DOUBLE MIXING STOPPED-FLOW METHOD FOR THE STUDY OF EQUILIBRIA AND KINETICS OF DIMER TETRAMER ASSOCIATION OF HEMOGLOBINS - STUDIES ON HB-CARP, HB-A, AND HB-ROTHSCHILD

A double mixing stopped-flow method is described for studying the dimer-tetramer equilibria of oxyhemoglobins and the kinetics of association of unliganded dimers. The three hemoglobins studied were: Hb Carp, Hb A, and Hb Rothschild (Trp β37(C3) → Arg). The new method reproduces the data obtained for oxyHb A by other established methods. In agreement with previous studies, the new method indicates little, if any, dissociation of oxyHb carp into dimers even in 2 m urea solutions (0.1 m Bis-Tris pH 7.0). OxyHb Rothschild, on the other hand, is extensively dissociated into dimers (K(Hb4L4 lrhar2 2Hb2) = 37.3 × 10-6 m) and the rate constant for the association of deoxy dimers of Hb Rothschild is about one-tenth of the value for Hb A indicating that the deoxy tetramer of Hb Rothschild is at least 10 times more dissociated into dimers than deoxyHb A. © 1991.