Stable octameric structure of recombinant hemoglobin α ₂ 83 Gly→Cys

We have engineered a recombinant hemoglobin (rHb βG83C) based on the variant Hb Ta‐Li, which oligomerizes through intertetramer disulfide bonds. Size exclusion chromatography and electrospray ionization mass spectrometry show that the rHb βG83C assembles into an oligomeric structure the size of a dimer of tetramers. The oligomer has carbon monoxide‐binding properties similar to those of natural human hemoglobin. Unlike HbA, the oligomer does not participate in dimer exchange. The CO kinetics, auto‐oxidation rate, and gel filtration experiments on the oligomeric βG83C did not show the usual concentration dependence, implying that it does not dissociate easily into smaller species. The octamer could be dissociated by the use of reducing agents. The action of reduced glutathione on oligomeric βG83C exhibited biphasic kinetics for the loss of the octameric form, with a time constant for the rapid phase of about 2 h at 1 mM glutathione. However, the size of oligomer βG83C was not modified after incubation with fresh plasma.