Photochemical studies of a-chymotrypsin and bovine serum albumin containing triplet photosensitizers

Abstract

A new and unusual photochemical method for achieving asymmetric synthesis which uses modified proteins as triplet photosensitizers and prochiral photolabile molecules as substrates is proposed. The modified protein, phenacyl α-chymotrypsin, was prepared by alkylation of the Met-192 residue of α-chymotrypsin with the affinity label, phenacyl bromide. Irradiation of this modified protein led to a partial regeneration of esterase activity. The initial rates of photoreactivation were dependent on the pH and reflected the pH-dependent equilibrium between the ylide and salt form of the phenacyl group. The photoreactivation of phenacyl a-chymotrypsin was only modestly quenched by potential competitive inhibitors which contain triplet-acceptor groups. 16 Irradiation of solutions containing [carbonyl- ¹⁴C] phenacyl α-chymotrypsin resulted in a partial loss of the radiolabel concomitant with the increase in esterase activity. These results were interpreted in terms of a dual photochemical behavior of phenacyl α-chymotrypsin involving (1) cleavage of the Met-192 sulfur-phenacyl α-carbon bond leading to liberation of α-chymotrypsin and a substituted phenone and (2) photoaffinitv labeling of one or more of the functional groups in the enzyme active site region. Bovine serum albumin was alkylated to various extents with p-benzoylbenzylbromide to yield modified proteins containing 13, 3, and 1 benzophenone chromophones per protein molecule. These modified proteins were subject to photoreduction by light of wavelengths greater than 320 nm. The observed photoreaction of each modified protein could be quenched by small molecule triplet acceptors. The quenching efficiency was found to depend qualitatively on the triplet acceptor's predicted binding constant to the modified proteins.