ANALYTICAL BIOCHEMISTRY 72,573-576 (1976)

Rapid Purification of Human Trypsin and Chymotrypsin I

DAVID A. JOHNSON ANDJAMES TRAVIS De~u~t~ent of Biochemistry, University of Georgia, Athens, Georgia .?06t?2

Received August II, 1975; accepted January 6. 1976

A one-step purification of both human cationic trypsin and chymotrypsin 1 is described. The method involves the adsorption of the enzymes onto a Sepharose- Trasylol affinity column. Subsequent elution and separation of the two enzymes is accomplished by a gradient of decreasing pH.

Evaluation of the properties of human pancreatic proteases, including their interactions with inhibitors, requires highly purified enzymes of maximal activity. This is particularly important when determining the stoichiometric relationship between the proteinases and their inhibitors. Earlier reports (1) have shown that the Kunifz bovine pancreatic trypsin inhibitor, commercially available as Trasylol, reversibly complexes with cationic human trypsin and chymotrypsin I but does not inhibit any of the other proteinases of human pancreas. Based on these observations we have developed a procedure for the one-step isolation of human chymo- trypsin I and cationic trypsin.

EXPERIMENTAL PROCEDURES

Materials

Sepharose 4-B was a product of Pharmacia Fine Chemicals. Trasylol (Kunitz bovine pancreatic protease inhibitor) was kindly furnished by Farbenfabriken Bayer AG. Human pancreas were obtained from Athens General Hospital, Athens, Georgia. All other chemicals used were reagent grade.

Methods

Trypsin esterase activity, with the substrate BzArgOEt (N-benzoyl-L- arginine ethyl ester), was measured by the method of Schwert and Takenaka (2) as described by Matlory and Travis (3). One unit of activity was defined as an absorbancy change of 1.0 optical density unit per minute at 253 nm. Chymotrypsin activity, using AcTyrOEt (N-acetyl-L-tyrosine ethyl ester), as substrate, was measured by the procedure of Coanet al. (4). Disc electrophoresis was performed according to Brewer and Ash- worth (5).

573 Copyright 0 1976 by Academic Press. Inc. All rights of reprodirction in any form reserved.

574 JOHNSON AND TRAVIS

Sepharose-Trasylol was prepared by coupling at pH 6.5 to cyanogen bromide activated (300 mg/ml gel) Sepharose 4-B (6). In a typical prepara- tion 100 ml ofwashed activated Sepharose was mixed with an equal volume of 0.1 M NaHCO, buffer, pH 6.5, containing 200 mg Trasylol. After coupling, the Trasylol-Sepharose conjugate was washed at both pH 2.0 and 8.0. These washes contained only a small amount of 280 nm absorb- ance, indicating that virtually all of the protein was coupled. Although this is a lysine active site inhibitor a product with excellent capacity is obtained.

RESULTS

A column (0.9 x 20 cm) was packed with the gel inhibitor conjugate and equilibrated with 0.05 M n-is-HCl, pH 8.0,0.5 ~Nacl. The 0.5 M salt concentration was utilized to rule out the possibility of ion exchange ef- fects. Human pancreatic acetone powder was extracted and activated by procedures described earlier (3). The solution (10 ml) was clarified by centrifugation and applied to the column followed by washing with equilibration buffer. After the absorbance at 280 nm had returned to less than 0.010, the column was developed with a convex exponential gradient from 0.05 M Tris, 0.5 M NaCl, pH 8.0 (50 ml lower reservoir) to 0.025 M citrate, 0.025 M CaCl,, pH 2.5 (upper reservoir) and finally washed with the latter buffer.

As shown in Fig. 1, the elution profile from the Sepharose-Trasylol derivative indicated one major protein peak which represented unadsorbed material. This peak was found to contain chymotrypsin esterase activity, which probably represents a mixture of chymotrypsin I and chymotrypsin II, as well as esterase activity due to an elastase-like enzyme which we have called protease E (7). Chymotrypsin II and protease E are not in-

5 10 15 20 25 30 35 40 Froct~on no

FIG. 1. Chromatography of activated human pancreatic extracts on Sepharose-Trasylol. Details as given in the text. 0 - 0, A,,, “,,,; A - A, chymotrypsin esterase activity: o- 0, trypsin esierase activity.

HUMAN TRYPSIN AND ~HYMOTRYPSIN 575

hibited by Trasylol (1,7) and some chymotrypsin I has no doubt been dis- placed by the more tightly bound cationic trypsin. Anionic trypsin autolyzes rapidly at alkaline pH and therefore was not seen (3).

Two distinct protein components were eluted under the gradient. The I%-st peak appeared at about pH 6.5 and was determined to be chymo- trypsin I. The amount of chymotrypsin I isolated was found to be highly variable, depending on the amount of trypsin in the activated extract applied to the column, as well as the size of the column, The second protein peak, eluted below pH 4.0, was found to be cationic trypsin. This protein was found to be quantitatively adsorbed and desorbed from the Sepharose-TrasyIol column. Examination of the two pooled protein peaks by disc electrophoresis showed each to be a single entity (Fig. 2). In addition, the specific activity of the chymotrypsin I was equivalent to that previously obtained (8), while the trypsin had a specific activity of 12, which is higher than the published value of 9.9 (9).

DISCUSSION

We have taken advantage of the properties of the Kunitz bovine pan- creatic protease inhibitor to produce an affinity column for the purifica- tion of human chymotrypsin 1 and cationic trypsin. The two were sepa- rable because the chymotrypsin inhibitor complex dissociates at a slightly

1 2 3

FIG. 2. Disc electrophoresis of human pancreatic enzymes at pH 2.3. 1. Activated human pancreatic extract; 2, human chymotrypsin 1; 3, human cationic trypsin. Direction of migration is from anode (top) to cathode (bottom). Gel concentration, 7.5%.

576 JOHNSON AND TRAVIS

higher pH than does the trypsin inhibitor complex. The dissociation of the proteinase-inhibitor complex does not result in a modified inhibitor. Since no peptide bond is cleaved, and since Trasylol is not easily denatured, the column can be used repeatedly (10). In addition, this column has been used in the purification of human leukocytic elastase (11) and should be of value in the purification of plasmin, plasmin activator, blood clotting factors, and kininogenases, all of which are reported to be inactivated by this inhibitor.

From a more practical standpoint, the use of Sepharose-Trasylol in repurifying commercial preparations of trypsin from various manufacturers cannot be overemphasized. For example, we have found that some samples of bovine trypsin with as little as 50% titratable active sites can be quantitatively retained on and subsequently desorbed from columns of Sepharose-Trasylol using the procedure described here; only inactive ma- terial passes through the column. The resultant trypsin preparation is chymotrypsin-free and has from 87-90% available active sites.

ACKNOWLEDGMENTS

We are deeply indebted to Dr. Ernst Truscheit for the gift of Trasylol. This research was sponsored in part by NIH Grant No. AL 14478 and by the Council

for Tobacco Research-USA. David Johnson is a NIH Postdoctoral Fellow (HL- 01978). James Travis is a Research Career Development Awardee (HL-70264).

REFERENCES

1. Coan, M. H., and Travis, J. (1971) in Proceedings of the International Research Confer- ence on Proteinase Inhibitors (Fritz, H., and Tschesche, H., eds.), pp. 294-298, Walter de Gruyter, Berlin-New York.

2. Schwert, G. W., and Takenaka, Y. (1955) Biochim. Biophys. Acta 16, 570-575. 3. Mallory, P. A., and Travis, J. (1973) Biochemistry 12, 2847-2851. 4. Coan, M. H., Roberts, R. C., and Travis, J. (1971) Biochemistry 10, 2711-2717. 5. Brewer, J. M., and Ashworth, R. B. (1%9)J. Chem. Educ. 46, 41-45. 6. Cuatrecasas, P. (197O)J. Biol. Chem. 245, 3059-3065. 7. Mallory, P. A., and Travis, J. (1975) Biochemisfry 14, 722-730. 8. Coan, M. H., and Travis, J. (1972) Biochim. Biophys. Acfa 268, 207-211. 9. Travis, J., and Roberts, R. C. (1%9) Biochemistry 8, 2884-2889.

10. Vogel, R., Trautschold. I., and Werle, E. (1968) in Natural Proteinase Inhibitors, pg. 76, Academic Press, New York.

11. Baugh, R., and Travis, J. (1975) Fed. Proc. 34, 484.