Separation of Trypsin and Peroxidase by UltraJiltration Using Crosslinked Soybean Trypsin Inhibitor

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1 BIOTECHNOLOGY AND BIOENGINEERING VOL. XVIII (1976) Separation of Trypsin and Peroxidase by UltraJiltration Using Crosslinked Soybean Trypsin Inhibitor INTRODUCTION In recent years a new technology has developed for the isolation and purification of enzymes and other proteins which takes advantage of biospecific interactions. One application of this principle, termed affinity chr~matography,l-~ is based on biospecific adsorption of proteins on ligands covalently attached to water insoluble polymeric carriers. The method has been applied to a wide variety of proteins including enzymes of virtually every class. Frequently, however, limitations are encountered with this technique. For example, the insoluble matrix must be chosen carefully so as to ensure proper flow characteristics and nonspecific protein adsorption must be avoided. Also, activation conditions for ligand attachment must be selected so as not to significantly alter the physical characteristics of the matrix particles. This limitation often excludes the use of organic solvents either in the reaction sequence (which places a severe constraint on synthetic methodology) or in the protein-ligand dissociation step. In addition, the use of cyanogen bromide activation of polysaccharide carriers, the most common method employed for ligand attachment to these polymers, can give rise to the operational problem of ligand leakage as a result of inherently unstable bonds formed between the ligand and ~arrier.~ We report here by way of example a method of protein separation based on biospecific interaction using a soluble affinity reagent in conjunction with an ultrafiltration membrane. The procedure represents an alternative to the classical approach with the potential for alleviating some of the problems discussed above. EXPERIMENTAL Bovine pancreatic trypsin, soybean trypsin inhibitor, horseradish peroxidase, tosyl arginine methyl ester, and N-ethyl-5-phenylisoxazolium-3'-sulfonate were purchased from Sigma Chemical Co., St. Louis, Mo. Benzidine dihydrochloride, ascorbic acid, and hydrogen peroxide (3Y) were purchased from Fisher Scientific Co., St. Louis, Mo. Spectral measurements were carried out with a Cary Model 15 spectrophotometer. Ultrafiltration membranes (Amicon 21 Centriflo cone; 5, mol wt cut-off) were purchased from Amicon Corporation, Lexington, Mass. Trypsin activity toward tosyl arginine methyl ester was measured according to the method of Hummel.5 Peroxidase activity toward benzidine w&s measured at 4 C by a modification of the method of Gregory.6 Thus,.2 ml of.25m benzidine dihydrochloride,.1 ml of.3yo (w/v) hydrogen peroxide, and.1 ml of.5m ascorbic acid were added to 2.5 ml of ph 4.25,.5M sodium citrate buffer. A.1 ml aliquot of a peroxidase solution in ph 8.3.1M Tris-HC1 buffer was added and the activity was calculated from the time necessary to produce the sudden appearance of a dark blue 1976 by John Wiley & Sons, Inc. 123

124 BIOTECHNOLOGY AND BIOENGINEERING, VOL. XVIII (1976) Preparation of' Crosslinked Soybean Trypsin Inhibitor The method of Woodward7 was used with modification. Fifty milligrams (2.

2 124 BIOTECHNOLOGY AND BIOENGINEERING, VOL. XVIII (1976) Preparation of' Crosslinked Soybean Trypsin Inhibitor The method of Woodward7 was used with modification. Fifty milligrams (2.12 pmol) of soybean trypsin inhibitor and 53.5 mg (64 pmol) of sodium bicarbonate were dissolved in 5 ml of water. To the solution was added 162 mg (64 pmol) of N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's Reagent K, equivalent amount for tenfold excess based on 3 carboxyl groups/mol of inhibitor)8 with stirring. The tube was stoppered and the clear solution was stirred at room temperature for 18 hr, during which time a white precipitate formed. The precipitate was dissolved by the addition of 25 ml of ph 8.3 O.1M Tris-HC1 buffer. The solution was transferred quantitatively to a.5 ml volumetric flask and diluted to the mark with buffer. Unreacted protein and crosslinked species of molecular weight less than 5, were removed by filtration of a 5 ml aliquot of the above solution using an ultrafiltration membrane. The residue on the filter (tan-colored gel) was redissolved in 5 ml of ph 8.3.1M Tris-HC1 buffer. The solution was again filtered and this procedure was repeated until no trypsin inhibitory activity appeared in the filtration. Separation of a Mixture of' Trypsin and Peroxidase The filtered and washed crosslinked soybean trypsin inhibitor described above was dissolved in 5 ml of ph 8.3.1M Tris-HC1 and mixed with.5 ml of a 1 mg/ml solution of trypsin dissolved in.1m HCI and 1. ml of a.5 mg/ml solution of horseradish peroxidase dissolved in water. The solution was filtered through the membrane and the filtrate was assayed for both peroxidase and trypsin activity (Table I). The residue was washed with additional 5 ml aliquots of the same buffer until no peroxidase activity appeared in the filtrate. The wash procedure was then continued using a.5m KCI-O.1M HCl solution9 and the filtrates were assayed for trypsin and peroxidase activity. This wash procedure was continued until trypsin activity no longer appeared in the filtrate (Table I). RESULTS AND DISCUSSION The varieties of techniques currently available for effecting separations of mixtures of proteins are most powerful when the mixture is composed of species which are significantly different in some physical property. For example, differences in charge characteristics can be exploited by application of electrophoresis or isoelectric focusing; differences in molecular weight can result in separation by gel permeation chromatography, etc. During the course of biochemical investigations, it is frequently necessary to separate protein species which are similar in such physical properties. This can place a restraint on the more classical procedures. Such separations can often be carried out more efficiently, though, by the application of the relatively new techniques based on affinities of the various components in the mixture for some biospecific reagent. While the use of a chromatography column consisting of chemically modified adsorbants (covalently attached ligands) is the more usual way of proceeding, this approach can give rise to operational difficulties as noted earlier. We report here a modification of the ligand specific chromatography technique using a water soluble affinity reagent. Our experiment called for the separation of a mixture of the enzymes horseradish peroxidase (mol wt 42,)1 and bovine pancreatic trypsin (mol wt

3 -I (STI),( retentate) Trvpsln (fll trate) COMMUNICATIONS TO THE EDITOR ,2).1* Considerations of an appropriate ligand for the separation led to the choice of soybean trypsin inhibitor (mol wt 23,)8 as the biospecific reagent for the proteolytic enzyme. While the classical approach involved covalent immobilization of this protein on a polysaccharide carrier (e.g., Sepharose 4B), it was found that a separation of the two enzymes could be achieved in solution using an ultrafiltration membrane in conjunction with a crosslinked derivative of the inhibitor (Fig. 1). Thus, using an ultrafiltration membrane and biospecifically complexing trypsin to a relatively high molecular weight derivative of the inhibitor, filtration resulted in its retention while the redox enzyme passed the filter. After removal of the peroxidase by a series of filtrations, a change in reaction conditions (ph), in turn, released the bound enzyme which was then separated from the soluble biospecific reagent. The crosslinked inhibitor was retained throughout by the membrane. Our results using such a scheme are summarized in Table I. Since the molecular weights of the protein species involved were nearly the same, good separation using membranes only was not realized. For reasons of economy and rapidity, a 5, molecular weight cut-off membrane was chosen for these experiments. While the three native proteins involved readily passed the membrane as a mixture, the inhibitor could be rendered impassable by increasing the molecular weight of the material via crosslinking the Woodward's Reagent K. This method of crosslinking 5 mg of STI protein produced sufficient yield of molecular weight species >5, to totally inhibit about 5 mg of trypsin. Thus, a mixture of soluble crosslinked inhibitor, trypsin, and peroxidase filtered through the membrane allowed the selective separation of peroxidase. This part of the separation was carried out at the optimal ph for trypsin-sti complexation. A simple six unit change in ph and also salt concentration then resulted in dissocia- STI Woodward's Reaeent recycle Trywin Peroxldase PH 8.3 UI trafll tration (STI )xm* i- Tn~sln ( retentate) Peroxldase (filtrate) Fig. 1. Separation by ultrafiltration of trypsin and peroxidase using crosslinked soybean trypsin inhibitor (STI),.

4 126 BIOTECHNOLOGY AND BIOENGINEERING, VOL. XVIII (1976) TABLE I Ultrafiltration of a Mixture of Peroxidase, Trypsin, and Crosslinked Soybean Trypsin Inhibitor Filtrate analysis Filtration Wash pgb pgb no. solutions HRPO Trypsin Totals Recovery 78 % 57% a A: ph 8.3,.1M Tris-HC1 buffer; B:.5M KC1-O.1M HCl. 5 pg present initially. tion of the proteolytic enzyme complex.9 Filtration of the resulting mixture resulted in the separation of trypsin. The fact that quantitative recoveries were not realized may have been due to proteolysis; nevertheless, a clean separation of the activities was achieved. For example, peroxidatic activity was not detected in the filtrate after wash no. 3 (Table I). The isolation and separation of enzymes based on manipulation of reaction conditions for precipitation and dissolution of molecular complexes have been reported. Dennis1* purified beef heart lactic dehydrogenase (LDH) via selective ammonium sulfate precipitation of the complex formed from LDH interaction with bovine serum albumin covalently linked to oxanylic acid. Sternberg13 reported the separation of an artificial mixture of lysozyme, 8-galactosidase, a-amylase, and protease by precipitation with increasing amounts of soluble poly(acry1ic acid). Recent advances in ultrafiltration membrane technology have made available filtration apparatus capable of, in principle, separating proteins differing in molecular weight by several thousand units. In practice, however, total selectivity is often not achieved. This is especially true, of course, when the species to be separated are of similar size. We suggest that by proper application of soluble polymeric ligand specific reagents, molecular weight discrimination by ultrafiltration membranes can be greatly improved. Perhaps most significant is the potential for constructing reagents by covalently attaching small molecule inhibitors to, for example, soluble polypeptide or synthetic polymers. Aqueous or nonaqueous preparative methodologies could then be used, the only limiting requirement being the solubility characteristics of the final product. We are

5 COMMUNICATIONS TO THE EDITOR 127 investigating the more general application of these techniques, the results of which will be reported elsewhere. This investigation was supported by National Science Foundation Grant GI References 1. P. Cuatrecasas and C. B. Anfinsen, Meth. Enzymol., 22, 345 (1971). 2. S. W. May and. R. Zaborsky, Sep. Purij. Meth., 3, 1 (1974). 3. J. Turkova, J. Chromutogr., 91, 267 (1974). 4. G. I. Tesser, H. U. Fisch, and R. Schwyzer, Helv. Chim. Acta, 57, 1718 (1974). 5. B. C. W. Hummel, Can. J. Biochem. Physiol., 37, 1393 (1959). 6. R. P. F. Gregory, Biochem. J., 11, 582 (1966). 7. R. B. Woodward, R. A. Olofson, and H. Mayer, J. Am. Chem. SOC., 83, 11 (1961). 8. T. Koide and T. Ikenaka, Eur. J. Biochem., 32,417 (1973). 9. H. F. Hixson and A. H. Niskikawa, Arch. Biochem. Biophys., 154, 51 (1973). 1. L. M. Shannon, E. Kay, and J. Y. Lew, J. Biol. Chem., 241,2166 (1966). 11. T. Hofmann, Biochemistry, 3, 356 (1964). 12. D. Dennis, Anal. Biochem., 24, 541 (1968). 13. M. Sternberg and D. Hershberger, Biochim. Biophys. Acta, 342,195 (1974). Biochemistry Section Cancer Research Center Business Loop 7 and Garth Avenue Columbia, Missouri 6521 GREG J. BARTLING CHARLES W. BARKER Accepted for Publication February 3, 1976

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BIOTECHNOLOGY AND BIOENGINEERING VOL. XIX (1977) Glucose Oxidase Pellets INTRODUCTION Considerable world-wide interest has arisen in the use of immobilized enzymes as catalysts in industrial process and