Oxygen equilibria of cathodic eel hemoglobin analysed in terms of the MWC model and Adair's successive oxygenation theory

Allosteric effects of erythrocytic NTP and proton concentrations on cathodic eel Hb were investigated by precise measurement of Hb-O₂ equilibria (including extreme saturation values) and analysis in terms of the MWC two-state model and the Adair four-step oxygenation theory. Stripped cathodic Hb shows a reverse Bohr effect and high sensitivities to ATP and GTP that extend to high pH values (>8.5). A decrease in pH raises K _T and lowers the allosteric constant L; compared to opposite effects in normal Bohr effect Hbs. Phosphates even at low concentrations (GTP/Hb=0.5) annihilate the reverse Bohr effect. GTP exerts a reater effect than ATP due to greater changes in K _T and L, and NTP slightly reduces K _R. In the absence of NTP, about 1.1 protons are released on deoxygenation at pH 8.15 (where most protons are released), indicating a pK value of the reverse Bohr group of approximately 8.2 (higher in oxy-Hb and lower in deoxy-Hb). The pH and NTP dependence of the Adair association constants and calculated fractional populations of Hb molecules in different oxygenation stages show that NTP effectors stabilise the T structure and postpone the T-R transition, whereas protons in the absence of NTP have the opposite effect. A molecular mechanism for the reverse Bohr effect is suggested.Abbreviations DPG 2,3 diphosphoglycerate - EPLC fast protein liquid chromatography - Hb hemoglobin - HEPES N-2-hydroxymethyl-piperazine-N-2-ethanesulfonic acid - K _T and K _R O₂ association equilibrium constants of Hb in the deoxy- and oxy-states, respectively - k ₁, k ₂, k ₃ and k ₄ Adair affinity constants for binding of the four O₂ molecules to Hb - L allosteric constant - NTP nucleoside triphosphate - P _m medium O₂ pressure - n ₅₀ Hill's cooperativity coefficient at P ₅₀ - P ₅₀ half-satutarion O₂ tension - TRIS tris(hydroxymethyl)aminomethane