The reactivity of the β-93 sulfhydryl groups with p-hydroxymercuribenzoate has been used as a kinetic probe to investigate conformational changes in globin, deoxyhemoglobin, and various liganded forms of hemoglobin using stopped-flow devices. The pH dependence and the energy of activation of the various reactions have been determined. Addition of heme to the a chains of globin causes only a very small change in the rate of reaction of the β-93 SH group. Further addition of heme which fills up the β-heme sites to form hemoglobin results in a tenfold decrease in the p-hydroxymercuribenzoate reaction rate, suggesting that the β-93 SH is less exposed in hemoglobin than in globin. The reaction of the β-93 SH group in isolated β-O2 chains is slightly faster than that of HbO2. This finding is consistent with the previous circular dichroic (CD) spectra and ligand-binding kinetic studies which indicated a difference between the heme environment in isolated β chains and in the β chains of liganded hemoglobin. The p-hydroxymercuribenzoate reaction rate is decreased nearly 70-fold at pH 7 when oxyhemoglobin is converted to deoxyhemoglobin. In the ligand-bound conformation of hemoglobin, the p-hydroxymercuribenzoate reaction was not sensitive to the nature of the ligand for the five forms studied (HbO2, HbCO, HbCN, HbN3, and methemoglobin). These results are consistent with a masking of the β-93 SH group when the hemoglobin molecule undergoes conformational changes from the oxy- to deoxyhemoglobin form, as indicated by crystallographic studies. The reaction of hemoglobin has been studied in dilute solution and in salt solutions thought to produce α-β dimers. In salt solution the kinetics remain essentially unchanged, indicating that if dimers are formed, they react with a rate similar to tetrameric hemoglobin. The studies in dilute solution indicate that at a heme concentration of 1.75 μM the fraction of the oxyhemoglobin dissociated into chains cannot be greater than 4-7%.
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