|Faibis, Valerie - USDA-ARS-SWSL|
|Jacobs, Judith - DARTMOUTH MEDICAL SCHOOL|
|Jacobs, Nicholas - DARTMOUTH MEDICAL SCHOOL|
Submitted to: Archives Of Biochemistry and Biophysics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 1, 1998
Publication Date: N/A
Interpretive Summary: We have found that the plant enzyme horseradish peroxidase converts deuteroporphyrin IX into a chlorin. Chlorins are pharmaceuticals that are expensive to synthesize. Characteristics of this enzymatic transformation were determined. This work is important because it leads the way to a better understanding of porphyrin metabolism in plants and other living organisms. Also, it demonstrates the possibility of enzymatically converting naturally occurring porphyrins into chlorins that may have important pharmaceutical applications.
Technical Abstract: Chlorins are cyclic tetrapyrrole derivatives of great interest for use in photodynamic therapy. These compounds are difficult to synthesize chemically. We have found that horseradish peroxidase (HRP) can convert deuteroporphyrin IX(Deutero) into a chlorin. Some characteristics of this enzymatic transformation were investigated. The formation of the chlorin was determined spectrophotometrically by monitoring the change in absorbance in the Q-band region (638 nm). The reaction occurred without addition of H2O2 and had a pH optimum of 7.5. The presence of thiol-containing reductants, with a great preference for reduced glutathione, was required and could not be substituted by adding H2O2. Ascorbic acid acted as a potent inhibitor of the reaction, while other organic acids (citric and benzoic) had little to no inhibitory effect. The complete inhibition of the reaction by sodium hydrosulfite demonstrated the requirement for O2. Though the reaction occurred without adding H2O2, low amounts of H2O2 (3 - 30 uM) were stimulatory to the assay. However, concentrations of 300 uM H2O2 or higher were inhibitory. Similarly, light was not required, but was stimulatory at low levels and inhibitory at high levels. Catalase and the ferric iron chelator deferroxamine were inhibitory, but superoxide dismutase and mannitol had no effect Kinetic analysis and respiratory studies suggest that HRP may initially react with reduced glutathione in a reaction that does not consume much oxygen. The ensuing steps, probably involving an oxygen free radical and porphyrin radical intermediates consume a large amount of O2 to oxidize Deutero into chlorin.