Quantitative evaluation for the role of beta 146 His and beta 143 His residues in the Bohr effect of human hemoglobin in the presence of 0.1 M chloride ion

Abstract

Two different methods were used to determine the number of Bohr protons released upon oxygenation of human hemoglobin (Hb A) and Hb A lacking beta 146 His (des-His Hb A) at the pH ranging from pH 5.0 to 9.0 in the presence of 0.1 M Cl- at 25 degrees C. One is the direct differential titration method, the other is based on the measurement of oxygen affinity as a function of pH. The results obtained for Hb A or des-His Hb A with two methods were completely mutually consistent. The number of Bohr protons released from des-His Hb A upon oxygenation at pH 7.5 was about 44% less than that from Hb A, while at pH 5.5 the number of Bohr protons taken up by des-His Hb A was 20% greater than that by Hb A. The differences in the number of Bohr protons between Hb A and des-His Hb A could not be simply ascribed to the lack of beta 146 His from Hb A. The pK alpha values, which were determined by the deuterium exchange method using 1H NMR, were 8.0 for beta 146 His of deoxy-Hb A and 6.5 for that of CO Hb A, while those of beta 143 His were 5.2 for deoxy-Hb A and 6.0 for CO Hb A. From these pK alpha values, in addition to those of alpha 1 Val proposed for the modified CO and deoxy-Hb A with carbamylated beta chains by Van Beek and De Bruin (Van Beek, G. M., and De Bruin, S. H. (1980) Eur. J. Biochem. 105, 353-360), it became evident that almost all (about 92%) of the alkaline Bohr protons released upon oxygenation of Hb A in the presence of 0.1 M Cl- could be accounted for by the protons from these 2 residues, although the involvement of other histidine residues could not be denied. About half the acid Bohr protons from Hb A, which corresponds to the higher pH part (above pH 5.0) of the acid Bohr effect, could be explained by the involvement of beta 143 His residue. The residual acid Bohr effect in the more acidic pH region was presumably contributed by an amino acid residue with pK alpha values of 4.05 and 5.95 for the deoxy- and CO Hb A, respectively, although the amino acid residue was unspecified.(ABSTRACT TRUNCATED AT 400 WORDS)