ISOLATION, ELECTROPHORETIC CHARACTERIZATION AND IMMUNO- LOGIC PROPERTIES

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1 Biochimica et Biophysica Acta, 379 (1975) Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA TRYPSIN INHIBITOR FROM COW COLOSTRUM ISOLATION, ELECTROPHORETIC CHARACTERIZATION AND IMMUNO- LOGIC PROPERTIES ANDRI~S PIIqEIRO a, FERNANDO ORTEGA a and JOSI~ URIEL b ~Fundaci6n F. Cuenca I/illoro, Gasc6n de Gotor, 4, Zaragoza (Spain) and blnstitut de Recherches Scientifiques sur le Cancer, l/illejuif (Francej (Received June 7th, 1974) SUMMARY Trypsin inhibitor from cow colostrum has been purified by affinity chromatography of colostral proteins on insolubilized trypsin. The method described compares favourably, in both simplicity and yield, with previous methods developed for the isolation of this inhibitor. Gel electrophoresis followed by characterization of antitrypsin activity allows the demonstration of four molecular forms of bovine colostral trypsin inhibitor in both crude colostral whey and purified preparations of the inhibitor. Immunoelectrophoresis of each of these materials with antisera specific for this inhibitor reveals a single precipitation line of broad anodic mobility. By immunodiffusion tests, the precipitation lines in preparations of purified inhibitor and colostral whey appear immunologically identical. In contrast, absence of crossed reactivity was observed between bovine colostral trypsin inhibitor and trypsin inhibitors of bovine serum. This strongly suggests the high specificity of this inhibitor as a colostral and milk constituent. INTRODUCTION A trypsin inhibitor in cow colostrum was originally identified by Laskowski and Laskowski [1], who also described several of its properties. More recently t~echov~i et al. [2] have demonstrated the microheterogeneity of this inhibitor after isolation by ion-exchange chromatography of at least three molecular forms with antitrypsin activity. In the present paper, we describe (a) a simple and rapid method of purification of this inhibitor by affinity chromatography of colostral whey proteins on insolubilized trypsin, and (b) the electrophoretic and immunological properties of the preparations obtained. MATERIALS AND METHODS Materials Bovine colostrum from Holstein cows was collected on the first day of delivery

202 and was stored frozen until use. Rennin (EC 3.4.99.19) and neuraminidase (EC 3.2.1. 18) were purchased from Sigma (St. Louis, U.S.A.) and trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) from Boehringer (Mannheim, G.

2 202 and was stored frozen until use. Rennin (EC ) and neuraminidase (EC ) were purchased from Sigma (St. Louis, U.S.A.) and trypsin (EC ) and chymotrypsin (EC ) from Boehringer (Mannheim, G.F.R.). N-benzoyl-DLarginine-p-nitroanilide (Bz-Arg-NAn) and N-acetyl-DL-phenylalanine-/~-naphtylesther (Ac-Phe-ONAP) were obtained from Merck (Darmstadt, G. F. R.) and Schwartz- Mann (Orangeburg, New York), respectively. CNBr-activated Sepharose 4B and Sephadex G-75 were from Pharmacia (Uppsala). Methods Trypsin insolubilization for affinity chromatography was achieved with CNBractivated Sepharose 4B following the method of AxOn et al. [3]. To minimize autodigestion of trypsin the coupling was performed at 4 C in 0.1 M sodium acetate-0.5 M NaCl buffer, ph 5, for 5 h. Under these conditions 120 mg of insolubilized trypsin were recovered from 350 mg of trypsin incubated with 10 g (dry weight) of activated Sepharose. The preparation was stored at 4 C in a 0.05 M glycine-hcl, 0.5 M NaCI buffer, ph 3. Trypsin and trypsin inhibitory activities were determined by the method of Erlanger et al. [4], using Bz-Arg-NAn as substrate (1/~g/ml of enzyme hydrolyzes 0.73 nmole of substrate/min). Specific inhibitory activity was expressed as/,g of trypsin inhibited per mg of protein. Molecular weight determinations were carried out by gel filtration on Sephadex G-75 according to the method of Whitaker [5]. Protein concentration was determined by the biuret method [6]. Electrophoretic analyses were carried out in acrylamide-agarose gel slabs [7]. After gel electrophoresis, constituents with trypsin or chymotrypsin inhibitory activity were characterized by the method of Uriel and Berges [8]. Antiserum to colostral trypsin inhibitor was prepared in rabbits by intradermal injection in the dorsal region of 0.5 ml (1 mg of protein) of the inhibitor homogeneized with an equal volume of Freund's complete adjuvant. 2 months later the animals were boosted with the same quantity of antigen on two successive days, the first day by intradermal injection and the second day via the intramuscular route. The animals were bled by cardiac puncture 8 to 10 days later and the serum obtained stored at --20 C. Further purification of the antiserum was achieved by immunoadsorption with bovine serum according to the method of Avrameas and Ternynck [9]. Immunoelectrophoresis was performed by the method of Grabar and Willians [101. RESULTS 1. Isolation of colostral trypsin inhibitor Defrosted colostrum samples were first defatted by centrifugation for 15 rain at 3000 x g. Casein was then precipitated by incubation with rennin for 30 rain at 30 C. After centrifugation (15 min, 3000 x g) the colostral whey was collected as the supernatant. To 100 ml of colostral whey, solid NaC1 was added to 0.5 M concentration and

203 TABLE I Purification step Total Specific Purification Yield activity* activity** rate (percent) Colostral whey (100 ml) 65 000 5 -- -- Affinity chromatography 40 200 1950 390 62 Trichloroacetic

10 g of trypsin-sepharose beads (see Methods) were suspended in the colostral solution and the mixture gently stirred for 30 min at room temperature.

3 203 TABLE I Purification step Total Specific Purification Yield activity* activity** rate (percent) Colostral whey (100 ml) Affinity chromatography Trichloroacetic acid precipitation */~g of trypsin inhibited. **!tg of trypsin inhibited per mg of protein. the solution adjusted to ph 7 with 1 M NaOH. 10 g of trypsin-sepharose beads (see Methods) were suspended in the colostral solution and the mixture gently stirred for 30 min at room temperature. The inhibitor-trypsin conjugate was removed from the mixture by filtration on a sintered glass filter and washed with 0.01 M potassium phosphate-0.5 M NaCI buffer, ph 7, until the absorbance at 280 nm was zero. The elution of enzyme-bound inhibitor was ph-dependent. Above ph 4, practically no elution of the inhibitor occurred. Best results were obtained by elution with 0.05 M glycine-hcl, 0.5 M NaCI buffer, ph ml of this buffer, added in several aliquots were necessary to complete elution of the inhibitor. The eluates were neutralized with 1 M NaOH and then dialyzed and concentrated by ultrafiltration through a UM 10 Diaflo membrane in an Amicon Cell. The trypsin inhibitor obtained (Preparation I) had a specific activity of 1950 which represents a 390-fold purification relative to colostral whey. Samples of purified inhibitor were used to prepare rabbit antisera. Subsequent immunologic analysis (see below) of Preparation 1 revealed the presence of traces of protein contaminants. Further purification of the inhibitor, without any significant loss in total inhibitory activity, was achieved by precipitation of the contaminants with the addition of an Fig. 1. Electrophoretic patterns in acrylamide-agarose of anti-trypsin activity: a, colostral whey (30 mg/ml of protein); b, co!ostral trypsin inhibitor (Preparation 2, 0.1 mg/ml); and c, same preparation as in b after treatment with neuraminidase (0.2 mg of inhibitor and 0.1 mg neuraminidase in 0.3 ml of 0.1 M sodium acetate buffer, ph 5, were left 2 h at 37 C, before electrophoresis).

204 equal volume of 5 ~ (w/v) trichloroacetic acid. After centrifugation the inhibitor recovered in the supernatant (Preparation 2) was neutralized and dialyzed as described above.

4 204 equal volume of 5 ~ (w/v) trichloroacetic acid. After centrifugation the inhibitor recovered in the supernatant (Preparation 2) was neutralized and dialyzed as described above. Table I summarizes the isolation procedure. The final yield was about 60 ~ of the total antitrypsin activity of colostral whey. Some loss of this anti-trypsin activity is caused by elution at acid ph since it has been reported previously [11 ], and confirmed by us, that cow colostrum contains another type of anti-trypsin activity which is acidlabile. The molecular weight of isolated inhibitor (Preparation 2) was estimated to be about by gel filtration in Sephadex G Electrophoretic and immunologic properties Gel electrophoresis followed by the characterization of anti-tryptic activity revealed the microheterogeneity of the cow colostrum trypsin inhibitor, four major bands with anti-trypsin activity being observed in the electrophoretic patterns of colostral whey (Fig. l a). The same method also revealed the presence of four isoinhibitors in the purified inhibitor (Fig. lb). No anti-chymotryptic activity was observed after electrophoresis of either colostral whey or purified inhibitor (Preparations 1 and 2). Treatment with neuraminidase of isolated trypsin inhibitor reduced the electrophoretic mobilities of the four isoinhibitors without any apparent change in their relative mobility and anti-trypsin activity (Fig. lc), or their antigenic properties (Fig. 2). The last was confirmed by the estimation of the total inhibitory activity of the sample Fig. 2. Immunoelectrophoresis of colostral whey trypsin inhibitors, a, Preparation 1 of the inhibitor; b, same preparation bovine trypsin (equimolar quantities were mixed before the immunoelectrophoretic run); c, bovine serum; d and e, colostral whey. Rabbit serum anti-colostral trypsin inhibitor (1); same after immunoadsorption with bovine serum (2).

5 205 Fig. 3. Immunodiffusion pattern in agarose gel. a, colostral trypsin inhibitor (Preparation 2); b, colostral whey; c, bovine serum. Rabbit serum anti-colostral trypsin inhibitor (1); same after immunoadsorption with bovine serum (2). before and after treatment with neuraminidase. No significant loss of anti-trypsin activity was observed. By immunoelectrophoresis, the antiserum against Preparation 1 revealed a single precipitation line of broad anodic electrophoretic mobility in both preparations of the inhibitor (Fig. 2a). Additional proof of the specificity of the immunoreaction was provided by treatment of the preparation with trypsin prior to the immunoelectrophoretic run. A single wide-spread precipitate with cathodic mobility was observed, probably due to the formation of trypsin-inhibitor complexes (Fig. 2b). Several precipitation lines were observed when the same antiserum was allowed to react with bovine serum and colostral whey (Figs 2c and 2d). After immunoadsorption of the antiserum on insolubilized bovine serum, antibodies other than antiinhibitor were removed as demonstrated by immunodiffusion tests (Figs 2e and 3). The adsorbed antiserum does not react with bovine serum proteins and gives a single line of precipitation with colostral whey. This line cross-reacts with that obtained from purified inhibitor, Preparation 2 (Fig. 3). DISCUSSION The affinity chromatography method described above compares favourably in both simplicity and yield with previous methods developed for the isolation of the cow colostrum trypsin inhibitor [1, 2]. The electrophoretic characterization of antitrypsin activity appears of good sensitivity since four molecular forms of this inhibitor were revealed instead of the three previously described by (~echov~ et al. [2]. Four isoinhibitors were also identified" by Kress et al. [12] in porcine colostrum. The preparations of purified inhibitor are acid-stable as are other swine and cow colostrum trypsin inhibitors already described [1, 2 12]. The molecular weight that we obtained by gel filtration is slightly higher than that reported previously by Laskowski et al. [13]. The difference is probably due to the use of different methods, especially as the inhibitor contains a significant proportion of carbohydrate which may affect gel filtration results. The four isoinhibitors contain sialic acid as shown by the reduced electrophoreric mobility after treatment with neuraminidase. Nevertheless, the enzyme has nc effect on the anti-trypsin activity in agreement with a similar observation reported for other trypsin inhibitors, including that of swine colostrum [12, 14, 15].

6 206 The origin of the microheterogeneity of colostrum trypsin inhibitor has been discussed by several authors [2, 12]. The question arises whether this heterogeneity is inherent or represents artifacts formed during the isolation procedure. As far as bovine colostrum trypsin inhibitor is concerned the four isoinhibitors identified appear to be native products since the same molecular forms are present in crude colostral whey. These forms possess identical electrophoretic and immunologic properties to the isolated preparations of the inhibitor. Immunodiffusion techniques show strong cross-reactivity between the four isoinhibitors. No antigenic differences among them were revealed with the antiserum used. The most important feature of the immunologic study presented here is the absence of cross-reactivity between this inhibitor and trypsin inhibitors of bovine serum which suggests the high specificity of cow colostrum trypsin inhibitor as a colostral and milk constituent. (~echov~i et al. [16] have determined the primary structure of the protein moiety of one component of this inhibitor and shown its homology with the basic pancreatic trypsin inhibitor. Additional work will be necessary to investigate the origin and the mechanism of storage and secretion of these particular trypsin inhibitors as well as their physiological functions. ACKNOWLEDGMENT The authors gratefully acknowledge the assistance of Professor E. Martin, Instituto Experimental de Cirugia y Reproducci6n, Facultad de Veterinaria, Zaragoza, for providing colostrum. REFERENCES 1 Laskowski, Jr, M. and Laskowski, M. (1951) J. Biol. Chem. 190, ~echov~i, D., Jon~ikov~i-~vetskova, V. and ~orm, F. (1970) Collect. Czech. Chem. Commun. 35, Axen, R., Porath, J. and Ernback, S. (1967) Nature 214, Erlanger, B. F., Kokowsky, N. and Cohen, W. (1961) Arch. Biochem. Biophys. 95, Whitaker, J. R. (1963) Anal. Chem. 35, Uriel, J. (1961) Biol. Med. (Paris) 50, Uriel, J. (1966) Bull. Soc. Chim. Biol. 48, Uriel, J. and Berges, J. (1968) Nature 218, Avrameas, S. and Ternynck, T. (1969) Immunochemistry 6, Grabar, P. and Williams, Jr, C. A. (1953) Biochim. Biophys. Acta 10, Barkholt Pedersen, V., Keil-Dlouha, V. and Keil, B. (1971) FEBS Lett. 17, Kress, L. F., Martin, S. R. and Laskowski, Jr, M. (1971) Biochim. Biophys. Acta 229, Laskowski, Jr, M, Mars, P. H. and Laskowski, M. (1952) J. Biol. Chem. 198, Feeney, R. E., Rhodes, M. B. and Anderson, J. S. (1960) J. Biol. Chem. 235, Schultze, H. E., Heide, K. and Haupt, H. (1962) Klin. Wochenschr. 40, (~echov~i, D., Jon~,kov~i, V. and ~orm, F. (1971) Collect. Czech. Chem. Commun. 36,

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