Role of alpha and beta carboxyl-terminal residues in the kinetics of human oxyhemoglobin dimer assembly.

Human oxyhemoglobin assembly was evaluated in the Soret region by rapidly mixing normal and carboxypeptidase-digested chains (1-10 x 10(-6) M, heme basis) in 0.1 M Tris-HCl, 0.1 M NaCl, 1 mM EDTA, pH 7.4, at 21.5 degrees C. Rate constants of 1.14 (+/- 0.09) and 2.11 (+/- 0.06) x 10(5) M-1 S-1 were measured for the association of Des(Arg-141) alpha with beta A and alpha A with beta A chains, respectively. The slower combination rate of Des(Arg-141) alpha with beta A chains is in agreement with that predicted solely on the basis of electrostatic considerations, as are the measured rate constants of 0.75 (+/- 0.12) and 1.86 (+/- 0.08) x 10(5) M-1 S-1 obtained for the combination of Des(Arg-141) alpha with variant beta S (Glu-6-->Val) and beta N Baltimore (Lys-95-->Glu) chains, respectively. However, the combination rates of alpha A and Des(Arg-141) alpha with beta A chains measured in the pH range from 7.0 to 9.0 demonstrated that the altered overall surface charge was not the only determinant of the assembly rates observed for Des(Arg-141) alpha chains. Furthermore, a rate constant of 11.3 (+/- 0.05) x 10(5) M-1 S-1 (which is 5.4-fold faster than the rate of alpha A beta A dimer assembly) was observed for Des(His-146,Tyr-145) beta chains. These kinetic studies suggest a critical role for the carboxyl-terminal domain in the assembly of human hemoglobin in vitro and perhaps in vivo.