HEMATOPORPHYRIN PHOTOSENSITIZATION OF SERUM ALBUMIN and SUBTILISIN BPN'

Abstract

Abstract —The photosensitized inactivation of subtilisin BPN' by free hematoporphyrin (HP) followed exponential kinetics with positive mechanistic tests for the involvement of singlet oxygen ( 1 O 2 ) as the principal intermediate. The photoinactivation quantum yield was 0.029 at 390 nm in oxygen‐saturated, D 2 O buffer at pH 7.0. The effects of HP binding were investigated for tryptophan oxidation in bovine serum albumin (BSA) and human serum albumin (HSA) at high protein:HP concentration ratios where the HP was > 97% complexed. The reaction kinetics were non‐exponential and mimick a second‐order process in the initial stages. The rate of HP photobleaching was 30‐fold faster for complexed HP compared with free HP, which was shown to account for the observed kinetics. Mechanistic tests showed that 1 O 2 was the dominant photooxidizing intermediate of tryptophan residues and that it was not involved in the accompanying photobleaching of HP. The quantum yield for tryptophan oxidation in BSA was 0.11 at 390 nm in oxygen‐saturated, D 2 O buffer at pH 8.0. The reactivity of HSA was approximately 2‐fold lower than BSA for equivalent conditions. Estimates of the reaction cross sections led to 3 Å 2 for the inactivation of subtilisin BPN' by 1 O 2 and 20 Å 2 for the oxidation of tryptophan in BSA.