V. Efforts at stabilization of rat liver phenylalanine hydroxylase.

The best from a series of matrices for the covalent coupling of rat liver phenylalanine (Phe H) was activated thiol-Sepharose 4B. An average of 0.38 mumoles of enzyme thiol was coupled per mumole of matrix thiol, representing 21 mg of protein per g of carrier. After thiolation with N-acetylhomocysteine thiolactone the enzyme, bound to the same matrix, was thermally more stable with a broad pH range of optimal activity. Thiolation with S-acetylmercaptosuccinic anhydride markedly reduced hydroxylase activity whereas succinylation with succinic anhydride had little or no effect. Treatment of the thiolated enzyme with various oxidants to form disulfide bridges did not increase the thermal stability. Efforts to improve stability by formation of new linkages while the enzyme was still bound to the matrix were unsuccessful. The untreated or thiolated enzyme was inactivated upon reaction with glutaraldehyde.