Catalase (CAT) in
solution or incorporated in erythrocytes and K562 leukemic cells is inactivated during
photosensitization with tetrasulfonated metallophthalocyanines (MePcS4). The effect of added scavengers and D2O showed that both
singlet oxygen and
free radical species are involved in this process. Evidence was found that direct interactions of ground or excited-stated
photosensitizer with CAT are not responsible for CAT inactivation. Specific techniques to probe early damage to the CAT structure involved optical and EPR spectroscopy, HPLC and
polyacrylamide gel electrophoresis analyses. Different primary events of photosensitized
protein damage included oxidation of
cysteine residues as well as other
amino acids, as demonstrated by the formation of
carbon-centered
free radicals and the loss of absorbance at lambda = 275 nm. In parallel, we detected degradation of the CAT
heme groups, accompanied by release of Fe(II)
ions in
solution. These combined phenomena initiate cross-linkages between CAT subunits and subsequent degradation of the
protein with formation of irreversible aggregates in
solution.
Phthalocyanine-mediated photoinactivation of cell-bound CAT results in loss of protection against accumulating H2O2, providing an additional pathway of
phototoxicity.