Location of the allosteric site for 2,3-bisphosphoglycerate on human oxy- and deoxyhemoglobin as observed by magnetic resonance spectroscopy

Gupta, R.K.; Benovic, J.L.; Rose, Z.B.

Journal of Biological Chemistry 254(17): 8250-8255


ISSN/ISBN: 0021-9258
PMID: 224047
Accession: 068524694

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2,3-Bisphosphoglycerate, the predominant phosphorylated metabolite of the human red blood cell [RBC] binds tightly to human deoxyhemoglobin and weakly, yet significantly, to human HbO2. To locate 2,3-bisphosphoglycerate binding site(s) in both oxy-and deoxyhemoglobins in solution, paramagnetic effects of Hb spin-labeled with a nitroxide radical at each of the 2 .beta.-93 cysteine S atoms/tetramer on the 31P-nuclei of 2,3-bisphosphoglycerate were measured and the effect of CrATP, a nonlabile paramagnetic analog of MgATP, bound at the allosteric site, on the EPR spectrum of the Hb-bound nitroxide spin label was studied. An appreciable paramagnetic effect of the Hb-bound spin label on the transverse and longitudinal relaxation rates of 31P-nuclei of 2,3-bisphophoglycerate was observed with both deoxy- and oxyHb; the effect was more than an order of magnitude greater on transverse relaxation rates. These observations were consistent with 2,3-bisphosphoglycerate binding to both Hb forms. From the magnitude of the paramagnetic effects on longitudinal and transverse nuclear relaxation rates, using correlation time of .apprx. 3 .times. 10-8 s obtained from the ratio of paramagnetic effects on longitudinal and transverse relaxation rates, which was of the same order as the rotational correlation time of the Hb molecule, similar NMR average distances of .apprx. 15 .ANG. were estimated for both forms of Hb between unpaired electron on nitroxide moiety of the spin label and either of the 2 31P-nuclei of the Hb-bound 2,3 bisphosphoglycerate molecule. A .apprx. 16 .ANG. distance was obtained between the Cr3+ atom of CrATP bound at the allosteric site and the spin label in oxyHb from dipolar interaction measured by EPR. Similar spatial location of the 2,3-bisphosphoglycerate binding site in both forms of Hb in solution was suggested.