Kinetic studies on the reconstitution of deoxyhemoglobin from isolated alpha and beta chains

The reconstitution reaction of deoxy hemoglobin (Hb) tetramer from isolated alpha and beta chains was kinetically studied by measuring circular dichroism (CD) changes in the Soret and the ultraviolet regions and optical absorbance change in the Soret region with a stopped-flow apparatus. The CD change in the Soret region was a fast reaction, followed by that in the ultraviolet region and the absorbance change in the Soret region. This fast reaction followed a simple second-order rate law with a rate constant of 6.4 x 10(5) M-1 . s-1. These results indicated that the combination of alpha and beta monomers into an alpha beta dimer accompanied the CD change in the Soret region and was the rate-limiting step of the overall reconstitution reaction of deoxyHb tetramer. On the other hand, the CD change in the ultraviolet region was ascribed to the combination of two alpha beta dimers into an Hb tetramer. The absorbance change in the Soret region was related to both the combination of alpha and beta monomers and that of two alpha beta dimers. From the analyses of these reactions the rate constant of the combination of two alpha beta dimers was determined to be 1.0 x 10(6) M-1 . s-1.