Weiss B, Hui M, Lajtha A
Res Commun Chem Pathol Pharmacol. 1977 Dec;18(4):709-21.
Optimal conditions have been determined for the coupling of rat liver phenylalanine hydroxylase (PheH) to activated CH-Sepharose-4B. When 12 mg of ligand was reacted with 100 mg of matrix, 20% of the initial enzyme activity was covalently bound along with 55% of the protein. The coupled enzyme showed greater thermal stability from 50 degrees to 60 degrees after heating for 15 min, a lower optimum pH, 5.8, slightly less inhibition by Ag+, Cu+2, and Hg+2, and greater resistance to hydrolysis by alpha-chymotrypsin and protease. The uncoupled enzyme, however, exhibited greater storage stability than the covalently linked enzyme at 25 degrees after 24 hrs and at 0 degrees after 21 days. Alteration of the microenvironment by the introduction of sulfhydryl groups and positive and negative charged carriers during coupling of the enzyme either had no effect or markedly reduced hydroxylase activity.
已确定大鼠肝脏苯丙氨酸羟化酶（PheH）与活化的CH - Sepharose - 4B偶联的最佳条件。当12毫克配体与100毫克基质反应时，20%的初始酶活性与55%的蛋白质共价结合。偶联酶在加热15分钟后，在50℃至60℃表现出更高的热稳定性，最佳pH较低，为5.8，受Ag +、Cu + 2和Hg + 2的抑制略少，并且对α - 胰凝乳蛋白酶和蛋白酶的水解具有更大的抗性。然而，未偶联的酶在25℃放置24小时后以及在0℃放置21天后，比共价连接的酶表现出更高的储存稳定性。在酶偶联过程中引入巯基以及带正电荷和负电荷的载体对微环境的改变要么没有影响，要么显著降低羟化酶活性。
