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Purification and characterization of a trypsin-like enzyme from the midgut gland of the Atlantic blue crab, Callinectes sapidus
Comp. Biochem. PhysioL Vol. 95B, No. 3, pp. 525-530, 1990 Printed in Great Britain
0305-0491/90 $3.00 + 0.00 © 1990 Pergamon Press plc
PURIFICATION A N D CHARACTERIZATION OF A TRYPSIN-LIKE ENZYME FROM THE M I D G U T G L A N D OF THE ATLANTIC BLUE CRAB, C A L L I N E C T E S SAPIDUS JAMES E. DENDINGER and KATHLEEN L. O'CONNOR Department of Biology, James Madison University, Harrisonburg, VA 22807, USA (Tel: (703) 433-6225)
(Recewed 14July 1989) Abstract--1. A trypsin-like enzyme from the midgut gland of the Atlantic blue crab, Callinectes sapidus, was purified and studied. 2. Purification was achieved by a combination of molecular sieving, ion exchange, and hydrophobic chromatography. Degree of purity was assessed by SDS-polyacrylamide electrophoresis. 3. Using N-p-tosyl-L-arginine methyl ester (TAME) as a substrate, the enzyme displays optimal activity in the absence of calcium at pH 8.2. 4. The tryptic activity is extremely stable between pH 7 and 8.5, but is rapidly inactivated below pH 6. Thirty-minute incubations above 50°C also inactivate the enzyme. 5. The tryptic activity has a molecular weight of 33,500 da and an isoelectric point of approximately 4. 6. The trypsin-like protease from Callinectes sapidus is similar to crayfish and bovine trypsins in that it is inhibited by phenylmethane sulfonylfluoride, tosyl-L-lysine chloromethyl ketone, and soybean trypsin inhibitor. Its pH optimum and its specificity for TAME and N-p-tosyl-L-lysine methyl ester (TLME) are also similar. It is concluded that the enzyme characterized in this study is trypsin.
INTRODUCTION In the numerous reports on digestive proteases found in Crustacea, almost all have included at least one trypsin-like enzyme (DeVillez and Buschlen, 1967). Some of the animals found to have this enzyme included copepods (Juhasz et al., 1980), several species of crayfish (DeVillez, 1965; Zwilling and Neurath, 1981), shrimp (Gates and Travis, 1969; Galgani et al., 1984), krill (Kimoto et al., 1983), lobster (Brockerhoff et al., 1970; Galgani and Nagayama, 1987b), and crabs (Brun and Wojtowicz, 1976; Galgani and Nagayama, 1986, 1987a; Dendinger, 1987). Some of these trypsin-like enzymes have been purified (Zwilling and Neurath, 1981; Titani et al., 1983) and although most of the characteristics of these enzymes are similar to vertebrate trypsin, some characteristics are markedly different. These differences include calcium ion requirements for the vertebrate enzyme catalysis and stability (Zwilling et al., 1969), and isoelectric point (Zwilling et al., 1969; K i m o t o et al., 1983). The Callinectes trypsin-like enzyme was first described in a previous paper (Dendinger, 1987) and the purpose of this study was to compare and contrast the Callinectes sapidus trypsin-like enzyme with that of other Crustacea and with the vertebrate form. MATERIALS AND METHODS
Animals and tissues Adult male blue crabs were obtained alive from commercial sources in the Chesapeake Bay of Virginia. Each midgut gland was removed and frozen until further processing in the laboratory. Frozen samples were homogenized for 5 min in
a Waring blender at 4°C in water. The homogenate was centrifuged at 10,000g for 30min at 4°C and the supernatant fluid decanted through cheesecloth to retain the thick lipid layer. This supernate was freeze-dried and stored desiccated at room temperature.
Materials The following were purchased from Sigma Chemical Company: bovine serum albumin, bovine pancreatic trypsin, p-toluene-sulfonyl-e-arginine methyl ester (TAME), p-toluene-sulfonyl-L-lysine methyl ester (TLME), benzoylarginine naphthylamide (BANA), benzoyl-tyrosine ethyl ester (BTEE), succinyl-(ala)3 nitroanalide (SANA), hippuryl-L-arginine (HLA), hippuryl-L-phenylalanine (HPLA), tosyl-L-lysine chloromethyl ketone (TLCK), iodoacetic acid, and phenylmethylsulfonyl fluoride (PMSDF) Sephadex G-75 Superfine, Q-Sepharose, Sephacryl S-300 Superfine, and Phenyl Sepharose CL-6B were obtained from Pharmacia LKB. All other chemicals were analytical grade. Assay of enzyme activity Trypsin-like activity was assayed using the method of Hummel (1959) with 1 mM TAME as substrate and the buffer modified to be 100mM Tris without CaCI2 at pH 8.2. This concentration of Tris gave optimum activity (data not shown). Assays were performed with a Varian DSM 100S spectrophotometer with a DS15 Data Station and its kinetics storage and calculation program. Assays, except for the temperature studies, were kept at 30°C with an ExaCal circulating water bath. For assay of the enzyme with substrates other than TAME, conditions were the same except for the substitution of the indicated substrate at a final concentration of 1 mM. For the inhibition studies inhibitors were incubated with the crab enzyme for 15 min at 30°C and then the enzyme activity was assayed in the presence of the same inhibitor at the same concentration in the assay mixture. All pH values are given for room temperature. 525
526
JAMESE. DENDINGERand KATHLEENL. O'CONNOR
Extraction of enzyme activity About 1 g of midgut gland powder was mixed with 80 ml and 10 mM Tris, pH 8.0 at 25°C (Buffer A), for 30min at room temperature. This mixture was centrifuged at 12,000g for 15min at 4°C and the supernatant fluid decanted and saved. The pellet was reextracted and centrifuged in the same manner and the supernatant fluids pooled. Solid ammonium sulfate was added to this pool to bring it to 70% saturation. This material was mixed for 30min at room temperature, centrifuged at 12,000g for 15 min at 4°C, and the supernate, which had no enzyme activity, discarded. The pellet was redissolved in Buffer A and chromatographed.
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Chromatography The sample was applied to a 2.5 x 30cm column of Sephadex G-75, which was equilibrated and eluted with Buffer A. Eluent was monitored at 280 nm with an I.S.C.O. UA-5 Absorbance Monitor, and fractions were collected with an I.S.C.O. Retriever III fraction collector. Fractions with trypsin-like activity against TAME (TAME activity) were pooled and freeze dried. The pool from the Sephadex G-75 column was resuspended in Buffer A and applied to a 1.5 x 23 cm column of Q-Sepharase equilibrated with the same buffer. Elution was with a linear gradient of 0-1 M NaC1 in the same buffer. The fractions with TAME activity were pooled and freeze dried to reduce volume. The pool from the Q-Sepharose column was loaded onto a 1.5 × 42 cm column of Sephacryl S-300 equilibrated and eluted with Buffer A. The fractions with TAME activity were pooled and freeze dried to reduce volume. The pool from the Sephacryl S-300 column was mixed with solid ammonium sulfate to 0.7 M and loaded onto a 1.5 × 18 cm column of Phenyl Sepharose equilibrated with 0.7 M ammonium sulfate in Buffer A. Elution was with a 0.74) linear gradient of ammonium sulfate in Buffer A and the fractions with TAME activity were pooled and freeze dried.
Temperature studies To determine the effect of temperature on the enzyme reaction rate, assays were performed at various temperatures maintained with an ExaCal circulating water bath. A 2 min lag time was allowed for the reaction mixture to reach the desired temperature before readings were taken. To determine stability of the enzyme at various temperatures, samples of the enzyme were incubated for 30 min at a particular temperature and then assayed at 30°C.
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0
150
Fig. 1. Chromatography of trypsin-like enzyme from
Callinectes sapidus midgut gland on Sephadex G-75. The column was eluted with 10mM Tris at pH 8.0. RESULTS
Purification The a m m o n i u m sulfate precipitate was serially c h r o m a t o g r a p h e d on ion exchange, molecular sieving and on h y d r o p h o b i c gels to achieve purification (Figs 1-4). Affinity c h r o m a t o g r a p h y on either Benzamadine Sepharose 6B or Soybean Trypsin Inhibitor Sepharose 4B was not successful because the enzyme could not be removed from the gel without inactivation. The blue crab enzyme was inactivated below p H 5 and could not be eluted from the gel with the HC1 as has been done for bovine and other trypsins (Hixson and Nishikawa, 1973). The enzyme was quite stable at p H 7-8.5. At r o o m temperature the activity was c o n s t a n t for several days, and at 4°C for several months. A summary o f the purification steps is s h o w n in Table 1. Figure 5 shows that we obtained only one protein b a n d with S D S - P A G E .
Enzyme characteristics Calcium ions seemed to have an inhibitory effect on the crab trypsin-like activity when either T A M E or T L M E was used as substrate (Fig. 6). As a result,
Electrophoresis Electrophoresis in 12% non-denaturing polyacrylamide gels (Davis, 1964) using a Hoffer Mighty Small Unit at 10 mA per gel at 4°C was used to determine enzyme purity. Electrophoresis in SDS-polyacrylamide gels (Laemmli, 1970) was used to determine both molecular weight and degree of enzyme purity. A Ferguson plot (Rodbard and Chrambach, 1974) ranging from 11 to 15% T was used to check the molecular weight determination. Sigma SDS-70L molecular weight standards were used and the proteins were stained with Coomassie Blue, destained with 10% acetic acid, and then scanned with a Gelman DCD-16 scanner.
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lsoelectric point Isoelectric point was determined using Serva Percoats (pH 3-10) on an Ephortec isoelectric Focusing Cell cooled to 10°C with a circulating water bath. Serva Protein Test Mixture was used as standard and staining was with Serva Blue W.
Protein determination Protein concentrations were measured using the Bio-Rad microassay procedure with bovine serum albumin as standard.
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50
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150
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250
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Fig. 2, Chromatography of trypsin-like enzyme from Callinectes sapidus midgut gland on Q-Sepharos¢, The column was eluted with a 0-1.0 M NaC1 linear gradients in 10 mM Tris at pH 8.0.
Callinectes trypsin-like enzyme
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Fig. 3. Chromatography of trypsin-like enzyme from Callinectes sapidus midgut gland on Sephacryl S-300. The column was eluted with 10 mM Tris at pH 8.0.
calcium was excluded from all solutions. The optim u m p H for the reaction was 8.2 with no apparent activity below p H 5.0 nor much activity above 9.0 (Fig. 7). The rate of the enzyme reaction increased linearly from 15°C to 70°C and above 70°C rapidly decreased (Fig. 8). When the enzyme was preincubated for 30 min at various temperatures and then assayed at 30°C, the activity was stable from 30°C to
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Fig. 4. Chromatography of trypsin-like enzyme from Callinectes sapidus midgut gland on Phenyl Sepharose CL4B. The column was eluted with a 0.7--0.0 M ammonium sulfate linear gradient in I0 mM Tris at pH 8.0.
50°C but above the latter temperature activity was rapidly lost (Fig. 8). The molecular weight was estimated to be 33,500 as determined by SDS electrophoresis against commercial standards (Fig. 9). Because this result was high for a trypsin-like enzyme, the samples were electrophoresed at polyacrylamide con-
Table 1. Purification of trypsin-like enzyme from Callinectes sapidus midgut gland Total activity Total protein Specific activity Method (/zmol TAME/min) (/z g) (,umol TAME/min/mg protein) Original 4808 423,748 11 Ammonium sulfate 4463 264,532 17 Q-Sepharose 3526 10,285 343 Sephacryl S-300 1981 1520 1304 Phenyl Sepharose 395 168 2350
Fig. 5. SDS-PAGE of the trypsin-like enzymes from Callinectes sapidus midgut gland and of molecular weight standards. A-D are the crab enzyme at 5, 10, 20 and 30 #1 samples, respectively, and S are the standards.
528
JAMESE. DENDINGERand KATHLEENL. O'CONNOR 5.0-
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Fig. 6. Effect of calcium on the activity of trypsin-like enzyme from Callinectes sapidus midgut gland with TAME or TLME as substrate. Each point represents the mean of seven assays __+SE. centrations ranging from 11 to 15% T and a linear Ferguson plot was constructed (Ferguson, 1964). A single protein band resulting from both SDS and non-denaturing electrophoresis indicated purity of the enzyme. However, in some enzyme preparations there was a minor band trailing the major protein. When these polyacrylamide gels were stained for trypsin activity (Zwilling et aL, 1969) both hands were positive (results not shown). Isoelectric focusing also resulted in a major and minor protein band and again both stained for trypsin activity (Fig. 10). Both proteins were anionic with an isoelectric point of approximately 4. When the purified crab enzyme was tested against a variety of synthetic substrates TAME and TLME were the only ones degraded. There was no detectable activity against BTEE, BANA, SANA, HLPA or HLA (Table 2). When TAME was used as substrate, the enzyme activity was inhibited by PMSF, as previously reported (Dendinger, 1987), TLCK, and soybean trypsin inhibitor (Table 2). There was also some inhibition by rather high concentrations of EDTA and iodoacetate.
Fig. 8. Effect of reaction temperature and 30 min preincubation temperature on the activity of trypsin-like enzyme from Callinectes sapidus midgut gland. For the reaction temperature (squares) each point represents the mean of five assays + SE. For the preincubation temperature (circles) each point represents the mean of eight + SE with the reactions measured at 30°C. DISCUSSION
Several of the properties of the trypsin-like protease from the blue crab, Callinectes sapidus, can be compared and contrasted with bovine vertebrate trypsin and the trypsin-like enzyme for other Crustacea. One of these properties is the extreme stability of the blue crab tryptic activity in the absence of calcium. This trait has also been noted in two genera of crayfish, Orconectes (DeVillez and Johnson, 1968) and Astacus (Zwilling et al., 1969). In contrast, bovine pancreatic trypsin undergoes rapid self-digestion in the presence of calcium, particularly in alkaline solutions (Zwilling et al., 1969). Limited ability of the crab enzyme to selfdigest indicates that the crab enzyme may be low in arginine and/or lysine residues. It has been reported that crayfish trypsin has only half the 4.703,
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Fig. 7. Effect of pH on the activity of trypsin-like enzyme from Callinectes sapidus midgut gland. Each point represents the mean of six assays +_SE.
Fig. 9. Molecular weight determination on SDS gels for the trypsin-like enzyme from Callineetes sapidus midgut gland. The standards are: (1) egg albumin tool. wt 45,000, (2) glyceraldehyde-3-Pdehydrogenase 36,000, (3) carbonic anhydrase 29,000, (4) trypsinogen 24,000, (5) trypsin inhibitor 20,100 and (6) alpha lactalbumin 14,200.
Callinectes trypsin-like enzyme
Fig. 10. Isoelectric focus of the trypsin-like enzyme (E) from Callinectes sapidus midgut gland. S indicates the standards with their isoelectric points shown on the right. number of lysine residues, as does bovine trypsin (Zwilling et aL, 1969). Inhibition of enzyme activity by calcium ions has been found not only in blue crabs, but also in crayfish (Zwilling et al., 1969). In contrast, calcium did not affect the tryptic activity of the shrimp (Gates and Travis, 1969; Galgani et al., 1984) or the spiny lobster (Galgani and Nagayama, 1987b). It might be presumed that calcium in the seawater which is ingested with food could inhibit the trypsin activity where it would interfere with digestion. Calcium concentration in full seawater is about 10mM (Prosser, 1973) and this concentration in our experiments gave
529
a decrease of about 12% of the trypsin activity, which is probably not physiologically significant. Acidic conditions quickly denatured the blue crab tryptic activity as has been found in crayfish (Zwilling et al., 1969), portunid crabs other than Callinectes (Galgani and Nagayama, 1968), spiny lobster (Galgani and Nagayama, 1987b), shrimp (Gates and Travis, 1969) and Arctic krill (Kimoto et aL, 1983). In contrast, bovine pancreatic trypsin has its greatest stability at low pH. Blue crab tryptic activity was maximal at pH 8.2, which is consistent with the results of pH 8.0-8.5 for other portunid crabs (Galgani and Nagayama, 1986) and 8.0 for Arctic krill (Kimoto et al., 1986). However, the maximum tryptic activity from spiny lobster has been reported to be pH 7.5 (Galgani and Nagayama, 1987b). Another comparable characteristic of the blue crab protease is its low isoelectric point of 4, which indicates its acidic nature. Other crustacean trypsinlike enzymes have been reported to have isoelectric points of 3.8 for crayfish (Zwilling et al., 1969) and 2.6 for Arctic krill (Kimoto et al., 1983). Bovine trypsin has a high isoelectric point between 9.3 and 10.1 (Walsh, 1970). This observation indicates that the crustacean enzyme is high in acidic amino acids. Indeed, it has been reported that both crayfish (Zwilling and Tomasek, 1970) and shrimp (Gates and Travis, 1969) trypsin have more than twice as many acidic amino acids as does the bovine form (Walsh and Neurath, 1964). Also note in Fig. 2 that the blue crab tryptic activity bound rather strongly to the anion exchange gel. We found the molecular weight of the blue crab trypsin-like enzyme to be 33,500, which is higher than has been reported for other crustacean trypsins. The following molecular weight values of trypsin-like enzymes have been reported: from crayfish, 24,700 (DeVillez and Johnson, 1968); shrimp, 24,000 and 25,000 (Gates and Travis, 1969; Galgani et aL, 1984, respectively); crabs, 16,700 and 20,500 (Brun and Wojtowicz, 1976); spiny lobster, 24,000 (Galgani and Nagayama, 1987b); and krill, 28,000-30,000 (Kimono et al., 1983). Specificity of the crab enzyme activity against synthetic substrates indicates that it cleaves next to arginine and lysine residues when coupled with methyl or ethyl esters, but does not cleave next to the arginine when coupled with naphthylamide (BANA) (Table 2). This lack of significant activity
Table 2. Activityof trypsin-likeenzymefrom Callinectes sapidus midgutglandwith varioussubstrates and inhibitors Substrate Inhibitor Average activity % (1 mM) I n h i b i t o r concentration (#mol TAME/min/ml+ SE) inhibition TAME Water -2.77__+0.23 0.0 TAME PMSF 2 mM 0.00 ~ 0.00 100.0 TAME EDTA 10mM 1.95 ± 0.04 29.6 TAME Iodoacetate 10mM 0.52 ± 0.099 81.2 TAME TLCK 2 mM 0.00__+0.00 100.0 TAME STI 50#g/ml 0.01 ___0.00 99.8 TLME None -1.25___0.02 -BTEE None -0.01 __+0.00 -BANA None 0 -SANA None -0 -HLPA None -0 -HLA None -0 -Inhibitorswere incubatedwith enzymefor 15min at 30°C before assay in inhibitors.
530
JAMESE. DENDINGERand KATHLEENL. O'CONNOR
against the latter substrate has been reported with both crustacean and bovine trypsin (Kimono et al., 1983). The activity with T A M E was more than twice that with T L M E as substrate. There was little or no "chymotryptic activity" against BTEE, no activity against the elastase substrate S A N A , or against the carboxypeptidase A and B substrates H L P A and H L A , respectively. The fact that the enzyme is inhibited by P M S F indicates that it is a serine protease rather than a metalo- or sulfhydryl-enzyme. That it is inhibited by both synthetic (TLCK) and natural (STI) trypsin inhibitors indicates that it is in fact a trypsin belonging to that family o f enzymes first identified and named from bovine pancreas. The evolutionary relationship of this blue crab trypsin to other serine proteases from Crustacea is still equivocal. We have found two serine proteases in the blue crab which appear to have chymotrypsin and elastase activity and they are still undergoing purification and characterization. Acknowledgement--This work was supported by a grant from the James Madison University Program of Grants for Faculty Research.
REFERENCES
Brockerhoff H., Hoyle R. J. and Hwang P. C. (1970) Digestive enzymes of the American lobster, Homarus americanus. J. Fish. Res. Bd Can. 27, 1357-1370. Brun G. L. and Wojtowicz M. B. (1976) A comparative study of the digestive enzymes in the hepatopancreas of Jonah crab (Cancer borealis) and rock crab (Cancer irroratus). Comp. Biochem. Physiol. 53B, 387-391. Davis B. J. (1964) Disc electrophoresis II: method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404-427. Dendinger J. E. (1987) Digestive proteases in the midgut gland of the Atlantic blue crab, Callinectes sapidus. Comp. Biochem. Physiol. 88B, 503-506. DeVillez E. J. (1965) Isolation of the proteolytic digestive enzymes from the gastric juice of the crayfish Orconectes virilis (Hagan). Comp. Biochem. Physiol. 14, 577-586. DeVillez E. J. and Buschlen K. (1967) Survey of a tryptic digestive enzyme in various species of Crustacea. Comp. Biochem. Physiol. 21, 541-546. DeVillez E. J. and Johnson D. R. (1986) Determination of the molecular weight of crustacean trypsin by gelfiltration. Comp. Biochem. Physiol. 24, 661-664. Ferguson K. A. (1964) Starch-gel electrophoresis.--Application to the classification of pituitary proteins and polypeptides. Metabolism 13, 985-1002. Galgani F. and Nagayama F. (1986) Characteristics of digestive proteolysis of the crabs Portunus trituberculatus,
Portuns sanguinolentus and Charybdis japonica. Bull. Jap. Soc. scient. Fish. 52, 2183-2188. Galgani F. and Nagayama F. (1987a) Digestive proteinases in five species of Lithodidae (Crustacea, Decapoda). Comp. Biochem. Physiol. $7B, 103 107. Galgani F. and Nagayama F. (1987b) Digestive proteinases in the Japanese spiny lobster Panulirus japonicus. Comp. Biochem. Physiol. $7B, 889-893. Galgani F., Benyamin Y. and Ceccaldi J. J. (1984) Identification of digestive proteinases of Penaeus kerathurus (Forskal): a comparison with Penaeus japonicus Bate. Comp. Biochem. Physiol. 76B, 355-361. Gates B. J. and Travis J. (1969) Isolation and comparative properties of shrimp trypsin. Biochemistry 8, 4483-4489. Hixson H. F., Jr. and Nishikawa A. H. (1973) Affinity chromatography: purification of bovine trypsin and thrombin. Arch. Biochem. Biophys. 154, 501-509. Hummel B. (1959) A modified spectrophotometric determination of chymotrypsin, trypsin and thrombin. Can. J. Biochem. Physiol. 37, 1393-1399. Juhasz S., Galfi P. and Molnar K. (1980) Data on the proteolytic enzyme system of Lernaea cyprinacea. Acta Vet. Acad. scient. Hung. 28, 57-69. Kimoto K., Kusama S. and Murakami K. (1983) Purification and characterization of serine proteinases from Euphausia superba. Agric. Biol. Chem. 47, 529-534. Kimoto K., Fukamizu A. and Murakami K. (1986) Partial purification and characterization of proteinases from abdomen part muscle of Antarctic krill. Bull. Jap. Soc. scient. Fish. 52, 745-749. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 227, 680-685. Prosser C. L. (1973) Comparative Animal Physiology, 3rd edn. W. B. Saunders, Philadelphia. Rodbard D. and Chrambach A. (1974) Quantitative polyacrylamide gel electrophoresis: mathematical and statistical analysis of data. In Electrophoresis and lsoelectric Focusing in Polyacrylamide Gel (Edited by Allen R. C. and Maurer H. R.), pp. 28-62. Walter de Gruyter, New York. Titani K., Sasagawa T., Woodbury R. G., Ericsson L. H., Dorsam H., Kraemer M., Neurath H. and Zwilling R. (1983) Amino acid sequence of crayfish (Astacus fluviatilis) trypsin I r. Biochemistry 22, 1459-1465. Walsh K. A. (1970) Trypsinogens and trypsins of various species. In Methods in Enzymology (Edited by Perlmann G. E. and Lorand L.), Vol. 19, pp. 41-63. Academic Press, New York. Walsh K. A. and Neurath H. (1964) Trypsinogen and chymotrypsinogen as homologous proteins. Proc. natn. Acad. Sci. USA 52, 884-889. Zwilling R. and Neurath H. (1981) Invertebrate proteases. In Methods in Enzymology (Edited by Lorand L.), Vol. 80, pp. 633-664. Academic Press, New York. Zwilling R. and Tomasek V. (1970) Amino acid composition of crayfish trypsin. Nature 228, 57-58. Zwilling R., Pfleiderer G., Sonnerborn H., Kraft V. and Stucky I. (1969) The evolution of endopeptidases. V. Common and different traits of bovine and crayfish trypsin. Comp. Biochem. Physiol. 28, 1275-1287.
0305-0491/90 $3.00 + 0.00 © 1990 Pergamon Press plc
PURIFICATION A N D CHARACTERIZATION OF A TRYPSIN-LIKE ENZYME FROM THE M I D G U T G L A N D OF THE ATLANTIC BLUE CRAB, C A L L I N E C T E S SAPIDUS JAMES E. DENDINGER and KATHLEEN L. O'CONNOR Department of Biology, James Madison University, Harrisonburg, VA 22807, USA (Tel: (703) 433-6225)
(Recewed 14July 1989) Abstract--1. A trypsin-like enzyme from the midgut gland of the Atlantic blue crab, Callinectes sapidus, was purified and studied. 2. Purification was achieved by a combination of molecular sieving, ion exchange, and hydrophobic chromatography. Degree of purity was assessed by SDS-polyacrylamide electrophoresis. 3. Using N-p-tosyl-L-arginine methyl ester (TAME) as a substrate, the enzyme displays optimal activity in the absence of calcium at pH 8.2. 4. The tryptic activity is extremely stable between pH 7 and 8.5, but is rapidly inactivated below pH 6. Thirty-minute incubations above 50°C also inactivate the enzyme. 5. The tryptic activity has a molecular weight of 33,500 da and an isoelectric point of approximately 4. 6. The trypsin-like protease from Callinectes sapidus is similar to crayfish and bovine trypsins in that it is inhibited by phenylmethane sulfonylfluoride, tosyl-L-lysine chloromethyl ketone, and soybean trypsin inhibitor. Its pH optimum and its specificity for TAME and N-p-tosyl-L-lysine methyl ester (TLME) are also similar. It is concluded that the enzyme characterized in this study is trypsin.
INTRODUCTION In the numerous reports on digestive proteases found in Crustacea, almost all have included at least one trypsin-like enzyme (DeVillez and Buschlen, 1967). Some of the animals found to have this enzyme included copepods (Juhasz et al., 1980), several species of crayfish (DeVillez, 1965; Zwilling and Neurath, 1981), shrimp (Gates and Travis, 1969; Galgani et al., 1984), krill (Kimoto et al., 1983), lobster (Brockerhoff et al., 1970; Galgani and Nagayama, 1987b), and crabs (Brun and Wojtowicz, 1976; Galgani and Nagayama, 1986, 1987a; Dendinger, 1987). Some of these trypsin-like enzymes have been purified (Zwilling and Neurath, 1981; Titani et al., 1983) and although most of the characteristics of these enzymes are similar to vertebrate trypsin, some characteristics are markedly different. These differences include calcium ion requirements for the vertebrate enzyme catalysis and stability (Zwilling et al., 1969), and isoelectric point (Zwilling et al., 1969; K i m o t o et al., 1983). The Callinectes trypsin-like enzyme was first described in a previous paper (Dendinger, 1987) and the purpose of this study was to compare and contrast the Callinectes sapidus trypsin-like enzyme with that of other Crustacea and with the vertebrate form. MATERIALS AND METHODS
Animals and tissues Adult male blue crabs were obtained alive from commercial sources in the Chesapeake Bay of Virginia. Each midgut gland was removed and frozen until further processing in the laboratory. Frozen samples were homogenized for 5 min in
a Waring blender at 4°C in water. The homogenate was centrifuged at 10,000g for 30min at 4°C and the supernatant fluid decanted through cheesecloth to retain the thick lipid layer. This supernate was freeze-dried and stored desiccated at room temperature.
Materials The following were purchased from Sigma Chemical Company: bovine serum albumin, bovine pancreatic trypsin, p-toluene-sulfonyl-e-arginine methyl ester (TAME), p-toluene-sulfonyl-L-lysine methyl ester (TLME), benzoylarginine naphthylamide (BANA), benzoyl-tyrosine ethyl ester (BTEE), succinyl-(ala)3 nitroanalide (SANA), hippuryl-L-arginine (HLA), hippuryl-L-phenylalanine (HPLA), tosyl-L-lysine chloromethyl ketone (TLCK), iodoacetic acid, and phenylmethylsulfonyl fluoride (PMSDF) Sephadex G-75 Superfine, Q-Sepharose, Sephacryl S-300 Superfine, and Phenyl Sepharose CL-6B were obtained from Pharmacia LKB. All other chemicals were analytical grade. Assay of enzyme activity Trypsin-like activity was assayed using the method of Hummel (1959) with 1 mM TAME as substrate and the buffer modified to be 100mM Tris without CaCI2 at pH 8.2. This concentration of Tris gave optimum activity (data not shown). Assays were performed with a Varian DSM 100S spectrophotometer with a DS15 Data Station and its kinetics storage and calculation program. Assays, except for the temperature studies, were kept at 30°C with an ExaCal circulating water bath. For assay of the enzyme with substrates other than TAME, conditions were the same except for the substitution of the indicated substrate at a final concentration of 1 mM. For the inhibition studies inhibitors were incubated with the crab enzyme for 15 min at 30°C and then the enzyme activity was assayed in the presence of the same inhibitor at the same concentration in the assay mixture. All pH values are given for room temperature. 525
526
JAMESE. DENDINGERand KATHLEENL. O'CONNOR
Extraction of enzyme activity About 1 g of midgut gland powder was mixed with 80 ml and 10 mM Tris, pH 8.0 at 25°C (Buffer A), for 30min at room temperature. This mixture was centrifuged at 12,000g for 15min at 4°C and the supernatant fluid decanted and saved. The pellet was reextracted and centrifuged in the same manner and the supernatant fluids pooled. Solid ammonium sulfate was added to this pool to bring it to 70% saturation. This material was mixed for 30min at room temperature, centrifuged at 12,000g for 15 min at 4°C, and the supernate, which had no enzyme activity, discarded. The pellet was redissolved in Buffer A and chromatographed.
B -
125
._=
m,
g
×
ku
- 63
~
Chromatography The sample was applied to a 2.5 x 30cm column of Sephadex G-75, which was equilibrated and eluted with Buffer A. Eluent was monitored at 280 nm with an I.S.C.O. UA-5 Absorbance Monitor, and fractions were collected with an I.S.C.O. Retriever III fraction collector. Fractions with trypsin-like activity against TAME (TAME activity) were pooled and freeze dried. The pool from the Sephadex G-75 column was resuspended in Buffer A and applied to a 1.5 x 23 cm column of Q-Sepharase equilibrated with the same buffer. Elution was with a linear gradient of 0-1 M NaC1 in the same buffer. The fractions with TAME activity were pooled and freeze dried to reduce volume. The pool from the Q-Sepharose column was loaded onto a 1.5 × 42 cm column of Sephacryl S-300 equilibrated and eluted with Buffer A. The fractions with TAME activity were pooled and freeze dried to reduce volume. The pool from the Sephacryl S-300 column was mixed with solid ammonium sulfate to 0.7 M and loaded onto a 1.5 × 18 cm column of Phenyl Sepharose equilibrated with 0.7 M ammonium sulfate in Buffer A. Elution was with a 0.74) linear gradient of ammonium sulfate in Buffer A and the fractions with TAME activity were pooled and freeze dried.
Temperature studies To determine the effect of temperature on the enzyme reaction rate, assays were performed at various temperatures maintained with an ExaCal circulating water bath. A 2 min lag time was allowed for the reaction mixture to reach the desired temperature before readings were taken. To determine stability of the enzyme at various temperatures, samples of the enzyme were incubated for 30 min at a particular temperature and then assayed at 30°C.
I
I
50
I
100 Eluent (ml)
0
150
Fig. 1. Chromatography of trypsin-like enzyme from
Callinectes sapidus midgut gland on Sephadex G-75. The column was eluted with 10mM Tris at pH 8.0. RESULTS
Purification The a m m o n i u m sulfate precipitate was serially c h r o m a t o g r a p h e d on ion exchange, molecular sieving and on h y d r o p h o b i c gels to achieve purification (Figs 1-4). Affinity c h r o m a t o g r a p h y on either Benzamadine Sepharose 6B or Soybean Trypsin Inhibitor Sepharose 4B was not successful because the enzyme could not be removed from the gel without inactivation. The blue crab enzyme was inactivated below p H 5 and could not be eluted from the gel with the HC1 as has been done for bovine and other trypsins (Hixson and Nishikawa, 1973). The enzyme was quite stable at p H 7-8.5. At r o o m temperature the activity was c o n s t a n t for several days, and at 4°C for several months. A summary o f the purification steps is s h o w n in Table 1. Figure 5 shows that we obtained only one protein b a n d with S D S - P A G E .
Enzyme characteristics Calcium ions seemed to have an inhibitory effect on the crab trypsin-like activity when either T A M E or T L M E was used as substrate (Fig. 6). As a result,
Electrophoresis Electrophoresis in 12% non-denaturing polyacrylamide gels (Davis, 1964) using a Hoffer Mighty Small Unit at 10 mA per gel at 4°C was used to determine enzyme purity. Electrophoresis in SDS-polyacrylamide gels (Laemmli, 1970) was used to determine both molecular weight and degree of enzyme purity. A Ferguson plot (Rodbard and Chrambach, 1974) ranging from 11 to 15% T was used to check the molecular weight determination. Sigma SDS-70L molecular weight standards were used and the proteins were stained with Coomassie Blue, destained with 10% acetic acid, and then scanned with a Gelman DCD-16 scanner.
-1
o
-0.s
45
×
lsoelectric point Isoelectric point was determined using Serva Percoats (pH 3-10) on an Ephortec isoelectric Focusing Cell cooled to 10°C with a circulating water bath. Serva Protein Test Mixture was used as standard and staining was with Serva Blue W.
Protein determination Protein concentrations were measured using the Bio-Rad microassay procedure with bovine serum albumin as standard.
I
50
I
I
150
I
I
0
250
Eluent (ml)
Fig. 2, Chromatography of trypsin-like enzyme from Callinectes sapidus midgut gland on Q-Sepharos¢, The column was eluted with a 0-1.0 M NaC1 linear gradients in 10 mM Tris at pH 8.0.
Callinectes trypsin-like enzyme
527
-0.7
D
w
<
I
115
o
<~
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o
95
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x
b
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0 50
75
Eluent (ml)
Fig. 3. Chromatography of trypsin-like enzyme from Callinectes sapidus midgut gland on Sephacryl S-300. The column was eluted with 10 mM Tris at pH 8.0.
calcium was excluded from all solutions. The optim u m p H for the reaction was 8.2 with no apparent activity below p H 5.0 nor much activity above 9.0 (Fig. 7). The rate of the enzyme reaction increased linearly from 15°C to 70°C and above 70°C rapidly decreased (Fig. 8). When the enzyme was preincubated for 30 min at various temperatures and then assayed at 30°C, the activity was stable from 30°C to
I
I
200 Eluent (ml)
100
300
Fig. 4. Chromatography of trypsin-like enzyme from Callinectes sapidus midgut gland on Phenyl Sepharose CL4B. The column was eluted with a 0.7--0.0 M ammonium sulfate linear gradient in I0 mM Tris at pH 8.0.
50°C but above the latter temperature activity was rapidly lost (Fig. 8). The molecular weight was estimated to be 33,500 as determined by SDS electrophoresis against commercial standards (Fig. 9). Because this result was high for a trypsin-like enzyme, the samples were electrophoresed at polyacrylamide con-
Table 1. Purification of trypsin-like enzyme from Callinectes sapidus midgut gland Total activity Total protein Specific activity Method (/zmol TAME/min) (/z g) (,umol TAME/min/mg protein) Original 4808 423,748 11 Ammonium sulfate 4463 264,532 17 Q-Sepharose 3526 10,285 343 Sephacryl S-300 1981 1520 1304 Phenyl Sepharose 395 168 2350
Fig. 5. SDS-PAGE of the trypsin-like enzymes from Callinectes sapidus midgut gland and of molecular weight standards. A-D are the crab enzyme at 5, 10, 20 and 30 #1 samples, respectively, and S are the standards.
528
JAMESE. DENDINGERand KATHLEENL. O'CONNOR 5.0-
=O-TAME Activity
1.0-
5
0.8"
4
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0.6"
3
E
0.4-
2
0.2-
1
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4.0 ._> co
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0.00
0.10 CaLcium Concentration (M)
10
0"20
20
30
40
50
60
70
80
Temperature (°C)
Fig. 6. Effect of calcium on the activity of trypsin-like enzyme from Callinectes sapidus midgut gland with TAME or TLME as substrate. Each point represents the mean of seven assays __+SE. centrations ranging from 11 to 15% T and a linear Ferguson plot was constructed (Ferguson, 1964). A single protein band resulting from both SDS and non-denaturing electrophoresis indicated purity of the enzyme. However, in some enzyme preparations there was a minor band trailing the major protein. When these polyacrylamide gels were stained for trypsin activity (Zwilling et aL, 1969) both hands were positive (results not shown). Isoelectric focusing also resulted in a major and minor protein band and again both stained for trypsin activity (Fig. 10). Both proteins were anionic with an isoelectric point of approximately 4. When the purified crab enzyme was tested against a variety of synthetic substrates TAME and TLME were the only ones degraded. There was no detectable activity against BTEE, BANA, SANA, HLPA or HLA (Table 2). When TAME was used as substrate, the enzyme activity was inhibited by PMSF, as previously reported (Dendinger, 1987), TLCK, and soybean trypsin inhibitor (Table 2). There was also some inhibition by rather high concentrations of EDTA and iodoacetate.
Fig. 8. Effect of reaction temperature and 30 min preincubation temperature on the activity of trypsin-like enzyme from Callinectes sapidus midgut gland. For the reaction temperature (squares) each point represents the mean of five assays + SE. For the preincubation temperature (circles) each point represents the mean of eight + SE with the reactions measured at 30°C. DISCUSSION
Several of the properties of the trypsin-like protease from the blue crab, Callinectes sapidus, can be compared and contrasted with bovine vertebrate trypsin and the trypsin-like enzyme for other Crustacea. One of these properties is the extreme stability of the blue crab tryptic activity in the absence of calcium. This trait has also been noted in two genera of crayfish, Orconectes (DeVillez and Johnson, 1968) and Astacus (Zwilling et al., 1969). In contrast, bovine pancreatic trypsin undergoes rapid self-digestion in the presence of calcium, particularly in alkaline solutions (Zwilling et al., 1969). Limited ability of the crab enzyme to selfdigest indicates that the crab enzyme may be low in arginine and/or lysine residues. It has been reported that crayfish trypsin has only half the 4.703,
4-
4.60 ° t ~ ' N ~
2
1
~°~ nknown
3-
--~
4.40 -
O'~ 4
~'
o
4.30
2-
-a 1
4 2
0
4.10 3.0
~ i , i , i , i , i , i 4.0 5.0 6.0 7.0 8.0 9 0 Migration Distance (cm)
0 5
6
7
8
9
i 10
pH
Fig. 7. Effect of pH on the activity of trypsin-like enzyme from Callinectes sapidus midgut gland. Each point represents the mean of six assays +_SE.
Fig. 9. Molecular weight determination on SDS gels for the trypsin-like enzyme from Callineetes sapidus midgut gland. The standards are: (1) egg albumin tool. wt 45,000, (2) glyceraldehyde-3-Pdehydrogenase 36,000, (3) carbonic anhydrase 29,000, (4) trypsinogen 24,000, (5) trypsin inhibitor 20,100 and (6) alpha lactalbumin 14,200.
Callinectes trypsin-like enzyme
Fig. 10. Isoelectric focus of the trypsin-like enzyme (E) from Callinectes sapidus midgut gland. S indicates the standards with their isoelectric points shown on the right. number of lysine residues, as does bovine trypsin (Zwilling et aL, 1969). Inhibition of enzyme activity by calcium ions has been found not only in blue crabs, but also in crayfish (Zwilling et al., 1969). In contrast, calcium did not affect the tryptic activity of the shrimp (Gates and Travis, 1969; Galgani et al., 1984) or the spiny lobster (Galgani and Nagayama, 1987b). It might be presumed that calcium in the seawater which is ingested with food could inhibit the trypsin activity where it would interfere with digestion. Calcium concentration in full seawater is about 10mM (Prosser, 1973) and this concentration in our experiments gave
529
a decrease of about 12% of the trypsin activity, which is probably not physiologically significant. Acidic conditions quickly denatured the blue crab tryptic activity as has been found in crayfish (Zwilling et al., 1969), portunid crabs other than Callinectes (Galgani and Nagayama, 1968), spiny lobster (Galgani and Nagayama, 1987b), shrimp (Gates and Travis, 1969) and Arctic krill (Kimoto et aL, 1983). In contrast, bovine pancreatic trypsin has its greatest stability at low pH. Blue crab tryptic activity was maximal at pH 8.2, which is consistent with the results of pH 8.0-8.5 for other portunid crabs (Galgani and Nagayama, 1986) and 8.0 for Arctic krill (Kimoto et al., 1986). However, the maximum tryptic activity from spiny lobster has been reported to be pH 7.5 (Galgani and Nagayama, 1987b). Another comparable characteristic of the blue crab protease is its low isoelectric point of 4, which indicates its acidic nature. Other crustacean trypsinlike enzymes have been reported to have isoelectric points of 3.8 for crayfish (Zwilling et al., 1969) and 2.6 for Arctic krill (Kimoto et al., 1983). Bovine trypsin has a high isoelectric point between 9.3 and 10.1 (Walsh, 1970). This observation indicates that the crustacean enzyme is high in acidic amino acids. Indeed, it has been reported that both crayfish (Zwilling and Tomasek, 1970) and shrimp (Gates and Travis, 1969) trypsin have more than twice as many acidic amino acids as does the bovine form (Walsh and Neurath, 1964). Also note in Fig. 2 that the blue crab tryptic activity bound rather strongly to the anion exchange gel. We found the molecular weight of the blue crab trypsin-like enzyme to be 33,500, which is higher than has been reported for other crustacean trypsins. The following molecular weight values of trypsin-like enzymes have been reported: from crayfish, 24,700 (DeVillez and Johnson, 1968); shrimp, 24,000 and 25,000 (Gates and Travis, 1969; Galgani et aL, 1984, respectively); crabs, 16,700 and 20,500 (Brun and Wojtowicz, 1976); spiny lobster, 24,000 (Galgani and Nagayama, 1987b); and krill, 28,000-30,000 (Kimono et al., 1983). Specificity of the crab enzyme activity against synthetic substrates indicates that it cleaves next to arginine and lysine residues when coupled with methyl or ethyl esters, but does not cleave next to the arginine when coupled with naphthylamide (BANA) (Table 2). This lack of significant activity
Table 2. Activityof trypsin-likeenzymefrom Callinectes sapidus midgutglandwith varioussubstrates and inhibitors Substrate Inhibitor Average activity % (1 mM) I n h i b i t o r concentration (#mol TAME/min/ml+ SE) inhibition TAME Water -2.77__+0.23 0.0 TAME PMSF 2 mM 0.00 ~ 0.00 100.0 TAME EDTA 10mM 1.95 ± 0.04 29.6 TAME Iodoacetate 10mM 0.52 ± 0.099 81.2 TAME TLCK 2 mM 0.00__+0.00 100.0 TAME STI 50#g/ml 0.01 ___0.00 99.8 TLME None -1.25___0.02 -BTEE None -0.01 __+0.00 -BANA None 0 -SANA None -0 -HLPA None -0 -HLA None -0 -Inhibitorswere incubatedwith enzymefor 15min at 30°C before assay in inhibitors.
530
JAMESE. DENDINGERand KATHLEENL. O'CONNOR
against the latter substrate has been reported with both crustacean and bovine trypsin (Kimono et al., 1983). The activity with T A M E was more than twice that with T L M E as substrate. There was little or no "chymotryptic activity" against BTEE, no activity against the elastase substrate S A N A , or against the carboxypeptidase A and B substrates H L P A and H L A , respectively. The fact that the enzyme is inhibited by P M S F indicates that it is a serine protease rather than a metalo- or sulfhydryl-enzyme. That it is inhibited by both synthetic (TLCK) and natural (STI) trypsin inhibitors indicates that it is in fact a trypsin belonging to that family o f enzymes first identified and named from bovine pancreas. The evolutionary relationship of this blue crab trypsin to other serine proteases from Crustacea is still equivocal. We have found two serine proteases in the blue crab which appear to have chymotrypsin and elastase activity and they are still undergoing purification and characterization. Acknowledgement--This work was supported by a grant from the James Madison University Program of Grants for Faculty Research.
REFERENCES
Brockerhoff H., Hoyle R. J. and Hwang P. C. (1970) Digestive enzymes of the American lobster, Homarus americanus. J. Fish. Res. Bd Can. 27, 1357-1370. Brun G. L. and Wojtowicz M. B. (1976) A comparative study of the digestive enzymes in the hepatopancreas of Jonah crab (Cancer borealis) and rock crab (Cancer irroratus). Comp. Biochem. Physiol. 53B, 387-391. Davis B. J. (1964) Disc electrophoresis II: method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404-427. Dendinger J. E. (1987) Digestive proteases in the midgut gland of the Atlantic blue crab, Callinectes sapidus. Comp. Biochem. Physiol. 88B, 503-506. DeVillez E. J. (1965) Isolation of the proteolytic digestive enzymes from the gastric juice of the crayfish Orconectes virilis (Hagan). Comp. Biochem. Physiol. 14, 577-586. DeVillez E. J. and Buschlen K. (1967) Survey of a tryptic digestive enzyme in various species of Crustacea. Comp. Biochem. Physiol. 21, 541-546. DeVillez E. J. and Johnson D. R. (1986) Determination of the molecular weight of crustacean trypsin by gelfiltration. Comp. Biochem. Physiol. 24, 661-664. Ferguson K. A. (1964) Starch-gel electrophoresis.--Application to the classification of pituitary proteins and polypeptides. Metabolism 13, 985-1002. Galgani F. and Nagayama F. (1986) Characteristics of digestive proteolysis of the crabs Portunus trituberculatus,
Portuns sanguinolentus and Charybdis japonica. Bull. Jap. Soc. scient. Fish. 52, 2183-2188. Galgani F. and Nagayama F. (1987a) Digestive proteinases in five species of Lithodidae (Crustacea, Decapoda). Comp. Biochem. Physiol. $7B, 103 107. Galgani F. and Nagayama F. (1987b) Digestive proteinases in the Japanese spiny lobster Panulirus japonicus. Comp. Biochem. Physiol. $7B, 889-893. Galgani F., Benyamin Y. and Ceccaldi J. J. (1984) Identification of digestive proteinases of Penaeus kerathurus (Forskal): a comparison with Penaeus japonicus Bate. Comp. Biochem. Physiol. 76B, 355-361. Gates B. J. and Travis J. (1969) Isolation and comparative properties of shrimp trypsin. Biochemistry 8, 4483-4489. Hixson H. F., Jr. and Nishikawa A. H. (1973) Affinity chromatography: purification of bovine trypsin and thrombin. Arch. Biochem. Biophys. 154, 501-509. Hummel B. (1959) A modified spectrophotometric determination of chymotrypsin, trypsin and thrombin. Can. J. Biochem. Physiol. 37, 1393-1399. Juhasz S., Galfi P. and Molnar K. (1980) Data on the proteolytic enzyme system of Lernaea cyprinacea. Acta Vet. Acad. scient. Hung. 28, 57-69. Kimoto K., Kusama S. and Murakami K. (1983) Purification and characterization of serine proteinases from Euphausia superba. Agric. Biol. Chem. 47, 529-534. Kimoto K., Fukamizu A. and Murakami K. (1986) Partial purification and characterization of proteinases from abdomen part muscle of Antarctic krill. Bull. Jap. Soc. scient. Fish. 52, 745-749. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 227, 680-685. Prosser C. L. (1973) Comparative Animal Physiology, 3rd edn. W. B. Saunders, Philadelphia. Rodbard D. and Chrambach A. (1974) Quantitative polyacrylamide gel electrophoresis: mathematical and statistical analysis of data. In Electrophoresis and lsoelectric Focusing in Polyacrylamide Gel (Edited by Allen R. C. and Maurer H. R.), pp. 28-62. Walter de Gruyter, New York. Titani K., Sasagawa T., Woodbury R. G., Ericsson L. H., Dorsam H., Kraemer M., Neurath H. and Zwilling R. (1983) Amino acid sequence of crayfish (Astacus fluviatilis) trypsin I r. Biochemistry 22, 1459-1465. Walsh K. A. (1970) Trypsinogens and trypsins of various species. In Methods in Enzymology (Edited by Perlmann G. E. and Lorand L.), Vol. 19, pp. 41-63. Academic Press, New York. Walsh K. A. and Neurath H. (1964) Trypsinogen and chymotrypsinogen as homologous proteins. Proc. natn. Acad. Sci. USA 52, 884-889. Zwilling R. and Neurath H. (1981) Invertebrate proteases. In Methods in Enzymology (Edited by Lorand L.), Vol. 80, pp. 633-664. Academic Press, New York. Zwilling R. and Tomasek V. (1970) Amino acid composition of crayfish trypsin. Nature 228, 57-58. Zwilling R., Pfleiderer G., Sonnerborn H., Kraft V. and Stucky I. (1969) The evolution of endopeptidases. V. Common and different traits of bovine and crayfish trypsin. Comp. Biochem. Physiol. 28, 1275-1287.