An investigation of human oxyhemoglobin beta tetramer dissociation using haptoglobin binding

Haptoglobin was used as a macromolecular probe to investigate the formation of human oxyhemoglobin beta chain dimers from tetramers in 0.1 M potassium phosphate buffer, 20 degrees C at pH 7 and pH 8. Monitoring of spectral changes upon mixing haptoglobin with beta heme chains (2.5 and 5 micromolar) revealed an overall decrease in absorbance accompanied by a shift of the Soret spectral peak from 415 to 417 nm. The magnitude of the absorbance decrease was proportional to the beta concentration; the time courses consistently yielded greater color at pH 8 than at pH 7. At pH 8, two exponential phases of 0.47 min-1 and 0.084 min-1 were seen whose rates remained invariant with concentration. In contrast, only one exponential process was evident at pH 7, yielding a first order rate constant of 0.21 min-1. We have spectrophotometrically followed the beta chain tetramer to dimer dissociation reaction, thus providing information about the contribution of this step to hemoglobin assembly.