title: Spectroscopic studies of the met form of the extracellular hemoglobin from Glossoscolex paulistus.
abstract: Sephadex G-200 chromatography of the extracellular hemoglobin from the giant earthworm G. paulistus in the met form presents a single peak at pH 7.0 and two peaks at pH 9.0 as a result of alkaline dissociation. SDS-PAGE shows that the polypeptide chains are very similar to those observed for the oxy form and the two peaks at pH 9.0 correspond to the trimer contaminated by linkers and monomers which seems to be quite pure. The aquomet acid form is stable as an oligomer of molecular mass 3.1 x 10(6) Da only in a narrow pH range around neutrality. Increasing the pH above 7.5 leads to an irreversible transition from aquomet to hemichrome I which is the low-spin bis-imidazole complex. At pHs above 9.5-10.0 a second reversible transition takes place from hemichrome I to hemichrome II, a high-spin complex which is associated with the weakening and possible disruption of the proximal Fe--N histidine bond. Thus, increase in pH above 8.0 induces changes in the heme pocket that involve both the distal and proximal sides of the heme. EPR measurements show a very sharp decrease of the aquomet high-spin signal in the range of pH 7.0-8.0 and a very small low-spin signal even at liquid helium temperatures. The transition to hemichrome I is also accompanied by the loss of heme optical activity monitored by CD, which is consistent with the weakening of heme--globin interaction. Hemichrome I in the presence of cyanide gives the typical cyanometHb derivative which has a transition to a hemichrome at much higher pHs. This observation suggests that the dissociation of the oligomer in alkaline medium as well as the stability of the heme on the proximal side, depend both upon the ligand present at the sixth coordination position on the distal side. Hence, we believe that hemi(hemo)chrome formation in G. paulistus Hb and other invertebrate hemoglobins is a common phenomenon, not associated with protein denaturation, which may provide a fine tuning mechanism to control subunit interactions through changes in the distal side of the heme pocket.
authors.affiliation: Departamento Química e Física Molecular, Instituto de Química de São Carlos, Universidade de São Paulo, Brazil.
authors.forename: S C
authors.initials: SC
authors.lastname: Agustinho
authors.forename: M H
authors.initials: MH
authors.lastname: Tinto
authors.forename: H
authors.initials: H
authors.lastname: Imasato
authors.forename: T T
authors.initials: TT
authors.lastname: Tominaga
authors.forename: J R
authors.initials: JR
authors.lastname: Perussi
authors.forename: M
authors.initials: M
authors.lastname: Tabak
mesh_terms: D000818:Animals; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D006420:Hemeproteins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008706:Methemoglobin / Q000737:chemistry*; D009835:Oligochaeta; D013053:Spectrophotometry
chemical_list: D006420:Hemeproteins; C001332:hemichrome; D008706:Methemoglobin