Van Beek G G, De Bruin S H
Eur J Biochem. 1980 Apr;105(2):353-60. doi: 10.1111/j.1432-1033.1980.tb04508.x.
The number of Bohr protons released upon oxygenation of human hemoglobin was measured at 25 degrees C and 37 degrees C as a function of the concentration of chloride ions. From the results obtained association constants could be evaluated for the binding of chloride ions to both deoxy and oxyhemoglobin at these two temperatures. Furthermore, pK values could be determined for those protonic groups involved in chloride ion binding to deoxy and oxyhemoglobin. From these data it was inferred that in oxyhemoglobin only imidazole groups are participating in chloride binding, whereas in deoxyhemoglobin the chloride binding site contained the alpha NH2 group of valine-1 alpha. The same conclusions were reached by measuring the pK values of the aminogroups of valine 1 alpha and valine-1 beta at different temperatures and ionic strengths. The pK values were measured by following the rate of reaction of 1-fluoro-2,4-dinitrobenzene with the alpha-NH2 group by spectrophotometric means. We further showed that binding of Cl-, Br- and I- to oxyhemoglobin follows the lyotropic or Hofmeister series, while this effect is much less for deoxyhemoglobin. This result indicates that for the binding of anions to oxyhemoglobin interactions with non-polar groups contribute to the free-energy change of binding.
在25摄氏度和37摄氏度下，测量了人血红蛋白氧合时释放的玻尔质子数量，该数量是氯离子浓度的函数。根据所得结果，可以评估在这两个温度下氯离子与脱氧血红蛋白和氧合血红蛋白结合的缔合常数。此外，可以确定参与氯离子与脱氧血红蛋白和氧合血红蛋白结合的质子基团的pK值。从这些数据可以推断，在氧合血红蛋白中只有咪唑基团参与氯离子结合，而在脱氧血红蛋白中，氯离子结合位点包含缬氨酸-1α的α-NH2基团。通过测量不同温度和离子强度下缬氨酸1α和缬氨酸-1β氨基的pK值，也得出了相同的结论。通过分光光度法跟踪1-氟-2,4-二硝基苯与α-NH2基团的反应速率来测量pK值。我们还表明，Cl-、Br-和I-与氧合血红蛋白的结合遵循离液序列或霍夫迈斯特序列，而对于脱氧血红蛋白，这种效应要小得多。这一结果表明，对于阴离子与氧合血红蛋白的结合，与非极性基团的相互作用对结合的自由能变化有贡献。
