Fluorescence studies of extracellular hemoglobin of Glossoscolex paulistus in met form obtained from Sephadex gel filtration

Chromatography in Sephadex G-200 of extracellular hemoglobin of the giant worm Glossoscolex paulistus in the met form presents an unique band at pH 7.0 and two bands at pH 9.0 as a result of alkaline dissociation. SDS-PAGE of the intact protein obtained at pH 7.0 is very similar to that for the oxyhemoglobin. Chromatography at pH 9.0 indicates complete dissociation of the oligomeric protein into two low molecular weight fractions corresponding to the trimers and monomers, showing that the oxidized extracellular hemoglobin is less stable than the oxyhemoglobin with respect to alkaline dissociation. Fluorescence quantum yields of different fractions obtained in the chromatography, as well as extinction coefficients at 280 nm and 415 nm, were estimated and compared to human methemoglobin. The fluorescence data are consistent with the high content of aromatic residues in G. paulistus hemoglobin. The increase in the fluorescence quantum yield upon both alkalinization and dissociation was correlated with the reduction of intramolecular quenching but the exposure of tryptophan residues to the solvent did not changed significantly as occurs for the oxy form. The intact native protein has a quantum yield of 0.36% at pH 7.0, increasing to 1.89% at pH 9.0 upon dissociation. The monomer has a fluorescence quantum yield of 1.1% at pH 7.0 increasing to 1.43% at pH 9.0. The maximum emission wavelength of the intact protein (330 nm) is consistent with tryptophan residues being relatively buried; they become more fluorescent upon dissociation into smaller subunits but not more exposed since the wavelength of maximum emission is essentially unchanged at pH 9.0. In the monomer, the tryptophan residues also remain buried inside the protein molecule at pH 9.0 (328 nm). The dependencies of fluorescence quantum yields on the pH show in a clear way the hemichrome transitions observed by optical absorption spectroscopy indicating that the formation of two types of hemichromes accompany the distinct increase in fluorescence quantum yield. One type of hemichrome is irreversibly formed around pH 7.5-8.0 and a second reversible hemichrome is formed above pH 9.5-10.0. They are associated with the bis-imidazole low spin hemichrome and with a high spin hemichrome where the weakening of the iron bond to proximal histidine takes place. Addition of cyanide to the metHb solution produces the cyanomet form of the protein which is considerably more stable towards alkaline dissociation and presents a smaller change in quantum yield as a function of pH. Circular dichroism suggests that the formation of hemichromes is not accompanied by significant protein denaturation. (C) 1997 Elsevier Science Inc.