Rate of allosteric change in hemoglobin measured by modulated excitation using fluorescence detection

We have measured the forward and reverse rates of the allosteric transition of hemoglobin A with three CO molecules bound by using modulated excitation coupled with fluorescence quenching of the DPG analogue, PTS (8-hydroxy-1,3,6 pyrene trisulfonic acid). This dye is observed to bind to the T state with significantly larger affinity than to the R state, and thus provides an unequivocal marker for the molecule's conformational change. The allosteric rates obtained with the fluorescent dye (pH 7.0, bis-Tris buffer) are (3.4 +/- 1.0) x 10(3)s-1 for the R to T transition and (2.1 +/- 0.5) x 10(4)s-1 for the T to R transition. This gives an equilibrium constant L3 of 0.16 +/- 0.06. These results provide good agreement with modulated difference spectra calibrated from model compounds, arguing that there is little if any difference in the kinetics observed by the heme spectra and the kinetics of the full subunit motion. The equilibrium constant between structures (L3) is smaller in the absence of phosphates than observed in phosphate buffer (0.33). However, the rates of the allosteric transition increase in the absence of phosphates as compared with the corresponding rates in phosphate buffer of 1.0 x 10(3)s-1 and 3.0 x 10(3)s-1. The effects of inorganic phosphates on the equilibrium can be separated from the effects on kinetics. We find that phosphates also affect the dynamic behavior of hemoglobin, and the presence of 0.15 M phosphate can be viewed as raising the transition state energy between R and T conformations by approximately 0.5 kcal/mol exclusive of the T state stabilization. Dissociation constants for the dye were measured to be 104 +/- 25 microM for unligated T state and 930 +/- 300 microM for the fully ligated R state. The best fit equilibrium constant (125 +/- 40 microM) for three ligands bound does not differ significantly from that measured without ligands bound. Incidental to the measurement technique is the determination of the rates of binding and release of the dye. The association rate for binding to the T state is large, (at least 4 x 10(9) M-1 s-1) and may be diffusion limited, while the association and dissociation rates for R state binding, while not determined with precision, are clearly much smaller, of the scale of 10(5) M-1 s-1 for association.