Effect of Carbamination on the Buffering Power of Purified Human Hemoglobin A Solutions at Two Temperatures

Abstract

The effect of COZ on hemoglobin buffering power was studied in purified human hemoglobin A solutions, native and specifically carbamoylated on N-terminal valines, at 26°C and 4 2 T , in the oxy and deoxy states. Titrations were performed by varying pco2 and by strong acid or base in the absence of C 0 2 . The participation of N-terminal valines to the total buffering power was estimated by subtracting the buffering value measured on carbainoylated hemoglobin solutions from that measured on native hemoglobin solutions.In the absence of COz the buffering value of native and modified hemoglobin increased slightly (Icss than lo:<) (a) on going from the deoxy to the oxy state, and (b) on raising the temperature from 26°C to 42°C.In the presence of COZ the buffering value of Hb increased from 9.1 to 16.6 mol mol HbT1 pH-' and that of HbOz from 10.1 to 19.6 mol mol H b i l pH-' when the temperature was raised from 26 ' C to 42 C. These figures correspond to a rise in the fraction of the total buffering value attributable to N-terminal valines from 1 1 7; to 25The present results point to a non-specific effect of COZ within the hemoglobin molecule independent of that of N-terminal valines. This effect nearly doubles the buffering value for CO2 when the temperature is raised, and contributes to pH regulation and COz removal in tissues with a high metabolic rate.for H b and from 3 'x to 33 yd for HbOz. Hemoglobin buffering power for non-volatile acids (Bny)is expressed differently from that for volatile acids (/jy), in this case C02. b,,, is defined as the slope of the titration curve of hemoglobin by strong base at a given pH, whereas /Iv is equal the negative slope of tlie hydrogen carbonate ion concentration versus pH curve of hemoglobin at a given pH. The buffering power of hernoglobin depends upon tlie presence of amino acids which ionize in the physiological pH range. These groups arc mainly tlie imidazole groups of the 22 titratable histidines of the polypeptide chains [I] and the a-amino groups of thc four N-terminal valines of the x and /i chains [2]. l n addition, oxygen (Or) and carbon dioxide ( C 0 2 ) binding to hemoglobin affccls the buffering value of the molecule. Indeed, in the physiological pH range, some specific groups (alkaline Bohr groups) undergo a lowering of their pk' upon hemoglobin oxygenation, which not only contributes to the alkaline Bohr effect but will also increase their buffcring power. These groups arc the two Val-lx, the two His-146P [ 3 ] and, to a still unsettlcd extent, other groups such as . The addition of C 0 2 to the solutions also influences hemoglobin buffcring power. In its presence, thc negative charge of the protein is increased and the pH is decreased owing to the binding of molecular CO2 to the x-amino groups of both valine-la and valinc-1B. This results in the formation of ionized carbamino compounds according to the following reactions [ S ] :HbNH:Ahhreviufions. Hb, deoxygenated hemoglobin A ; H b 0 2 , oxygenated hemoglobin A ; Hbc, dcoxygenated hemoglobin A reacted wit...