Poyart C F, Guesnon P, Bohn B M
Biochem J. 1981 May 1;195(2):493-501. doi: 10.1042/bj1950493.
We have used isoelectric focusing to measure the differences between the pI values of various normal and mutant human haemoglobins when completely deoxygenated and when fully liganded with CO. It was assumed that the DeltapI(deox.-ox.) values might correspond quantitatively to the intrinsic alkaline Bohr effect, as most of the anionic cofactors of the haemoglobin molecule are ;stripped' off during the electrophoretic process. In haemoglobins known to exhibit a normal Bohr coefficient (DeltalogP(50)/DeltapH) in solutions, the DeltapI(deox.-ox.) values are lower the higher their respective pI(ox.) values. This indicates that for any particular haemoglobin the DeltapI(deox.-ox.) value accounts for the difference in surface charges at the pH of its pI value. This was confirmed by measuring, by the direct-titration technique, the difference in pH of deoxy and fully liganded haemoglobin A(0) (alpha(2)beta(2)) solutions in conditions approximating those of the isoelectric focusing, i.e. at 5 degrees C and very low concentration of KCl. The variation of the DeltapH(deox.-ox.) curve as a function of pH (ox.) was similar to the isoelectric-focusing curve relating the variation of DeltapI(deox.-ox.) versus pI(ox.) in various haemoglobins with Bohr factor identical with that of haemoglobin A(0). In haemoglobin A(0) the DeltapI(deox.-ox.) value is 0.17 pH unit, which corresponds to a difference of 1.20 positive charges between the oxy and deoxy states of the tetrameric haemoglobin. This value compares favourably with the values of the intrinsic Bohr effect estimated in back-titration experiments. The DeltapI(deox.-ox.) values of mutant or chemically modified haemoglobins carrying an abnormality at the N- or C-terminus of the alpha-chains are decreased by 30% compared with the DeltapI value measured in haemoglobin A(0). When the C-terminus of the beta-chains is altered, as in Hb Nancy (alpha(2)beta(Tyr-145-->Asp) (2)), we observed a 70% decrease in the DeltapI value compared with that measured in haemoglobin A(0). These values are in close agreement with the estimated respective roles of the two major Bohr groups, Val-1alpha and His-146beta, at the origin of the intrinsic alkaline Bohr effect [Kilmartin, Fogg, Luzzana & Rossi-Bernardi (1973) J. Biol. Chem.248, 7039-7043; Perutz, Kilmartin, Nishikura, Fogg, Butler & Rollema (1980) J. Mol. Biol.138, 649-670]. In other mutant haemoglobins it is demonstrated also that the DeltapI(deox.-ox.) value may be decreased or even suppressed when the substitution affects residues involved in the stability of the tetramer. These results support the interpretation proposed by Perutz, Kilmartin, Nishikura, Fogg, Butler & Rollema [(1980), J. Mol. Biol.138, 649-670] for the mechanism of the alkaline Bohr effect, and also indicate that the transition between the two quaternary configurations is a prerequisite for the full expression of the alkaline Bohr effect.
我们利用等电聚焦来测量各种正常和突变型人血红蛋白在完全脱氧状态以及与一氧化碳完全结合状态下的等电点（pI）值之间的差异。假定ΔpI（脱氧-氧合）值可能在数量上与内在碱性玻尔效应相对应，因为在电泳过程中血红蛋白分子的大多数阴离子辅因子会被“去除”。在已知在溶液中表现出正常玻尔系数（ΔlogP(50)/ΔpH）的血红蛋白中，其各自的pI（氧合）值越高，ΔpI（脱氧-氧合）值越低。这表明对于任何特定的血红蛋白，ΔpI（脱氧-氧合）值说明了在其pI值的pH下表面电荷的差异。通过直接滴定技术测量在接近等电聚焦条件（即5℃和极低浓度的氯化钾）下脱氧和完全结合一氧化碳的血红蛋白A(0)（α₂β₂）溶液的pH差异，证实了这一点。ΔpH（脱氧-氧合）曲线随pH（氧合）的变化类似于等电聚焦曲线，该曲线表示在各种与血红蛋白A(0)具有相同玻尔因子的血红蛋白中ΔpI（脱氧-氧合）相对于pI（氧合）的变化。在血红蛋白A(0)中，ΔpI（脱氧-氧合）值为0.17个pH单位，这对应于四聚体血红蛋白的氧合态和脱氧态之间1.20个正电荷的差异。该值与在反滴定实验中估计得到的内在玻尔效应的值相当。与在血红蛋白A(0)中测得的ΔpI值相比，在α链的N端或C端携带异常的突变型或化学修饰型血红蛋白的ΔpI（脱氧-氧合）值降低了30%。当β链的C端发生改变时，如在血红蛋白Nancy（α₂β(Tyr-145→Asp)₂）中，我们观察到与在血红蛋白A(0)中测得的值相比，ΔpI值降低了70%。这些值与在内在碱性玻尔效应起源处两个主要玻尔基团Val-1α和His-146β的估计各自作用密切一致[Kilmartin、Fogg、Luzzana和Rossi-Bernardi（1973年）《生物化学杂志》248，7039 - 7043；Perutz、Kilmartin、Nishikura、Fogg、Butler和Rollema（1980年）《分子生物学杂志》138，649 - 670]。在其他突变型血红蛋白中还表明，当取代影响参与四聚体稳定性的残基时，ΔpI（脱氧-氧合）值可能会降低甚至被抑制。这些结果支持了Perutz、Kilmartin、Nishikura、Fogg、Butler和Rollema[(1980年)，《分子生物学杂志》138，649 - 670]对碱性玻尔效应机制提出的解释，并且还表明两种四级结构之间的转变是碱性玻尔效应充分表达的先决条件。
