemoglobin, which could be found in homogenates of adult worms was first reported in 1959 (Timms & Bueding, 1959); a recent communication (Grant & Senft, 1971) has given additional details. These studies indicate the importance of hemoglobin as a substrate for worm metabolism. In this work we report the purification of the proteolytic enzyme and some of its physical and chemical properties. Kinetic studies were also performed on the enzyme-globin complex in the absence and in the presence of a competitive inhibitor. Reaction products, using globin and hemoglobin, were studied in order to obtain information on the cleavage specificity of the enzyme on these substrates. MATERIALS AND METHODS

Enzyme source Dried and lyophilized adult schistosome worms, which were collected by perfusing the mesenteric and portal veins of infected mice, were kindly provided by Dr. Shirley E. Maddison of the Center for Disease Control, Atlanta. The worms, in separate stocks of about 80-120 mg each, were kept in a deep freeze at - 1 2 5 ° C until use.

Substrate and chemicals Globin was used as the substrate in the routine assay of proteolytic activity as well as in the kinetic studies and in the determination of reaction products. The globin was prepared from adult human hemoglobin by Dr. C. T. Grant, following the method of Kistler et al. (1953). H u m a n hemoglobin, twice recrystallized and lyophilized as supplied by Sigma, was also used in the detection of reaction products. Sephadex G-200 was obtained from Pharmacia. Agarose-L-phenylalanine (14"5/xm/ml resin), agarose-L-tyrosine (5/zm/ml resin) and agarose-L-tryptophan (13/~m/ml resin) were purchased from MilesYeda. Sodium acetate and glacial acetic acid reagent grade were used without further purification in the preparation of buffers. Hydrochloric acid 5"7 N, used in the hydrolysis of proteins and peptides, was purified by glass distillation of commercial hydrochloric acid. Solutions of trichloroacetic acid (10%) were obtained from Fisher.

Assay of proteolytic activity Globin in 0"2 M sodium acetate buffer of pH 3"94 + 0"02 was used in a concentration of 10 mg/ml for the assay of enzymic activity. One ml of this solution was incubated with 0"5 ml of enzyme solution at 37°C. The reaction was stopped after 1 hr by the addition of an equal volume of 10% T C A and precipitated protein was removed by centrifugation at 4°C. Enzymic activity was determined by measuring the absorbance of the supernatant at 280 rim. Control samples containing enzyme or globin were treated in the same way as the mixture samples, and these supernatant absorbancies were subtracted from those of the reaction. Although the measurement of absorbance at 280 n m is not as sensitive as the ninhydrin reaction generally employed for the detection of amino nitrogen, the spectrophotometric technique is faster and more convenient. In the present work, a unit of activity is expressed as 1"00.D. in a mixture incubated for 60 rain at 37°C.

A P R O T E O L Y T I C ENZYME FROM S C H I S T O S O M A

207

MANSONI

Purification of the enzyme A weighed quantity of lyophilized worms, usually about 80-100 mg, was homogenized at 0°C and suspended in about 10 ml of 0.2 M acetate buffer at pH 3-94. It was found that by keeping the ratio of buffer volume to weight of enzyme source fairly constant, 90-97 per cent of the activity remains in the supernatant. Thus, a second extraction of the precipitate is not needed. The suspension was centrifuged at 22,000g for 30 min at 4°C. The clear supernatant was then transferred to an Amicon ultrafiltration cell and concentrated to 1"0-1"5 ml at 4°C, using a P-10 membrane, having an exclusion limit of 10,000 mol. wt. The filtration cell is driven by an applied nitrogen pressure of 60-70 lb/in2. The concentrate was next layered on a 0"9 x 40 cm Sephadex G-200 column previously equilibrated with pH 3"94 acetate buffer at 4°C. Figure 1 shows a typical plot of the eluate absorbanee at 280 nm. Fraction II, containing all of the enzymic activity, was further

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F[o. 1. Fractionation of a centrifuged extract of adult S. mansoni worms. A 0.9 x 40 cm Sephadex G-200 column was equilibrated with 0.2 M sodium acetate buffer at pH 3'95 at 4°C. Gravity-driven flow rate was 2 ml/hr. Fraction size = 1 ml. All of the proteolytic activity was found in fraction II. Fraction I contains high mdecular weight components from the worm homogenate; fraction I I I contains free amino acids and small peptides. concentrated by ultrafihration. The new concentrate was then passed through a 0"5 x 5 cm agarose-L-phenylalanine column previously equilibrated with pH 3"94 acetate buffer. Elution of the enzyme is effected with 0.2 M acetic acid solution. Figure 2 is a plot of the absorbance of this eluate. The protein content of the various preparations was determined by the method of Lowry et al. (as reported by Needleman, 1970), using bovine serum albumin as a standard.

Molecular weight The molecular weight of the enzyme was estimated by chromatography on Sephadex G-200, using a 1.2 x 55 cm column equilibrated with acetate buffer, pH, 3"95 at 4°C. Hemoglobin, ovalbumin, chymotrypsinogen A and ribonuclease A were used as markers. The void volume of the column was determined by using dextran blue. As a second procedure, the molecular weight was determined by electrophoresis through a polyacrylamide gel. Using the technique of Weber & Osborn (1969), a sample of fraction I I b obtained by aEmity chromatography was incubated at 37°C for 3 hr in 0"01 M sodium phosphate containing 1% sodium dodecyl sulfate and 1% ~-mercaptoethanol. Other chosen

208

~ . C. V. SAUERAND A. W. SENFT

marker proteins of known molecular weight were simultaneously incubated. The mixture was then separated by disc electrophoresis. The molecular weight of the enzyme was calculated from the plotted position of the enzyme on the slope of migration.

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FIc. 2. Affinity chromatography of the enzyme on agarose-L-phenylalanine. A 0"5 x 5 cm column was equilibrated with 0"2 M sodium acetate buffer, pH 3"95, at 4°C. Fraction II from Step 1 (see Fig. 1) was passed through the column with buffer at pH 3"95. IIa, representing a non-active companion protein, was not retained by the matrix. When the pH of the elution buffer was lowered, fraction IIb, containing all of the enzymic activity appeared. Fraction size = 2 ml. Flow rate = 20 ml/hr. The arrow indicates the elution of the enzyme with 0'2 M acetic acid.

Amino acid analysis Analyses were performed on a Beckman Model 121 Amino Acid Analyser, using a 0"9 x 5"5 cm column with PA-35 resin and pH 5"28 sodium citrate buffer for basic amino acids and PA-28 resin in a 0"9 x 55 cm column, using pH 3"25 and 4"25 sodium citrate buffers for analysis of acidic and neutral amino acids. Samples containing 0"1-0"4 mg of pure enzyme were hydrolyzed under vacuum with 5-7 N HCI for 22, 40, and 70 hr at 107°C. After evaporation of the liquid under vacuum, the hydrolyzates were redissolved in 0"8 ml of citrate buffer, pH 2"2, and analyzed.

RESULTS

Recovery of the enzyme Purification of this enzyme by Sephadex G-200 and affinity chromatography (Cuatraeasas et al., 1968) provides a convenient and rapid recovery method. Using these techniques a seventyfold purification of the enzyme can be achieved, along with about 50 per cent recovery. T h e preliminary separation of the enzyme from the worm homogenate is shown in Fig. 1, while the specific purification of the enzyme, based on affinity chromatography, is shown in Fig. 2. A stepwise procedure for this purification is given in Table 1.

209

A PROTEOLYTIC ENZYME FROM S C H 1 S T O S O M A ! k l A N S O N 1

TABLE 1 - - T Y P I C A L ENZYME PURIFICATION OBTAINED FROM 1 0 0 m g LYOPHILIZED WORMS

Procedure 1. Crude suspension homogenized worms (pH 3"95)

Volume (ml)

Activity Protein (units) (mg/ml)

Sp. act. (units/mg protein)

Yield (%)

Purification (-fold) --

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13-4

2.45*

0.55

100

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* A considerable amount of the protein in the original homogenate is precipitated when a suspension is made at pH 3.95. t When one refers the final weight of enzyme to the weight of the lyophilized worms, a purification of about 71"4 times is realized. Figure 3 shows the electrofocusing profile of fraction II from Sephadex G-200 and fractions IIa and IIb obtained from the affinity chromatography system, using agarose-L-phenylalanine as the substrate. It can be seen that fraction IIb (Fig. 2) contains all of the enzymic activity and may be focused into one peak having a pI of 3.55. Fraction IIa resolves into one or more peaks, according to the number of times it is chromatographed on agarose-L-phenylalanine (Fig. 3c and 3d). The resolving capacity of the agarose-L-phenylalanine decreases after being used several times. This is probably due to absorption of contaminants by the agarose gel under our experimental conditions. Washing the column with 8 M urea after every run will help to maintain the ability of the gel to separate these fractions. The enzyme is very stable as the partially purified fraction from Sephadex G-200, and does not appear to lose activity or denature by fi'eezing and lyophilization. However, the pure enzyme obtained by affinity chromatography is quickly inactivated at room temperature, and is denatured when frozen and lyophilized. It seems possible, therefore, that the presence of a companion protein, having an isoelectric point of 4.6 (Fig. 3d), has a stabilizing effect on the enzyme.

Molecular weight The results of the experimental procedures are shown in Figs. 4 and 5. When the elution volume (EV) of the enzyme was plotted as a function of the logarithm of molecular weight of proteins eluted from Sephadex (Fig. 4) a value of 27,000 daltons was obtained. During gel eleetrophoresis, the enzyme moved as a single band through polyacrylamide and migrated to a position which indicated a molecular weight of about 28,000 (Fig. 5).

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FIG. 4. Estimation of molecular weight of the enzyme by chromatography. A Sephadex G-200 column (1"2 x 55 cm) was calibrated with dextran blue (tool. wt. = 2,000,000), hemoglobin (68,000), ovalbumin (45,000), chymotrypsinogen A (25,000) and ribonuclease A (13,700). Elution volumes (EV) were plotted against the log of the molecular weight. T h e enzyme was eluted under identical conditions as the reference compounds. Molecular weight was estimated from a plot. EV = Ve/Vo, where V0 is the void volume of the column as determined by dextran blue. T h e enzyme molecular weight is approximately 27,000.

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A PROTEOLYTIC ENZYME FROM SCHISTOSOMA MANSON1

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FIG. 5. Estimation of molecular weight of the enzyme by gel electrophoresis. A 50-/zg sample of affinity-chromatography-purified enzyme was electrophoresed in polyacrylamide gel along with marker proteins of creatine kinase, pepsin, chymotrypsinogen A and ribonuclease. The position of each band was determined by staining with Amido Schwarz. A plot of the mol. wt. : mobility indicates a value of 28,000 daltons for the schlstosome enzyme. The sample was pretreated at 37°C for 3 hr in 0"01 M sodium phosphate buffer, pH 7"0, containing 1% sodium dodeeyl sulfate and 1% /3-mercaptoethanol. Electrophoresis was carried out in 0"1 M Na phosphate buffer containing 0"1% Sodium dodecyl sulfate at a current strength of 8 mA.

Amino acid composition Since several criteria, gel filtration, electrofocusing and SDS gel electrophoresis, indicated that the purified enzyme was essentially homogeneous, a determination of the amino acid composition of such preparation became meaningful. Table 2 shows the amino acid composition of the enzyme. It can be seen that aspartate and glutamate account for approximately 20 per cent of the residues of this protein. T h e proportion of these residues which, prior to hydrolysis, were actually glutamine or asparagine is believed to be low, based on the small amount of ammonia liberated during hydrolysis.

Ultraviolet spectra Ultraviolet spectra of the enzyme in acetate buffer, pH 3.95, and in 0.1 N N a O H solution were recorded using a Cary 14 spectrophotometer and also a Spectronic 505 in the range of 230-320 nm. Molar extinction coefficients were estimated: pH 3.95 : e~so= 24-2 x l0 s, pH 13: e2a0= 37.5 x l0 s,

A2so/A2so = 1.4.

212

~"I. C. ~ r SAUER AND A. W . SENFT TABLE 2 - - A M I N O ACID COMPOSITION OF THE ENZYME (HYDROLYZATE)

Residues/mole enzyme *

Nearest integer

Lysine Histidine Arginine Tryptophan

14"1 5'4 9.1 4 ?

14 5 9 4

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine ~-Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine

25"7 15"8, 24"4 ~ 26"8 11 '1 20" I 18"1 2'4t 14' 4 § 4'6 10'2 § 21 "3 11 "2+, 8"8

26 16 24 27 11 20 18 2 14 5 10 21 1l 9

Amino acid

Total

2 51

* Based on mol. wt. = 28,000. t Spectrophotometric determination. ++Extrapolated to time 0 of hydrolysis. § Value at 70 hr of hydrolysis.

Effect of pH on enzymic activity Figure 6 shows the effect of the p H of the reaction mixture on enzymic activity against globin at 37°C. T h e m a x i m u m activity of the enzyme lies in a range f r o m p H 3.9 to 4.5, with no sharp m a x i m u m observed experimentally. Partially purified enzyme f r o m Sephadex G-200 was used for this purpose.

Initial velocity studies T h e kinetics of the proteolytic reaction using partially purified enzyme f r o m Sephadex G-200 and pure enzyme f r o m agarose-L-phenylalanine were studied at 37°C in various globin concentrations f r o m 2.5 to 10 mg/ml. T h e enzyme concentration was in the range of 0.07 to 0.1 mg/ml. T h e velocity of the reaction was calculated on the basis of the change in absorbance that occurs during the first 15 min. Figure 7 shows the L i n e w e a v e r - B u r k plot with an extrapolated value. K,,~ at 37°C = 0.137 × 10 -a M.

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A PROTEOLYTIC ENZYME FROM SCHISTOSOMA M.qNSON1 CO

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Effect of free amino acids on enzymic activity Proline, histidine, arginine and phenylalanine were added in a range of concentration of 0.8-2.5 mM to incubation mixtures of enzyme and globin. Only in the case of phenylalanine was an inhibitory effect noted. Tyrosine and tryptophan have not yet been directly studied as possible inhibitors, since they have exceedingly high spectrophotometrie blanks, even at very low concentrations. However, both agarose--L-tyrosine and agarose-L-tryptophan were tested as possible complexing substances for this protease. It was found that agarose-L-tyrosine binds the enzyme much as does agarose-L-phenylalanine. A

214

M.C.V. SAUERANn A. W. S~.NFT

change in pH will release the enzyme from the tyrosine column. Agarose-Ltryptophan appeared to retain the protein without evidence of specificity. These results suggest that tyrosine undergoes the formation of a reversible complex with the enzyme and may therefore also be an inhibitor of the enzyme.

Initial velocity studies in the presence of phenylalanine In order to estimate the equilibrium constant (Kt) for the dissociation of the enzyme--phenylalanine complex, kinetic studies of the enzyme-globin reaction were carried out in the presence of phenylalanine. Figure 8 shows the Lineweaver-Burk plots at various concentrations of phenylalanine. As can be seen, the results indicate a competitive inhibition. A replot of the slope of the reaction velocity versus inhibitor concentration leads to an estimate: K l = 3.5 x 10 -3 M at 37°C. I _ = Zl_.]t (rain) v AA 150

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Products from the enzymic digestion of globin and hemoglobin Purified enzyme (0.6--0.8 mg) was incubated at 37°C for 4-16 hr with 80-100 mg of globin or hemoglobin in 10 ml 0.2 M acetate buffer, pH 3.95. Before use, the globin and hemoglobin were purified on a 1.2 x 55 cm Bio-Gel P-60 column, since both were found to contain a small quantity of low molecular weight contaminants. After enzymic digestion of the purified globin, the mixture was chromatographed on the same Bio-Gel P-60 column. Figure 9 shows the absorbance at 280 nm of the eluate. As can be seen, the products of the reaction consist primarily of large peptides (PI) and small peptides (PII). An electrofocusing pattern of fraction PI shows that it contains at least six to seven peptides having tyrosine or tryptophan content (absorbance recorded at 280 nm as shown in Fig. 10). A similar electrofocusing run was attempted with fraction PII. The results shown in Fig. 11 may be somewhat misleading for several

A PROTEOLYTICENZYMEFROM SCHISTOSOMA MANSON1

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215

216

M. C. V. SAUV,n AND A. W. SENFT

reasons. First, peak Pn may be contaminated with material from the overlapping tail portion of PI; secondly, the quantity of peptide material is estimated on the basis of 280 nm u.v. absorption. Any small peptides having little or no tryptopha~ or tyrosine content would not have been detected.

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An analysis of fraction Pn with the amino acid analyzer indicated the presence of free leucine and traces of tyrosine and phenylalanine. In addition, when Pn was analyzed prior to hydrolysis (using a 55-cm column) the presence of about ten peptides was estimated. The relative frequency of amino acids in these peptides was determined after hydrolysis (Table 3). It can be noted that alanine, leucine and glycine contribute most of the residues of these peptides. A preliminary attempt was made to characterize individual peptides from both PI and PH fractions. These fractions were separately chromatographed on a 0.5 x 60 cm column of Dowex 50 X4, using pyridine-acetic acid-water buffer as the eluent, according to methods described by Bchroeder (1967). Unfortunately, the individual peptides were present in very small concentration. The application of the Banger reaction, in an effort to identify the terminal amino acid, was not altogether conclusive. The presence of free leucine in fraction PII was confirmed by formation of the dinitrophenyl derivative and its identification by paper chromatography. It was also possible to identify phenylalanine as the DNP-aminoterminal amino acid in five of the peptides analyzed. However, it is felt that more evidence is needed concerning the carboxy and amino terminal amino acids of each of the peptides produced in the digestion before one could describe the cleavage specificity of this enzyme.

217

A PROTEOLYTIC ENZYME FROM SCHISTOSOMA M A N S O N I TABLE 3 - - A M I N O ACID ANALYSIS OF FRACTION

Source Fraction Ptt Hydrolyzate of PII

Amino acid Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Aspartic acid Serine Threonine Glutamic acid Proline Glycine Alanine Valine Leucine Tyrosine Phenylalanine

P~

AND HYDROLYSIS OF FRACTION P n

/zM in Fraction Pn Residues 0"037 0"004 0"016 0"68 0"50 0'81 0"78 0"84 0"92 1-22 0"43 1"52 2"12 1"13 1"64 0.51 1"00

1 0"1 0"4 2 1 2 2 2 2 3 1 4 5 3 4 1 2

DISCUSSION The physical and chemical characteristics found for this enzyme indicate that it belongs to a class of peptide peptidohydrolases having a low pH optimum similar to pepsin. Our experiments indicate that the enzyme exhibits a somewhat broader optimal pH curve against globin than had been previously reported. The fact that the enzyme has an unusually low isoelectric pH is consistent with the fact that its content of aspartate and glutamate is quite high. While it is not unusual for a digestive enzyme to have a low pH optimum, it is tempting to speculate that in this enzyme the carboxyl groups of aspartate and glutamate might be involved in the catalytic center. The pH range associated with maximum activity could indicate that ionization of aspartic acid (--COO-) and protonation of glutamic acid (--COOH) in or near the active center could strongly influence the attachment of the enzyme to the substrate. The pKa for the fl-carboxyl group of free aspartate is 3"86 and that of the y-carboxyl group of free glutamate is 4"25. However, a definitive study of the variation of Km and VmaX with pH is required in order to establish the p K a values involved in the active state and also the contribution of substrate ionization. From the Michaelis constant obtained at pH 3.95 and 37°C, it is calculated that 90% saturation by the substrate occurs at globin concentrations above 22 mg/ml; this value agrees with experimental observations previously reported (Grant & Senft, 1971). When Km is compared with K~tphe~ (0.137 : 3.5 raM), one might predict that phenylalanine is likely to be a weak competitive inhibitor of the reaction. However, since there may be some twenty cleavage points in globin, the

218

M.C.V.

SAUER AND A. W. SENFT

effective concentration of the substrate is considerably higher than its actual molar concentration. (Thus, K,, for equivalent cleavage sites = 20 × K,, observed = 2-74 mM.) On this basis it is possible that phenylalanine binds the enzyme as tightly as globin. The apparent binding specificity of this enzyme for aromatic amino acids (phe, tyr, try) may serve as a guideline for the development of specific and potent inhibitors of this enzyme. If it can now be shown, as is currently our belief, that this enzyme acts within the lumen of the gut, then analogs of phenylalanine might represent potential agents which could block this metabolic activity. Thus a new point of vulnerability of this parasite to chemotherapeutic attack may become evident. A preliminary search (Zussman, 1971) has recently shown that N-tosyl-Lphenyalanyl chloromethane is not effective against this enzyme, whereas its lysyl counterpart has some inhibiting potential Several concepts are suggested on the basis of these studies concerning the importance of hemoglobin digestion by S. mansoni worms. First, unless subsequent degradation of peptides can be shown, it would appear that hemoglobin is not a source of free amino acids for these worms. Second, if free amino acids are not eventually produced from the digestion of hemoglobin, then a rational use of these peptides must be presented. It has previously been postulated (Senft, 1969) that peptides may be used directly in the synthesis of yolk granules within the vitelline gland of these worms. Such a system, presumably, would require a protein synthesizing pathway which is different from the tRNA-activated-amino-acid system. The need for stringent amino acid sequence control may not be critical in yolk, since this protein is destined for quick destruction in order to meet the nutritional needs of the growing embryo worm. In consequence, yolk might possibly be assembled (by a mechanism as yet not described) from various peptides, rather than from amino acids. The resulting protein might be expected to contain peptide sequences which are identical to those derived from globin by the digestion of that substrate by this enzyme. Since products of digestion of globin can now be obtained in pure form by electrofocusing, this hypothesis may now be investigated in some detail. While free amino acids do not appear to be produced as a general product of digestion, we have noted the appearance of a limited amount of free leucine. The mechanism of its release cannot, at present, be satisfactorily explained. L e u - C O N H - p h e or l e u - C O - N H - t y r combinations do not appear in either alpha or beta chains of human hemoglobin. Thus, it is not possible for leucine to be released by virtue of a l e u - C O i - N H - p h e break. Accordingly, it seems clear that, while N-terminal phenylalanine accounts for most of the presently identified hydrolysis sites, other loci not involving phenylalanine must be included in the proteolytic attack of this enzyme. It is known that leucine is represented 18/141 times in the alpha chain and 18/146 times in the beta chain of human hemoglobin (Perutz et al., 1960). Leucine, thus, is the most abundant amino acid in globin. Since it also is the only amino acid liberated in the digestion of globin, it could have a fate somewhat different

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219

from that of the many peptides set free by enzymic hydrolysis. Therefore, the recent study of Zussman (1970), in which leucine-tagged hemoglobin was used to follow the metabolic route of digested hemoglobin, may require a modified interpretation. Although we report both the molecular weight and data concerning the amino acid composition of this enzyme, it should be made clear that these are preliminary findings. Ordinarily, achieving homogeneity and specificity of an enzyme after only a seventyfold purification would be a most unusual occurrence. In the case of these schistosomes, a concentration of proteolytic enzyme around 1.6% of the dry weight of the worms is suggested by the evidence presented. Such a concentration represents an almost unbelievable amount of a single enzyme. Thus this evidence requires further confirmation before it can be accepted at face value. Nevertheless, the three extraction techniques reported in the present study (homogenization at low pH, Sephadex column separation of molecular sizes and elution from the specific substrate agarose gel) all tend to be additive in the selection of proteins of specific size and characteristics. Thus these findings of a rather impressive enzyme concentration per worm cannot be easily dismissed. Acknowledgements--We wish to thank Dr. K. Agarwal for performing the SDS-gel electrophoresis of the enzyme for the determination of molecular weight. Dr. Sungman Cha was particularly helpful in critically reviewing the experimental data and the manuscript. The kindness of Dr. J. O. Edwards and Dr. R. E. Parks, Jr. in suggesting technical procedures is acknowledged.

REFERENCES CH~WR A. & WELLERT. (1958) Observations on the growth and nutritional requirements of Sehistosoma mansoni in vitro. Am. 3. Hyg. 68, 322-339. CLEGGJ. A., SMXTHERSS. R. & TERRY R. J. (1971) Acquisition of human antigen by Schistosoma mansoni during cultivation in vitro. Nature, Lond. 232, 653-654. CUATR~CASASP., WmCh'~CKM. & AtCFL~S~ C. (1968) Selective enzyme purification by affmity chromatography. Proe. Nat. Acad. Sd. 61, 636-643. GRANT C. T. & Sm~'r A. W. (1971) Schistosome proteolytic enzyme. Comp. Biochem. Physiol. 38B, 663-678. KISTI~R P., Bum A. & NITSCHMAt~ H. (1953) Crber Gewinnug und Eigenschaften von Humanem Globin. Helv. chim. Acta 36, 1058-1070. NEEDLEMAN S. B. (1970) Protein Sequence Determination, p. 187. Springer-Verlag, New York. PERUTZM. F., ROSSMANM. G., CULLISA. F., MUmH~ADH., WmL G. & NORTHA. C. T. (1960), The structure of hemoglobin. Nature, Lond. 185, 416--422. ROGERSW. P. (1940) Hematological studies on the gut contents of certain nematode and trematode parasites. •. Helminth. 18, 53-62. SCHROEDERW. A. (1967) Methods in Enzymology, Vol. XI, Sect. IV, p. 351. Academic Press, New York. SmCFTA. W. (1969) Considerations of schistosome physiology in the search for antibilharziasis drugs. Ann. N . Y . Aead. Sei. 160, 571-592. TIMMSA. ~ BUEDINOE. (1959) Studies of a proteolytic enzyme from Sehistosoma mansoni. Br. jY. Pharmacol. 14, 68-73.

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WEBER K. & OSBORNE M. (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. ~t. B. C. 244, 4406-4412. WICHSER J. A. (1970) Some aspects of protein metabolism in Schistosoma mansoni. Master's thesis at Brown University, Providence, R.I. ZUSSMAN R. A. • BAUMAN P. M. (1971) Schistosome hemoglobin protease; search for inhibitors. 3t. Parasitol. 57, 233-234. ZUSSMAN R. A., BAUMANP. M. & PETRUSKAJ. C. (1970) The role of ingested hemoglobin in the nutrition of Schistosoma mansoni, jY. Parasitol. 56, 75-79. Key Word Index.--Schistosoma mansoni; protease; phenylalanine; digestion; hemoglobin ; pigment.