ALLOSTERIC KINETICS AND EQUILIBRIA OF TRILIGATED, CROSS-LINKED HEMOGLOBIN

Using modulated excitation, we have measured the forward and reverse r ates of the allosteric transition between relaxed (R) and tense (T) qu aternary structures for triply ligated hemoglobin (Hb), cross-linked b etween the a chains at Lys 99. Oxygen, carbon monoxide, and water were used as ligands and were studied in phosphate and low Cl- bis-Tris bu ffers at neutral pH. Since the cross-link prohibits disproportionation , triply ligated aquomet Hb species with ferrous beta chains were spec ifically isolated by isoelectric focusing. Modulated excitation provid es rate pairs and therefore gives equilibrium constants between quater nary structures. To coordinate with that information, oxygen binding c urves of fully ferrous and tri-aquomet Hb were also measured. L3, the equilibrium constant between three liganded R and T structures, is det ermined by modulated excitation to be of order unity for 02 or CO (1. 1 to 1.5 for 3O2 and 0.7 for 3CO bound), while with three aquomet subu nits it is much greater (greater-than-or-equal-to 23). R --> T convers ion rates are similar to those found for HbA, with weak sensitivity to changes in L3. The L3 values from Hb(XL)O2 were used to obtain a uniq ue allosteric decomposition of the ferrous O2 binding curve in terms o f K(T), K(R), and L3. From these values and the O2 binding curve of tr i-aquomet Hb(XL), L3 was calculated to be 2.7 for the tri-aquomet deri vative. Consistency in L3 Values between equilibrium and modulated exc itation data for tri-aquomet-Hb(XL) can be achieved if the equilibrium constant for O2 binding to the alpha chains is six times lower than t hat for binding to the beta chains in the R state, while the cooperati ve properties remain homogeneous. The results are in quantitative agre ement with other studies, and suggest that the principal effect of the cross-link is to decrease the R state and T state affinity of the a s ubunits with almost no change in affinity of the beta subunits, leavin g the allosteric parameters L and c unchanged.