Abstract
The association of the isolated cytoplasmic fragment of band 3 protein with human hemoglobin was studied by rate zonal centrifugation in sucrose density gradients, by quenching of fragment fluorescence by hemoglobin, and by flash photolysis of carbon monoxidebound hemoglobin as a function of fragment concentration. The centrifugation results showed that both proteins interact and that the interaction is abolished upon addition of glyceraldehyde-3-phosphate dehydrogenase. The fractions eluted from the density gradient were analyzed further by spectrophotometric and gel electrophoretic methods. Two types of complexes could be identified, one containing the equivalent of 1 hemoglobin tetramer/dimer of cytoplasmic fragment and another containing 2 tetramers of hemoglobin/dimer of fragment. Flash photolysis and fluorescence-quenching experiments showed that liganded hemoglobin is stabilized as the alpha beta dimer when bound to the fragment, a result almost identical with that seen for membranebound hemoglobin in previous studies. The results further suggest that there are two binding sites for the alpha beta dimer of hemoglobin on one monomer of the cytoplasmic fragment but only one mutually exclusive hemoglobin tetramer binding site, suggesting the possibility of conformational isomerism when the fragment with two dimers bound isomerises to a fragment monomer with one hemoglobin tetramer bound. Finally, despite the stabilization of the dimeric state of liganded hemoglobin when bound to the fragment, estimates of the hemoglobin dimer and tetramer binding constants suggest that the hemoglobin tetramer binds more tightly by about 2 orders of magnitude.
Highlights
Moglobin, and by flash photolysis of carbon inonoxide- The interactionsof hemoglobin with the erythrocytemembound hemoglobin as a function of fragment concentra- branehave been studied for manyyears [2]
Flash photolysis and fluorescence-quench- stilbene disulfonate to the anion transport domain of band 3 ing experiments showed that liganded hemoglobin is [7] all strongly suggest, but do noptrove, that thecytoplasmic stabilized as thea/3dimer when bound to the fragment, domain of band 3 is the high affinitybinding site for hemogloa result almost identical with that seen for membrane- bin
Salhany et al ( 7 ) did show that the isolated plasmic fragment but only one mutually exclusive he- cytoplasmic fragment of band 3 protein causes an increase in moglobin tetramer binding site, suggesting the possi- the proportionof high affinity hemoglobin dimers in solution, bility of conformational isomerizations when the frag- mimicking the effect seen earlier [8] on the membrane, ment with two dimers bound isomerizes to a fragment monomer with one hemoglobin tetramer bound
Summary
Bennett and Steinbuck [11]. The ghosts were depleted of most of from the erythrocyte membraneA. mixture of oxyhemoglobin their extrinsic proteinsand digestedwith chymotrypsin. Thehemolysate cifically to BsF [15], the hemoglobin molecule was displaced wasdialyzed against 20 mM phosphate, 130 mM NaCI, pH 7, and filtered on along Sephadex column equilibrated with the samebuffer This purification step was necessaryin order to eliminate endogenous fluorescence which wasalways present in thecrude hemolysate. Carboxy, deoxy, met) have characteristicoptical spectra with a constant absorbance ratiowhen measured inthe visible and ultraviolet region of the spectrum a t a given set of wavelengths. The scheme was integrated numerically for different BsF concentrations on a Minc 11 digital computer according to the Runge-Kutta method [14] using arbitrary on and off FRACTIO NN UMBER rate constants only compatible with the values of the equilibrium
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