The Chemistry of the Bohr Effect I. THE REACTION OF N-ETHYL MALEIMIDE WITH THE OXYGEN-LINKED ACID GROUPS OF HEMOGLOBIN*

One of the most intriguing phenomena associated with the function of hemoglobin is the reversible dissociation of protons which accompanies its oxygenation (2-4). This oxygen-linked ionization, which is commonly referred to as the Bohr effect, results in the liberation of about 2.7 protons per mole when human hemoglobin is oxygenated at physiological pH. Moreover, a “reverse Bohr effect” occurs in the region of pH 5.5, at which oxygenation is accompanied by the absorption of one proton per mole. It should be kept in mind that this latter dissociation is in a pH region in which partial splitting into half molecules occurs. It was shown by Wyman (5) that the Bohr effect between pH 6 and 9 can be quantitatively accounted for in terms of four oxygen-linked acid groups per mole of hemoglobin with a pK of 7.93 in reduced hemoglobin and 6.68 in oxyhemoglobin. The chemical identity of these ionizing groups is, however, still not settled. The groups which have been suggested are amino groups (6), sulfhydryl groups (7)) and imidazole groups (5,8-10). Amino groups are unlikely to be involved in this ionization, since their pK and, especially, their heat of ionization, is too high. A number of considerations would seem to rule out Riggs’ recent suggestion that the reactive -SH groups are the source of the “Bohr protons.” Since there are only two to two and one-half reactive -SH groups in human hemoglobin (7, II), they could not account for the oxygen-linked acid production (about 2.7 moles per mole), even if they were completely unionized in the reduced and completely ionized in the oxygenated form. In any case, this would require -SH groups which change their dissociation constant by four orders of magnitude on oxygenation. As a matter of fact, it has been reported previously (1,12) that the protons of the reactive -SH groups of hemoglobin can be quantitatively displaced by mercaptide formation or alkylation without any change in the Bohr effect. More detailed experiments of this nature will be reported below. The imidazole hypothesis remains the most likely explanation for the Bohr effect, since both the heat of dissociation and the pK of histidine residues, of which hemoglobin contains over 30, fall within the correct range. A reaction of the oxygen-linked acid groups of hemoglobin which is in excellent agreement with the imidazole hypothesis will be reported in this paper, and a molecular mechanism for the Bohr effect will be proposed.