ABSTRACT
The affinity constants for the binding of NADPH to human hemoglobin A were directly determined by fluorescence analyssis since nucleotide fluorescence is quenched on binding to the protein. The binding constants 6.1 x 10**5, 5.02 x 10**5 and 1.2 x 10**5 were found for deosyhemoglobin at pH 6.5, 7.0,respectively. Oxyhemoglobin does not bind NADPH significantly. These results are consistent with those found in oxygen-hemoglobin equilibrium experiments. The human hemoglobin variant, Providence-Asp, which has a marked decrease in 2,3 DPG affinity was also investigated. NADPH does not bind to the variant suggesting that the Lys B 82 residues is of fundamental importance to nucleotide binding and showing that the binding site is the same as that of 2,3 DPG or other organic polyphosphate, aloosteric modulators of hemoglobins. Experiments of inositol hexaphosphate (IHP)-NADPH site competition corroborate these results
Subject(s)
Humans , Binding Sites , Hemoglobin A/metabolism , Hemoglobin J/metabolism , Hemoglobins, Abnormal/metabolism , NADP/metabolism , Spectrometry, FluorescenceABSTRACT
Liophis miliaris hemoglobins in the stripped form exhibit a high oxigen affinity, a small alkaline Bohr effect, a pronounce organic polyphosphate effect and a pH-dependent Hill coefficient, close to 2 below pH 7.5 and near 1 at higher pHs. Molecular weight determinations indicate 2 forms, a dimeric form of MW 32000 d. and a tetrameric form of about 64000 d. The deoxyhemoglobin is tetrameric. Ion-exchange chromatography shows two components which may bind to each other being eluted as a single component of MW 32000 d. The osygen alkaline Bohr effect observed for the stripped hemoglobin may be explained by the transition from the tetramer to the dimer during oxygen addition experiments. The phenomenon appears to be unique among animals. ß-Chain sequencing determinations show that abnormal human hemoglobin with similar properties has a glutamic acid residue substituted at the alfa1-ß2 interface by valine in snake hemoglobin