The reaction of normal adult human haemoglobin with heavy-metal reagents

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Abstract
Adult human hemoglobin has 2.2 reactive SH groups and 3.8 unreactive SH groups. The reactive SH groups can be titrated with heavy-metal reagents in the same way as simple thiols. The unreactive SH groups can only be titrated in this way after the protein has been denatured at pH reduced hemoglobin>carboxyhemoglobin. Phenylmercuric hydroxide and mercuric chloride react with all the unreactive SH groups but p-chloromercuribenzenesulphonate reacts with only 2 groups. The mercaptides formed by the reactive SH groups with phenylmercuric hydroxide or mercuric chloride show little tendency to bind additional reagent, but those formed by the unreactive SH groups each bind up to one molecule of reagent. This additional reagent can be removed by dialysis against thiosulphate. Carboxyhemoglobin with all 6 SH groups combined with phenylmercuric hydroxide or mercuric chloride can be prepared by this method. The tendency of the unreactive SH group to bind more than one molecule of heavy-metal reagent made it impracticable to apply a kinetic treatment to the reaction. Attempts to overcome this difficulty by carrying out the reactions in the presence of sulphite or thiosulphate were unsuccessful. Phenylmercuric hydroxide forms diphenylmercury in the presence of these reagents. Mercuric chloride reacts with only two unreactive SH groups in the presence of sulphite and with none in the presence of thiosulphate.