Subunit interactions in hemoglobin probed by fluorescence and high-pressure techniques.
The dissociation of the subunits of human adult oxyhemoglobin has been investigated by using steady-state fluorescence anisotropy, multifrequency phase fluorometry, and high hydrostatic pressure. Human hemoglobin obtained by using two purification procedures (bulk preparation by centrifugation or further fractionation using anion-exchange chromatography) was labeled with an extrinsic fluorescent probe, 5-(dimethylamino)naphthalene-1-sulfonyl chloride (DNS-Cl). The long fluorescence lifetime of this probe allows for the observation of the macromolecular tumbling, and thus provides a method for observing changes in the size of the complex upon subunit dissociation under differing solution conditions of proton and organic phosphate concentration. At pH 7, the dansylated preparations of bulk and fractionated hemoglobin showed a concentration-dependent decrease in the anisotropy which though not identical can only arise from the tetramer to dimer dissociation. We observed primarily the dimer at pH 9 and a small destabilization of the tetramer in the presence of saturating inositol hexaphosphate (IHP). High-pressure experiments allowed for the observation of the dissociation of the hemoglobin dimer into monomers. From these measurements, we estimate the dimer dissociation constant to be between 0.1 and 1 nM. We compare the present results on the subunit affinities in hemoglobin obtained from steady-state and time-resolved fluorescence data with those obtained previously by using gel filtration, sedimentation, and kinetic techniques. These comparisons are indicative of a certain degree of conformational heterogeneity in the hemoglobin preparations.
Pin
S
S
Department of Physics, University of Illinois, Urbana 61801.
Royer
C A
CA
Gratton
E
E
Alpert
B
B
Weber
G
G
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003619:Dansyl Compounds; D005453:Fluorescence; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006874:Hydrostatic Pressure; D007700:Kinetics
D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D006454:Hemoglobins