III. Covalent coupling of rat liver phenylalanine hydroxylase.
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.
III. Covalent coupling of rat liver phenylalanine hydroxylase. 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.