Isolation and Characterization of the Triply Oxidized Derivative of a Cross-Linked Hemoglobin

Hemoglobin A, cross-linked between Lys99α₁and Lys99α₂, was used to obtain a partially oxidized tetramer in which only one of the four hemes remains reduced. Because of the absence of di-merization, asymmetric, partially oxidized derivatives are stable. This is evidenced by the fact that eight of the ten possible oxidation states could be resolved by analytical isoelectric focusing. A triply oxidized hemoglobin population HbXL+3 was isolated whose predominant componeftt was (α⁺α⁺,β⁺βΰ). This triferric preparation was examined as a possible model for the triliganded state of ferrous HbA. The aquomet and cyanomet derivatives were characterized by their CD spectra and their kinetic reactions with carbon monoxide. CD spectra in the region of 287 nm showed no apparent change in quaternary structure upon binding ligand to the fourth, ferrous heme. The spectra of the oxy and deoxy forms of the cyanomet and aquomet derivatives of HbXL+3 differed insignificantly and were characteristic of the normal liganded state. Upon addition of inositol hexaphosphate (IHP), both the oxy and deoxy derivatives of the high-spin triaquomet species converted to the native deoxy conformation. In contrast, IHP had no such effect on the conformation of the low-spin cyanomet derivatives of HbXL+3. The kinetics of CO combination as measured by stopped-flow and flash photolysis techniques present a more complex picture. In the presence of IHP the triaquomet derivative does bind CO with rate constants indicative of the T state whether these are measured by the stopped-flow technique or by flash photolysis. However, CO binding characterized entirely by R-state rate constants is observed only with the tricyanomet derivative in the absence of IHP and only when examined by flash photolysis. Under all other conditions, complex kinetics are observed which suggest the presence of more than one functional state of the protein.