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 pota ssium phosphate buffer, pH7.5, at 5 degrees C over a protein concentra tion range from 1 to 50 mu M. Analysis of quenching dynamics revealed a strong dependence on acrylamide concentration for the intrinsic fluo rescence of both normal and sickle beta apohemoglobins, suggesting tha t one tryptophanyl residue [presumably that at position 37(C3)], was m ore accessible to collisional quencher than the other beta tryptophany l residue [15(A12)]. Additional studies, which altered viscosity and s ubunit assembly experimental parameters, supported the assignment of r esidue 37 as the more dynamically accessible residue. Finally, the que nching 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 quench ing constants at low concentration of acrylamide were used to estimate the dimer-monomer equilibrium dissociation constants of normal and si ckle beta apohemoglobins, and were found to be 5.6 and 2.4 mu M, respe ctively, thus demonstrating distinct self-association properties of be ta(A) and beta(S) apohemoglobins.