Characterization of Amynthas Gracilis Hemoglobin (HbAg) and its Subunits by AUC and MALDI-TOF-MS

Abstract

The giant extracellular hemoglobin (HbAg) of the annelid Amynthas gracilis has a molecular mass (MM) of 3400kDa. In the current work, the characterization of MM values of HbAg and its subunits is presented. Electrophoresis, MALDI-TOF-MS and AUC show that the MM values of HbAg subunits are very close, but not identical to those of Glossoscolex paulistus (HbGp) and Rhinodrilus alatus (HbRa) hemoglobins. Analytical ultracentrifugation (AUC) sedimentation velocity experiments were performed to obtain M for HbAg in oxy- form. value of 59.3 ± 0.2 S was obtained for native HbAg. From the ratio between sedimentation and diffusion coefficients values for M of approximately 3400 ± 100 kDa for oxy-HbAg was obtained. MALDI-TOF-MS data gave MM for HbAg subunits. Monomer d is found to exist in, at least, four isoforms with MM 16,244±3Da, 16,459±5Da, 16,667±5Da and 16,855±3Da, as noticed for HbGp, and not observed for HbRa. Furthermore, the trimer subunit presents two isoforms ((abc)1 and (abc)2) with MM 51,415±20Da and 51,610±14Da, respectively. This might indicate that the monomers a, b and c do have isoforms, as found for HbGp and not for HbRa. The monomeric chains a, obtained from the trimer abc reduction, present three isoforms with MM 17,015Da, 17,061Da and 17,138Da, differing from HbGp that presents four isoforms. A less intense species is observed at 67,717, and is due to the tetramer abcd contribution. Finally, AUC and MALDI-TOF-MS data are very close as compared to that obtained for HbGp and HbRa. Our results show total consistency between M obtained by AUC and recent partial characterization by mass spectrometry. Finantial support: FAPESP and CNPq Brazilian agencies.