Abstract
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.
Highlights
In the present work, we have undertaken the study of the subunit interactions
Heterogeneity in hemoglobin preparations has been reported from observations of the intrinsic tryptophan fluorescence in which the long-lived tryptophan decay is essentially eliminated by anion exchange (Bucci et al, 1988a,b;Szabo et a!., 1989)
It is interesting to note that for each HB preparation the apparent calculated labeling ratio was largest for the solutions dialyzed in the presence of inositol hexaphosphate (IHP) (1.98 f 0.40) and smallest for those dialyzed at pH 7 (1.56 f 0.43)
Summary
Thereon) in hemoglobin using fluorescence spectroscopy and high hydrostatic pressure. These methods permit the direct observation of variation in the size of the macromolecular complex upon dissociation through the measurement of the depolarization of fluorescencedue to brownian tumbling. The fluorescence lifetime of the covalent probe 5-(dimethylamino)naphthalene- 1-sulfonyl chloride (DNS-Cl) is sufficiently long for the observation of the global tumbling of proteins with the molecular weight of hemoglobin. The combination of fluorescence techniques with high hydrostatic pressure has made possible the direct observation of the dissociation of the dimers into monomers. The use of hydrostatic pressure to study highaffinity equilibria in oligomeric proteins is based on Le Chatelier's principle of equilibrium perturbation (Paladini & Weber, 1981a; Heremans, 1982; Weber & Drickamer, 1983, Weber, 1987). One consisted of hemoglobin purified by standard procedures, HB, and the second from a further fractionation using anion-exchange chromatography, MC
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