Separation and identification of the regioisomers of verdoheme by reversed-phase ion-pair high-performance liquid chromatography, and characterization of their complexes with heme oxygenase

We report an HPLC method for separating the four regioisomers of verdoheme formed in the coupled oxidation of hemin with oxygen and ascorbate in aqueo us pyridine. The reversed-phase ion-pair system uses hexafluoroacetone and pyridine as ion-pair agents. The regiochemistry of the separated isomers wa s established both by HPLC of the corresponding biliverdin IX derivatives a nd by H-1 NMR of each isomer. Optical spectra of the pyridine verdohemochro me isomers were similar to each other, but showed differences in the absorp tion maxima in the red region, which appear at 680, 663, 648 and 660 nm for the alpha, beta, gamma, and delta -isomers, respectively. Each of the four isomers was incorporated anaerobically into heme oxygenase-l, yielding the corresponding verdoheme-enzyme complex. The ferrous forms had absorption m axima at 690, 667, 655, and 663 nm, and their GO-bound forms had maxima at 638, 624, 616, and 626 nm for alpha, beta, gamma, and delta -isomer, respec tively. Addition of ferricyanide to the alpha -verdoheme-heme oxygenase com plex brought about a ferric low-spin heme-like signal, which is identical w ith the ferric alpha -verdoheme complexed with the heme oxygenase that was observed in the heme oxygenase reaction. (C) 2000 Elsevier Science BN. All rights reserved.