HORSERADISH PEROXIDASE-DEPENDENT OXIDATION OF DEUTEROPORPHYRIN-IX INTO CHLORINS

Chlorins are cyclic tetrapyrrole derivatives of great interest for use in photodynamic therapy, We have found that horseradish peroxidase (E C 1.11.1.7) (HRP) can convert deuteroporphyrin IX (Deutero) into chlor ins, Some characteristics of this enzymatic transformation were invest igated, The formation of chlorins was determined spectrophotometricall y by monitoring the change in absorbance in the Q-band region (638 nn) , The reaction occurred without addition of H2O2 and had a pH optimum of 7.5. The presence of thiol containing reductants, with a great pref erence for reduced glutathione, was required and could not be substitu ted by adding H2O2. Ascorbic acid acted as a potent inhibitor of the r eaction, while other organic acids (citric and benzoic) had little to no inhibitory effect, The requirement for O-2 was suggested by the inh ibitory effect of sodium hydrosulfite and was confirmed by carrying th e assay in nitrogen-saturated solutions, Though the reaction occurred without adding H2O2, low amounts of H2O2 (3-30 mu M) were stimulatory to the assay, However, concentrations of 300 mu M H2O2 Or higher were inhibitory, Similarly, light was not required, but was stimulatory at low levels and inhibitory at high levels, Catalase and deferoxamine we re inhibitory, but superoxide dismutase and mannitol had no effects. K inetic analysis and respiratory studies suggest that HRP may initially react with reduced glutathione in a reaction that does not consume mu ch oxygen, The ensuing steps, probably involving an oxygen free radica l and porphyrin radical intermediates, consume a large amount of O-2 t o oxidize Deutero into chlorin. (C) 1998 Academic Press.