MECHANISTIC STUDIES ON UROPORPHYRIN I-INDUCED PHOTOINACTIVATION OF SOME HEME-ENZYMES

Abstract Aerobic and anaerobic studies have demonstrated that uroporphyrin I-induced inactivation of δ-aminolevulinic acid dehydratase, porphobilinogenase, deaminase and uroporphyrinogen decarb-oxylase was dependent on oxygen and mediated by reactive oxygen species. The mechanism of photoinactivation of those heme-enzymes from human erythrocytes by uroporphyrin I by u.v. light was investigated. Enzymes of the heme pathway were preincubated in the presence of specific scavengers for several reactive oxygen species and then exposed to uroporphyrin I and u.v. light. Upon exposure of the enzymes to the porphyrin under u.v. light, and in an aerobic atmosphere, the percentage of enzyme activities with respect to the corresponding controls were 50.2 ± 5.1 (SD, n = 6), 25.3 ± 3.0 (SD, n = 6), 25.9 ± 2.8 (SD, n = 6) and 49.7 ± 7.5 (SD, n = 8) for δ-aminolevulinic acid dehydratase, porphobilinogenase, deaminase and uroporphyrinogen decarb-oxylase, respectively. The presence of sodium azide, histidine or superoxide dismutase did not protect the enzymes against the effects of uroporphyrin I. However, both cysteine and potassium ferrycyanide prevented the enzyme photoinactivation induced by uroporphyrin I. In the presence of either catalase or GSH, the enzyme photoinactivation was lower. Ethanol, glucose and dimethylsulfoxide had no effect on enzyme activity, while ion chelators had variable effects. This study shows that the type II mechanism is not the predominant reaction mediating the uroporphyrin I effect and enzyme photoinactivation would involve an electron transfer. Hydrogen peroxide and hydroxyl radicals could possibly mediate the uroporphyrin I-induced enzyme photoinactivation.