Mechanistic studies on uroporphyrin I-induced photoinactivation of some heme-enzymes.

Aerobic and anaerobic studies have demonstrated that uroporphyrin I-induced inactivation of delta-aminolevulinic acid dehydratase, porphobilinogenase, deaminase and uroporphyrinogen decarboxylase 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 delta-aminolevulinic acid dehydratase, porphobilinogenase, deaminase and uroporphyrinogen decarboxylase, 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.

Further evidence for an essential histidyl residue at the active site of pig liver 5-aminolevulinic acid dehydratase.

Photoxidation with methylene blue and rose bengal and chemical modification by diethylpryrocarbonate of pig liver 5-aminolevulinic acid dehydratase produced strong inactivation of the enzyme which was concentration dependent. Loss of enzyme activity by both photoxidation and ethoxyformylation was pH and time-dependent and protected by the presence of the substate and competitive inhibitors. The rate of inactivation was directly related to the state of protonation of histidyl groups, the unprotonated from being modified at a much faster rate than the protonated form. Plots of the pseudo-first order rate constants for 5-aminolevulinic acid dehydratase inactivation against pH resulted in typical titration curves showing inflection points at about pH 6.4 for methylene blue and rose bengal and 6.8 for diethylprocarbonate providing further and unequivocal evidence for the existence of critical histidyl groups at the active centre of the enzyme.

Photodynamic and non-photodynamic action of several porphyrins on the activity of some heme-enzymes.

The action of porphyrins, uroporphyrin I and III (URO I and URO III), pentacarboxylic porphyrin I (PENTA I), coproporphyrin I and III (COPRO I and COPRO III), protoporphyrin IX (PROTO IX) and mesoporphyrin (MESO), on the activity of human erythrocytes delta-aminolevulinic acid dehydratase, porphobilinogenase, deaminase and uroporphyrinogen decarboxylase in the dark and under UV light was investigated. Both photoinactivation and light-independent inactivation was found in all four enzymes using URO I as sensitizer. URO III had a similar action as URO I on porphobilinogenase and deaminase and PROTO IX exerted equal effect as URO I on delta-aminolevulinic acid dehydratase and uroporphyrinogen decarboxylase. Photodynamic efficiency of the porphyrins was dependent on their molecular structure. Selective photodecomposition of enzymes by URO I, greater specificity of tumor uptake by URO I and enhanced porphyrin synthesis by tumors from delta-aminolevulic acid, with predominant formation of URO I, underline the possibility of using URO I in detection of malignant cells and photodynamic therapy.