PORPHYRIN-INDUCED PROTEIN STRUCTURAL ALTERATIONS OF HEME ENZYMES II -PROTECTION OF 5-AMINOLEVULINIC ACID DEHYDRATASE AND PORPHOBILINOGEN DEAMINASE FROM THE PHOTODYNAMIC AND NON-PHOTODYNAMIC EFFECTS OF URO ANDPROTO

Background and aims: Uroporphyrin and protoporphyrin produce alteratio ns 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 porphyrin s effect on the proteins, some chemicals and the enzyme substrates wer e assayed. Methods: Enzymes were pre-incubated with the protecting age nts (beta-mercaploethanol, dithiotreitol, hydroxylamine, succinic anhy dride) 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 group s, 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 t o the aromatic amino acids on the enzymes and blockage of their free a mino groups did not prevent the direct effect of porphyrins, but these agents protected the enzyme activities from the photodynamic action o f the tetrapyrroles, and also prevented the formation of molecular agg regates. However, an increased amount of free amino groups was observe d, probably due to protein fragmentation. Conclusions: Porphyrins main ly affected the SH groups at or near the active site of the enzymes. M ost of the free amino groups on the treated enzymes were involved in t he formation of cross-links among the protein molecules. Protein fragm entation induced by porphyrins under UV light, and the consequent incr eased amount of free amino groups, were observed. (C) 1998 Published b y Elsevier Science Ltd. All rights reserved.