FLUORESCENCE STUDIES OF HUMAN SEMI-BETA-HEMOGLOBIN ASSEMBLY

The intrinsic fluorescence properties of human alpha apohemoglobin at protein concentrations from 1 to 5 mu M in 0.1 M potassium phosphate b uffer, pH 7 or 8 at 5 degrees C were monitored in the absence and pres ence of a fixed concentration (5 mu M) of a fluorescence quenching hem e-containing native or Des (146-His, 145-Tyr) beta chain partner. Thes e ''reverse quenching'' studies revealed that the emission intensity c hanges observed correlated well with protein concentration and theoret ical extent of semi-beta-hemoglobin assembly. Furthermore, the relativ e quenching efficiencies were calculated to be 0.32, 0.25 and 0.61 for beta (pH 7), beta (pH 8) and Des beta (pH 7) chains, respectively. Th us, heme-mediated quenching was sensitive to the expected pH induced a lpha apohemoglobin conformational change and to alteration in beta cha in structure. Intramolecular changes induced by carboxylterminal modif ication (decreased ''beta chain self-quenching'') appeared to enhance the intermolecular rearrangements (increased ''alpha chain partner que nching'') seen upon subunit assembly. (C) 1998 Academic Press.