Cooperative oxygen binding in beta-semihemoglobins caused by a chemical modification in the alpha1beta1 interface

A beta-semihemoglobin is an alpha-beta dimer of hemoglobin (Hb) in which the beta-subunit carries heme, while the alpha-subunit is heme-less, in apo form. It is characterised by displaying a high affinity for oxygen, and absence of cooperative binding of oxygen. We have modified chemically the residue beta112Cys (G14), located adjacent to the alpha1beta1 interface, and studied the impact of such a modification on the oligomeric state and oxygenation properties of the derivatives. We also studied the impact of modifying beta93Cys (F9) since its modification was unavoidable. For this, we used N-Ethyl maleimide and iodoacetamide. For the alkylation of beta112Cys (G14) in isolated subunits, we used N-Ethyl maleimide, iodoacetamide, or additionally, 4,4'-Dithiopyridine. Seven native and chemically modified beta-subunit derivatives were prepared and analysed. Only those derivatives treated with iodoacetamide showed oxygenation properties that were indistinguishable from those of native beta-subunits. These derivatives were then converted into their respective semihemoglobin forms, and four additional derivatives were prepared and analysed .in terms of ligation-linked oligomeric state, and oxygenation function, and contrasted against native Hb and unmodified beta-subunits. Strikingly, beta-semiHbs with modifications in beta112Cys showed indications of cooperative oxygen binding in various degrees, which suggested the possibility of assembly of two beta-semiHbs. The derivative modified with 4-Thiopyridine in beta112Cys showed a highly cooperative binding of oxygen (nmax = 1.67). A plausible allosteric scheme that could explain allostery in beta-semiHb system is suggested.