A simple preparation of β-trypsin based on a calorimetric study of the thermal stabilities of α- and β-trypsin

Abstract

Bovine trypsin preparations contain, in addition to the single chain form of the enzyme, an active two-chain autolysis product (Schroeder, D. D., and Shaw, E., J. Biol. Chem. (1968), 243, 2943–2949). Differential scanning calorimetric (DSC) studies showed that the single chain form, β-trypsin, is more stable to thermal denaturation than the two-chain form, α-trypsin. Rate constants and activation energies for the thermal denaturation of β-trypsin are 5 × 10 −5 sec −1 and 69 kcal/mole and of α-trypsin are 5 × 10 −3 sec −1 and 38 kcal/mole at pH 4.4 and 48 °C. Preparation of pure β-trypsin can be greatly simplified by prior thermal denaturation of the α form. At least 75% of the α form is denatured by heating a 10–15% solution of commercial crystalline trypsin for 30–45 min at 48 °C, pH 4.4, 0.02 m Ca 2+. The native β-trypsin is then easily isolated from the denatured α-trypsin by batchwise adsorption onto ovoinhibitor-agarose at pH 8. After elution at pH 2, dialysis, and lyophilization an average preparation contained approximately 85% β-trypsin, 10% α-trypsin, and 5% inactive material. Benzamidine was used during the isolation to decrease the rate of conversion of β- to α-trypsin. Because the separation of active β-trypsin from heat-denatured α-trypsin is relatively easy, the total preparation time has been reduced to 1 day.