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

Aerobic and anaerobic studies have demonstrated that uroporphyrin I-in duced inactivation of delta-aminolevulinic acid dehydratase, porphobil inogenase, deaminase and uroporphyrinogen decarboxylase was dependent on oxygen and mediated by reactive oxygen species. The mechanism of ph otoinactivation of those heme-enzymes from human erythrocytes by uropo rphyrin I by u.v. light was investigated. Enzymes of the heme pathway were preincubated in the presence of specific scavengers for several r eactive oxygen species and then exposed to uroporphyrin I and u.v. lig ht. Upon exposure of the enzymes to the porphyrin under u.v. light, an d in an aerobic atmosphere, the percentage of enzyme activities with r espect 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 +/- 75 (SD, n = 8) for delta-aminolevulinic acid dehydratase, porphobilinogenase, deaminase and uroporphyrinogen decarboxylase, respectively. The presen ce of sodium azide, histidine or superoxide dismutase did not protect the enzymes against the effects of uroporphyrin I. However, both cyste ine 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 dimethyls ulfoxide had no effect on enzyme activity, while ion chelators had var iable effects. This study shows that the type II mechanism is not the predominant reaction mediating the uroporphyrin I effect and enzyme ph otoinactivation would involve an electron transfer. Hydrogen peroxide and hydroxyl radicals could possibly mediate the uroporphyrin I-induce d enzyme photoinactivation. Copyright (C) 1996 Elsevier Science Ltd.