Studies on Human Hemoglobin Treated with Various Sulfhydryl Reagents

Abstract

Abstract The reactive sulfhydryl groups of human oxyhemoglobin (at position 93 of the β chains) form compounds with cystine, cystamine, dimercaptoethanol, and iodoacetamide. No reaction occurs with dithioglycolic acid, dithiobutyric acid, or diformylcystine. Some reaction occurs with iodoacetic acid but only under special conditions. The general properties (stability, spectra, molecular weight) of the modified hemoglobins described in this paper (except perhaps for the hemoglobin treated with iodoacetic acid) are very similar to those of unreacted hemoglobin. The oxygen equilibrium of these compounds has been studied. In all cases, the value of n is similar to that of normal hemoglobin (namely about 2.9) and is approximately invariant with pH. The oxygen Bohr effect is substantially reduced, and the pattern of all the results is similar in spite of the different chemical structures of the blocking reagents. The hemoglobin treated with cystine shows differential binding of the HPO4=ion as between the oxy and deoxy forms at concentrations of HPO4= in the neighborhood of 0.3 m. This explains the discrepancy between the directly measured Bohr effect for this compound and for hemoglobin treated with cystamine and that calculated from differential titrations. On the other hand, in the case of hemoglobin treated with iodoacetamide, where the newly introduced group carries no charge, there is good agreement between the Bohr effect measured by the two methods. The kinetics of combination of deoxyhemoglobins with carbon monoxide and of the dissociation of O2 from oxyhemoglobins has been studied. The kinetic behavior of all the compounds described in the present work is qualitatively similar to that of normal hemoglobin, but different from that of hemoglobin treated with p-mercuribenzoate.