A simple procedure for the purification of the zymogens and active ...

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trypsins (Malvano et al., 1979), and human cationic trypsinogen. (Brodrick et al., 1978). Additionally, synthetic ligands such as proflavin (Brantner et al., 1976) ...
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603rd MEETING, LIVERPOOL

A simple procedure for the purification of the zymogens and active forms of canine anionic trypsin, cationic trypsin and chymotrypsin DAVID A. WILLIAMS,* ROGER M. BATT" and ROBERT J. BEYNONt *Department of Veterinary Pathology and ?Department of Biochemistry, University of Liverpool, P.O. Box 147, Liverpool L69 JBX, U.K. In order to prepare canine trypsins (EC 3.4.21.4) for use in radioimmunoassay, a convenient method for their purification was required. Amnity chromatography has been used with considerable success in the purification of serine proteinases. Naturally occurring inhibitors such as chicken ovomucoid, bovine pancreatic trypsin inhibitor and lima-bean trypsin inhibitor have facilitated the resolution of bovine u- and &trypsins (Robinson et al., 1971), human anionic and cationic trypsins (Malvano et al., 1979), and human cationic trypsinogen (Brodrick et al., 1978). Additionally, synthetic ligands such as proflavin (Brantner et al., 1976) and 4-aminobenzamidine (Schmer, 1972) have been shown to bind serine proteinases, and thus several potential options exist for the purification of canine trypsins. The present communication reports that 4-aminobenzamidine, covalently linked to agarose beads via a 6amino-n-hexanoic acid spacer (Schmer, 1972), is a suitable aflinity matrix for this purpose, and in addition may be used to purify the trypsinogens. The matrix also retards the passage of chymotrypsin (EC 3.4.21.1) and chymotrypsinogen. Pancreas (1OOg) stored frozen at -2OOC was homogenized in 0.125 M-H,SO,, and the supernatant was fractionated between 0 . 8 ~ -and 3.0hi-(NH4),S04 (Travis & Roberts, 1969). The precipitate was dissolved in water, dialysed exhaustively against ImM-HCI and freeze-dried to yield approx. 1.5g of crude extract. Gel filtration on a 90cm x 3.8cm2 column of Sephadex G-75 in 0.2 M-NaCI/O.OS M-CaCl, medium pH 2.6, yielded two major elution peaks. The second peak contained the zymogens, and these fractions were dialysed and freeze-dried. Pancreatic secretory trypsin inhibitor (Fritz, 1967) was eluted in a minor peak immediately after the zymogens. The fractions containing the pancreatic trypsin inhibitor were pooled and adjusted to pH9.0 by addition of NaOH to give a crude inhibitor solution. The partially purified zymogens were further resolved by dissolving the freeze-dried residue in solution containing the pancreatic trypsin inhibitor and applying this extract in SOmM-Tris buffer, pH9.75, containing 2m~-CaC1, to the aflinity matrix in a 12cm x 0.8cm2 column. Chymotrypsinogen was retarded, but was eluted rapidly with buffer containing 0.1 M-NaCl. Cationic trypsinogen was desorbed with buffer containing 0.2 M-NaCl. Anionic trypsinogen was eluted slowly by high-ionic-strength solutions, but was desorbed readily with 50mM-sodium acetate buffer, pH 3.75, containing 2mM-CaC1,. To resolve active enzymes the partially purified zymogens obtained by gel filtration were allowed to autoactivate for 12h at 4°C in 0.1 M-TI-~sbuffer, pH8, containing 4 m ~ - C a C l ,in the absence of the pancreatic trypsin inhibitor. This extract was applied to the affinity matrix in SOmM-Tris buffer, pH8.0, containing 21AM-CaC1,. Chymotrypsin was desorbed with the same buffer containing O.SwNaC1. Cationic trypsin was eluted with 50mM-sodium acetate buffer, pH4.25, containing 2 mMCaCl,, and anionic trypsin was eluted with 5OmM-sodium acetate buffer, pH 3.25, containing 2mwCaC1,. Typical purification schemes for the zymogen and active forms of anionic and cationic trypsin are shown in Table 1. Trypsin and chymotrypsin were assayed with benzoylarginine ethyl ester and acetyltyrosine ethyl ester respectively (Schwert &

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Table 1. Purijication of the zymogens and active forms of canine anionic and cationic trypsins Total Specific Recovery Total enzyme protein activity of trypsin (&t) (mg) (pkathg) activity (%) (NH,),SO, precipitation 63.9 250 0.26 100 60.9 I20 Sephadex G-75 0.5 1 95.4 Affinity chromatography (a)

Zymogens

Anionic trypsinogen Cationic trypsinogen (b) Active enzymes Anionic trypsin Cationic trypsin

7.5 36.3

15.7 23.3

0.48 1.56

11.7 56.8

11.0 44.9

19.2 26

0.57 1.73

17.3 70.3

Takenaka, 1955). Before assay, zymogen-containing fractions were activated at 30°C with 0.32 units of pig enteropeptidase (EC 3.4.21.9) (Sigma Chemical Co.)/ml or approx. 20nkat of crude canine trypsin/ml. Protein was determined by the method of Schacterle & Pollack (1973), with bovine serum albumin as standard. Trypsinogen activity could not be reliably determined in crude tissue preparations, and the recoveries are based on the activity in the (NH,),SO, precipitate. The presence of the pancreatic trypsin inhibitor in this fraction probably caused some underestimation of the trypsinogen activity at this stage of the purification, but this error is unlikely to be greater than 5% of the measured activity (Fritz, 1967). Discontinuous sodium dodecyl sulphate/polyacrylamide-gelelectrophoresis in 15% gels (Studier, 1973) showed single bands for the zymogens and for chymotrypsin. Anionic and cationic trypsins both migrated as two closely spaced bands. Active-site titration with pnitrophenyl pguanidinobenzoate hydrochloride (Chase & Shaw, 1967) showed the trypsins to be 90-95% pure, Affinity chromatography with the use of 4-aminobenzamidine is therefore a useful procedure for separation of the zymogens and active forms of canine trypsins and chymotrypsin. Careful selection of elution conditions allows a single inexpensive affinity matrix to be used in the preparation of these proteins. We are grateful to the Wellcome Trust for their financial support. Brantner, J. H., Medicus, R. G. & McRorie, R. A. (1976) J. Chromatogr. 129,97-105

Brodrick, J. W., Largman, C., Johnson, J. H. & Geokas, M. C. (1978) J. Biol. Chem. 253,2732-2736

Chase, T. & Shaw, E. (1967) Biochem. Biophys. Res. Commun. 29, 508-5 14

Fritz, H., Hutzel, M., Huller, I., Wiedemann, M., Stahlheber, H., Lehnert, P. & Forell, M. (1967) Hoppe-Seyler's Z. Physiol. Chem. 348,1575-1578 Malvano, R., Marchisio, M., Massaglia, A., Giacosa, P. A., Zannino, M.. Andriulli. A. & Burlina, A. (1979) Scand. J. Gasteoenterol. SUPPI.62,3-10. Robinson. N. C.. Tve. R. W.. Neurath, H. & Walsh, K. A. (1971) Biochemistry 10, i743-2747 Schacterle, G. R. & Pollack, R. L. (1973) Anal. Biochem. 51,654-655 Schmer, G. (1972) Hoppe-Seyler's Z . Physiol. Chem. 353,810-814 Schwert, G. W. & Takenaka, Y. (1955) Biochim. Biophys. Acta 16, 570-575 Studier, F. W. (1973) J. Mol. Biol. 19, 237-248 Travis, J. & Roberts, R. C. (1969) Biochemistry 8,2884-2889