Fluorescence studies of normal and sickle beta apohemoglobin self-association.

The acrylamide quenching of the intrinsic tryptophanyl fluorescence of normal and sickle beta apohemoglobins has been studied in 0.05 M potassium phosphate buffer, pH 7.5, at 5 degrees C over a protein concentration range from 1 to 50 microM. Analysis of quenching dynamics revealed a strong dependence on acrylamide concentration for the intrinsic fluorescence of both normal and sickle beta apohemoglobins, suggesting that one tryptophanyl residue [presumably that at position 37(C3)], was more accessible to collisional quencher than the other beta tryptophanyl residue [15(A12)]. Additional studies, which altered viscosity and subunit assembly experimental parameters, supported the assignment of residue 37 as the more dynamically accessible residue. Finally, the quenching data were also found to be dependent on protein concentration, implying that this difference in the mobility between the two residues is a sensitive probe of self-aggregation. Extrapolated dynamic quenching constants at low concentration of acrylamide were used to estimate the dimer-monomer equilibrium dissociation constants of normal and sickle beta apohemoglobins, and were found to be 5.6 and 2.4 microM, respectively, thus demonstrating distinct self-association properties of beta A and beta S apohemoglobins.