Schilling R J, Mitra A K
Department of Industrial and Physical Pharmacy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana 47907.
Pharm Res. 1991 Jun;8(6):721-7. doi: 10.1023/a:1015893832222.
The rate and extent of insulin degradation by trypsin and alpha-chymotrypsin were examined in vitro, and the initial sites of cleavage by alpha-chymotrypsin were identified. The apparent Km for both enzymes was approximately the same but the apparent Vmax for alpha-chymotrypsin was 8.6 times greater. At a molar ratio of 172:1 (insulin:enzyme), chymotrypsin caused near-total loss of insulin within 40 min, while very little insulin was degraded by trypsin. Chymotrypsin appeared to cleave initially at the carboxyl side of the B26-Tyr and A19-Tyr residues, and additional cleavage at the B16-Tyr, B25-Phe, and A14-Tyr residue sites also occurred rapidly. Only two to three other susceptible bonds, which are not exposed at the surface of the insulin molecule, remained intact after the quenching of initial cleavage. Four of the amino acids involved in initial cleavage are essential for receptor binding ability, making it difficult to modify insulin chemically to achieve greater stability without losing activity.
在体外检测了胰岛素被胰蛋白酶和α-糜蛋白酶降解的速率和程度，并确定了α-糜蛋白酶的初始切割位点。两种酶的表观Km大致相同，但α-糜蛋白酶的表观Vmax大8.6倍。在172:1（胰岛素:酶）的摩尔比下，糜蛋白酶在40分钟内导致胰岛素几乎完全丧失，而胰蛋白酶对胰岛素的降解很少。糜蛋白酶似乎最初在B26-酪氨酸和A19-酪氨酸残基的羧基侧切割，并且在B16-酪氨酸、B25-苯丙氨酸和A14-酪氨酸残基位点的额外切割也迅速发生。在初始切割被淬灭后，胰岛素分子表面未暴露的另外两到三个敏感键保持完整。参与初始切割的四个氨基酸对于受体结合能力至关重要，这使得在不丧失活性的情况下对胰岛素进行化学修饰以实现更高的稳定性变得困难。
