Porphyrin-induced protein structural alternations of heme enzymes. II: Protection of 5-aminolevulinic acid dehydratase and porphobilinogen deaminase from the photodynamic and non-photodynamic effects of URO and PROTO.
OBJECTIVE
Uroporphyrin and protoporphyrin produce alterations on 5-aminolevulinic acid dehydratase and porphobilinogen deaminase, as a result of a direct effect of porphyrins on the protein structure. With the aim of assessing the possible protection from the porphyrins effect on the proteins, some chemicals and the enzyme substrates were assayed.


METHODS
Enzymes were pre-incubated with the protecting agents (beta-mercaptoethanol, dithiotreitol, hydroxylamine, succinic anhydride) or the corresponding substrates (delta-aminolevulinic acid and porphobilinogen), and then exposed to the porphyrins. All experiments were performed in the enzyme solutions after removing the porphyrins.


RESULTS
The presence of sulfhydryl reagents partially protected both the enzyme activities and the content of total SH and free amino groups, but they did not prevent the appearance of molecular aggregates in the electrophoresis. Similar results were obtained in the presence of the corresponding substrates. Nucleophilic addition of hydroxylamine to the aromatic amino acids on the enzymes and blockage of their free amino groups did not prevent the direct effect of porphyrins, but these agents protected the enzyme activities from the photodynamic action of the tetrapyrroles, and also prevented the formation of molecular aggregates. However, an increased amount of free amino groups was observed, probably due to protein fragmentation.


CONCLUSIONS
Porphyrins mainly affected the SH groups at or near the active site of the enzymes. Most of the free amino groups on the treated enzymes were involved in the formation of cross-links among the protein molecules. Protein fragmentation induced by porphyrins under UV light, and the consequent increased amount of free amino groups, were observed.
Afonso
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S
Centro de Investigaciones sobre Porfirinas y Porfirias (CIPYP) CONICET, University of Buenos Aires-Argentine, Argentina.
Enríquez de Salamanca
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Batlle
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D000818:Animals; D002417:Cattle; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D011163:Hydroxymethylbilane Synthase / Q000737:chemistry* / Q000378:metabolism; D000623:Porphobilinogen Synthase / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011524:Protoporphyrins / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D014578:Uroporphyrins / Q000737:chemistry* / Q000378:metabolism
D011524:Protoporphyrins; D014578:Uroporphyrins; D006418:Heme; C034103:uroporphyrin I; D011163:Hydroxymethylbilane Synthase; D000623:Porphobilinogen Synthase