Fluorescence studies of extracellular hemoglobin of Glossoscolex paulistus obtained by gel filtration

Abstract

Chromatography in Sephadex G-200 of extracellular hemoglobin of the giant worm Glossoscolex paulistus presents a unique band at pH 7.0 and several bands at pH 9.0 as a result of alkaline dissociation. SDS-polyacrylamide gel electrophoresis (PAGE) of the intact protein obtained at pH 7.0 shows the existence of five different bands with molecular weights of 12 ± 1, 26 ± 3, 28 ± 2, 34 ± 1 and 53 ± 1 kDa. In the presence of β-mercaptoethanol, six distinct bands are obtained with molecular weights 13 ± 1, 14.8 ± 0.3, 15.8 ± 0.2, 16.6 ± 0.2, 35.1 ± 0.3 and 40.5 ± 0.5 kDa. Reduction of disulfide bonds of a trimer of molecular weight 53 kDa leads to the appearance of monomeric subunits of 14.8, 15.8 and 16.6 kDa. The molecular weights obtained from chromatography in Sephadex G-200 at pH 9.0 are different from those from SDS-PAGE. An intense band at 110 ± 12 kDa due to the tertiary complex of two disulfide-linked trimers and monomer chain (trimer + monomer), (abcd)2, is observed in addition to a small amount of high molecular weight fraction in the exclusion volume, as well as poorly resolved trimer (42 ± 2 kDa) and monomer (15 ± 2 kDa) bands. Fluorescence quantum yields of different fractions obtained in the chromatography as well as extinction coefficients at 280 and 415 nm were estimated and compared with human hemoglobin. The fluorescence data presented are consistent with the high content of aromatic residues in the G. paulistus hemoglobin. The increase in the fluorescence quantum yields upon both alkalinization and dissociation were correlated with the exposure of tryptophan residues to the solvent. The intact native protein has a quantum yield of 0.25% at pH 7.0, assigning 14% to tryptophan solution at pH 7.0 and increasing to 0.8% at pH 9.0 upon dissociation. The monomer has a fluorescence quantum yield of 0.8% at pH 7.0, increasing to 2.1% at pH 9.0. The maximum emission wavelength of the intact protein (327 nm) is consistent with tryptophan residues buried, quenched and becoming more exposed and more fluorescent upon dissociation into smaller subunits (332-335 nm). In the monomer the tryptophan residues remain buried inside the protein molecule at pH 9.0.