Effect of low and high temperatures on chymotrypsin from atlantic cod (Gadus Morhua L.); comparison with bovine α-chymotrypsin

Comp. Biochem. Physiol. Vol.97B, No. 1, pp. 145-149, 1990

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EFFECT OF LOW AND HIGH TEMPERATURES ON CHYMOTRYPSIN FROM ATLANTIC COD (GADUS MORHUA L.); COMPARISON WITH BOVINE -CHYMOTRYPSIN ARNT J. RAAE University of Bergen, Department of Biochemistry, Arstadveien 19, N-5009 Bergen, Norway (Tel: 291700) (Received 27 February 1990)

Abstract--1. Cod chymotrypsin displays higher enzyme activity compared to bovine ct-chymotrypsin when assayed at low temperatures (3-15°C). 2. Both enzymes are inactivated when incubated at temperatures between 60 and 70°C. 3. When incubated at 99°C the cod enzyme retains about 50% of the initial activity measured at room temperature. 4. Preincubation at boiling temperature renders the cod chymotrypsin active at 70°C whereas the bovine enzyme is rapidly inactivated.

INTRODUCTION It has been well established that enzymes isolated from organisms adapted to different environmental conditions possess different catalytic properties (Hochacha and Somero, 1984). One of the most studied environmental effectors is temperature. Several authors have found that enzymes from cold-adapted organisms are characterized by having a higher turnover number at low temperatures, lower Km values and slightly lower free energies of activation when compared to homologous enzymes from warm-blooded organisms (Cowey, 1967; Assaf and Graves, 1969; Pesce et al., 1967; Low and Somero, 1974). However, the majority of such studies have dealt with intracellular enzymes such as dehydrogenases and phosphorylases. Only few comparative studies have so far been concerned with temperature effects on proteolytic enzymes. Racicot and Hultin (1987) have compared the properties of dogfish chymotrypsin with those of bovine chymotrypsin. They investigated parameters such as enthalpies of association, association constants and the free energies of association. The authors relate the observed differences between the two enzymes to a greater hydrophobicity in the active site of the dogfish chymotrypsin. A general explanation for the differences in catalytic behaviour between enzymes from low- and warm-temperature-adapted organisms has also been suggested by Low and Somero (1974). They propose that enzymes from cold-adapted organisms possess a loosely fit tertiary structure and thus enables the enzyme to lower the activation energy when binding to the substrate. Cod chymotrypsin differs in several criteria from the homologous bovine ~-chymotrypsin such as pI and intramolecular s-s-bridges (Raae and Walther, 1989). This also seems to include differences in

polypeptide chain construction as demonstrated by differences in mobility when analyzed by gel filtration under different pressure conditions. At elevated pressures the cod enzyme was strongly retarded. We assume that these observations are in favour with a more flexible polypeptide construction which is sensitive to pressure changes. In this study, data is presented where the catalytic effectiveness of the chymotrypsins from cod and bovine sources has been compared at low and high temperatures. In addition comparisons have been made of enzyme stabilities at high temperatures. MATERIALS AND METHODS Materials

All inorganic salts were of analytical grade. Benzoyltyrosine-ethylester (BTEE), bovine serum albumin (BSA) type V, bovine ~-chymotrypsin and N-N-N-tetra methyl ethylene diamine were purchased from Sigma Chemical Company (St Louis, MO). Acrylamide and 2-mercaptoethanol were from Merck. Cod chymotrypsin, PBAfraction 2, were purified from cod pyloric caeca as previously described (Raae and Walther, 1989). Methods

Chymotrypsin activity was assayed according to the modified Hummel procedure after Rao and Lombardi (1975). The standard reaction mixture contained in a total vol of 1 ml, 75 mM Tris-HCl pH 7.8, 0.2% Triton X-100, 0.I M CaCI2, I% methanol and 0.5mM BTEE. The reaction was initiated by adding the enzyme and the change in absorbance was monitored at 256 nm with a PerkinElmer 554 spectrophotometer equipped with a thermostated cuvetteholder. One unit of enzyme hydrolyzes I gmol of substrate/min at 23°C. .Low temperature studies

The enzyme activity of cod and bovine chymotrypsins in the temperature range 3-20°C were determined directly in

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the cuvette. The reaction mixtures were equilibrated for 10 min at each reaction temperature and the reactions were initiated by the addition of 0.5 #g of the respective enzymes.

High temperature studies The enzyme stability at elevated temperatures was determined as the residual enzyme activity, both esterolytic and proteolytic, after incubation at various temperatures. Also the ability to hydrolyze BSA at different high temperatures was investigated. (A) Residual esterolytic activity. The enzymes, 5~g in 20 #1 of 10 mM Tris--HCl 40% glycerol, were incubated for 30 min in the temperature range 40-99°C. Aliquots of 1/~1 were withdrawn at various times and the residual enzyme activity was determined in the standard BTEE-hydrolyzing assay at 23°C. (B) Residual proteolytic activity. The enzymes, I/~g of protein in 10#1 10mM Tris-HCl pH7.8, 20raM CaC12 40% glycerol, were incubated for 10 min at different temperatures in the range between 40 and 99°C. After equilibration for 5 min at room temperature the enzyme solutions were added to 0.5 mg BSA and the reaction mixtures were made up with 75 mM Tris-HC1 pH 7.8, 20 mM CaC12 and 5% glycerol in a total of 20/al. Proteolysis was carried out for 10 min at room temperature and the reaction mixtures were subsequently analyzed by SDS-polyacrylamide gel electrophoresis. (C) Proteolytic activity at high temperatures. The proteolytic activity of cod chymotrypsin was measured in the temperature range between 50 and 99°C. The reaction mixture, a total vol of 20 #1, contained 75 mM Tris-HCl pH 7.8, 50 mM CaCI 2 and 0.5 mg BSA. After equilibration for 5 min at the different temperatures, 1 2 pg of enzyme was added and proteolysis was carried out for I0 min. The reactions were stopped by immediately adding 20/~1 of 0.25 M Tris-HC1 pH 6.8, 4% SDS, 20% glycerol, 0.005% Bromphenol Blue and 0.7 M 2-mercaptoethanol. Aliquots of I0/~1 of the reaction mixtures were subsequently analyzed by polyacrylamide gel electrophoresis.

Gel eleetrophoresis

inactivation of the enzymes when they were incubated for periods of 50 min at various temperatures. Little or no enzyme inactivation was detected at temperatures below 50°C (data not shown). Figure 2A shows that cod chymotrypsin practically loses no enzyme activity when incubated for 50 min at 50°C, whereas bovine ~t-chymotrypsin retains ca 70% of the initial activity during the same period. When incubated at 68°C both enzymes were rapidly inactivated. The bovine enzyme was inactivated more rapidly than the cod enzyme which displayed 65% of initial activity after 0.5 min of incubation compared to 20% for the bovine enzyme. When incubated at 85°C both enzymes were rapidly inactivated, however, the residual activity of cod chymotrypsin stabilized at ca 30% whereas the bovine enzyme slowly lost the final 25% of initial activity within 15 min of incubation. The cod chymotrypsin residual activity at 99°C, was rapidly stabilized to ca 40% of the enzyme activity at 23°C. After 2 0 m i n of incubation the enzyme activity slowly decreased and the total inactivation was observed after another 60min of incubation. The reaction characteristics of the bovine enzyme were similar to those of the cod enzyme at this temperature, the major difference being a lower level or residual enzyme activity. Figure 3 summarizes the effect of high temperatures on enzyme activity. The residual enzyme activities after 10 min of incubation are plotted against the respective temperatures. The figure shows that both enzymes are totally inactivated when incubated at 68°C. The bovine ~-chymotrypsin, however, was inactivated more rapidly and was in addition inactive over a wider range of temperatures than the cod enzyme. The cod chymotrypsin retains ca 38% of its initial activity after 10 min of incubation at 85°C. At 99°C the cod enzyme was found to have between 40 and 50% of

Gel electrophoresis was carried out essentially according to Laemmli (1970) for 4 hr at room temperature at 35 mA per slab. Protein bands were visualized by silver staining using the procedure of Marshall and Lattner (1981).

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Protein Protein was determined according to the procedure of Lowry et al. (1951). RESULTS

Effect o f low temperatures Enzyme activity. The enzyme activities for the cod and bovine chymotrypsins were compared at temperatures ranging from 2.8 to 25°C. The results are shown in Fig. 1. As can be seen from the figure, cod chymotrypsin is ca 1.5-fold more active than the bovine enzyme in the range between 2.8 and 8°C. At higher assay temperatures the differences in enzyme activities are gradually reduced and reach the same level as at room temperature (20-25°C). The figure also shows that both enzymes exhibit accelerated activity at incubation temperatures above 15~'C. Effect o f high temperatures Enzyme stability. The effect of higher temperature on cod chymotrypsin was investigated in the range from 25 to 99°C. Figure 2 shows time courses of

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TEMPERATURE (°C) Fig. I. Effect of low temperature on the enzyme activities of cod and bovine chymotrypsins. The figure presents the results of two parallel experiments. Cod chymotrypsin enzyme activity is marked with filled circles whereas bovine chymotrypsin is represented with filled triangles. Experimental conditions are as described in Materials and Methods.

Cod and bovine chymotrypsins initial activity whereas the bovine enzyme retained between 21 and 39% of its initial activity. In order to investigate the residual enzyme activity at 99°C, both enzymes were first incubated for l0 min at 99°C and then transferred to 70°C. Figure 4 shows a time course of such an analysis. The cod chymotrypsin stabilized at 40% of the initial activity when incubated at 99°C. When the reaction mixture was transferred to 70°C the enzyme was slowly inactivated in a linear fashion and was only 50% inactivated after 25 rain. The bovine enzyme activity was reduced to 18% after l0 min at 99°C and was totally inactivated after a 2.5 min incubation at 70°C. The effect of high temperature on the proteolytic activity of cod chymotrypsin was investigated using bovine serum albumin as substrate. Figure 5 shows a SDS-polyacrylamide slab gel analysis of the reaction mixtures after incubation at 23.7 and 99°C, In lanes D, E and F BSA and cod chymotrypsin were added simultaneously to the reaction mixtures and in lanes G, H and I cod chymotrypsin was preincubated for 10min at the various temperatures before the substrate was added. At 23°C, Fig. 5 lane D, no high molecular weight BSA can be seen whereas the cod chymotrypsin band at 28,000 D is clearly visible. When BSA and cod chymotrypsin were incubated at 70°C, lane E, the BSA gel band pattern is virtually unchanged compared to the control (lane D) whereas the chymotrypsin band is missing. When the enzyme

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Fig. 3. Effect of different temperatures on the stabilities of cod and bovine chymotrypsin activities. The enzyme activities are expressed as the residual activity after 10 min of incubation at the different temperatures. Symbols are as in Fig. l. and substrate were incubated at 99°C hydrolysis of BSA was seen as an accumulation of low molecular weight material and the chymotrypsin band is present. Lanes F, H and I in Fig. 5 represent reactions where cod chymotrypsin was preincubated for l0 min before BSA was added after the reaction temperature was shifted to 23°C. The resulting protein patterns from SDS-polyacrylamide analysis are similar to those in lanes D, E and F. Virtually all of the BSA-substrate was hydrolyzed when the enzyme was preincubated at room temperature (lane G) whereas minor amounts of BSA were hydrolyzed by cod chymotrypsin which had been preincubated at 70°C. Lane I shows that BSA was hydrolyzed by cod chymotrypsin which had been preincubated at 90°C. The band corresponding to the enzyme is also present.

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Fig. 2. Time courses of cod and bovine chymotrypsin enzyme activities at various temperatures. The enzyme activities at 23°C for both enzymeswere set to 100% and all other activities are expressed relative to those values. The symbols are as described in Fig. 1 and the experimental conditions are as described in Materials and Methods.

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Fig. 4. The effect of preincubation at boiling temperature on the enzyme activity at 70°C. The enzymes were first incubated at 100°C for l0 rain before the incubation mix'tures were transferred to ?0°C. Residual enzyme activities were measured as described in Materials and Methods. Symbols are as in Fig. I.

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Fig. 5. SIS-PAGE analysis of cod chymotrypsin catalyzed hydrolysis of BSA at w.rious temperatures. Lane A; cod chymotrypsin (1.5 #g), B; mol. wt standards, C; standard BSA. Lanes D F shows analysis of reactions where enzyme and substrate were incubated simultaneously at 23°C (lane D), 70°C (lane E) and 100°C (lane F). Lanes G I show analysis of reactions where cod chymotrypsin was preincubated for 10 rain at various temperatures before the substrate was added. Lane G; 23°C, H; 70°C and I; 100°C.The proteolytic reactions were carried out at 23°C before SDS PAGE analysis. Other details of the experiments are as described in Materials and Methods. DISCUSSION

The observation that cod chymotrypsin is more active at low temperatures than bovine ~-chymotrypsin is in accordance with other studies which have dealt with the comparison of enzymes from cold-adapted organisms with enzymes from warm-blooded animals (Hochacha and Somero, 1984). The difference in enzyme activity is gradually reduced when the reaction temperature is raised. At temperatures from 20 to 25°C the enzymes display the same activity. Also the kinetics of enzyme activity vs temperature tend to be different for the two enzymes. The cod enzyme roughly increases in activity in a linear fashion in two phases, the first at temperatures between 2.8 and 15°C, and the second between 15 and 21°C. The bovine enzyme, in the same temperature range, shows an increase in activity in a hyperbolic fashion. Similar differences in low temperature enzyme kinetics were also observed when cod elastase was compared to porcine elastase (Raae, unpublished data). An interpretation of these observations is that the cod enzymes more readily adapt their configuration to changes in temperature due to a loosely fit tertiary structure. When incubated at moderately high temperatures, between 30 and 50°C, both the cod and bovine chymotrypsin showed little or no decrease in enzyme activity. However, at a temperature of c a 70°C both enzymes were rapidly inactivated. When the incubation temperature was increased to 80°C and above the enzymes were not totally denatured and the

cod enzyme was found to display nearly 50% of its initial enzyme activity after 10rain at boiling temperature. In order to investigate this phenomenon more closely the enzymes were preincubated for 10 min at boiling temperature and then transferred to 70°C. This resulted in rapid inactivation of the bovine enzyme whereas the cod enzyme showed improved stability and was only slowly inactivated during an incubation period of 25 min. The rapid inactivation of the native enzymes observed at 70°C could therefore be due to thermal distortion of the 3-dimensional polypeptide structure which renders the enzymes susceptible to auto-digestion. One could further speculate that the residual enzyme activity observed at higher temperatures is due to a transformation of polypeptide structure to a configuration that retains enzyme activity but allows less auto-proteolysis. Such a structure seems to be better stabilized in the cod chymotrypsin as indicated by the improved stability at 70°C. This hypothesis was further investigated by slab gel analysis of the reaction mixtures using BSA as substrate. The gel pictures (Fig. 5) show that the band corresponding to the cod chymotrypsin polypeptide was absent when the reaction had been carried out at 70°C. However, after preincubation at 99°C, the enzyme band was visible after incubation at both 99 and 70°C. These results support the hypothesis that the structure of the cod chymotrypsin polypeptide is modified when the enzyme is heated up to 99°C, and that this structure retains enzymatic activity and is stabilized against the auto-digestion which is normally observed at 70°C,

Cod and bovine chymotrypsins

Acknowledgement--I wish to thank Mrs M. Gundersen for excellent technical assistance and Dr I. F. Pryme for correcting the manuscript. REFERENCES

Assaf S. A. and Graves D. J. (1969) Structural and catalytic properties of lobster muscle glycogen phosphorylase. J. biol. Chem. 244, 5544-5555. Cowey C. B. (1967) Comparative studies on the activity of D-glyceraldehyde-3-phosphate dehydrogenase from coldand warm-blooded animals with reference to temperature. Comp. Biochem. Physiol. 23, 969-976. Hochacha P. W. and Somero G. N. (1984) Biochemical Adaptation. Princeton University Press, NJ. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 227, 680-685. Lowry O, H., Rosebrough N. J., Farr A. L. and Randall

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R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Marshall T. and Lattner A. L. (1981) Incorporation of methylamine in an ultrasensitive silverstain for detecting protein in thick polyacrylamide gels. Electrophoresis 2, 228-235. Pesce A., Fondly T. P., Stolzenbach F., Castillo F. and Kaplan N. O. (1967) The comparative enzymology of lactic dehydrogenases. J. biol. Chem. 242, 2151-2167. Raae A. J. and Walther B. (1989) Purification and characterization of chymotrypsin, trypsin and elastase like proteinases from Cod (Gadus morhua L.) Comp. Biochem. Physiol. 93B, 317-324. Racicot F. R. and Hultin H. O. (1987) A comparison of dogfish and bovine chymotrypsins. Arch. Biochem. Biophys. 256, 131-143. Rao K. N. and Lombardi B. (1975) Substrate solubilization for the Hummel ~-chymotrypsin assay. Analyt. Biochem. 65, 548-551.