Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 1994 Nov;67(5):2096–2109. doi: 10.1016/S0006-3495(94)80693-9

Hemoglobin affinity for 2,3-bisphosphoglycerate in solutions and intact erythrocytes: studies using pulsed-field gradient nuclear magnetic resonance and Monte Carlo simulations.

A J Lennon 1, N R Scott 1, B E Chapman 1, P W Kuchel 1
PMCID: PMC1225585  PMID: 7858147

Abstract

The diffusion coefficient (D) of 2,3-bisphosphoglycerate (DPG) was measured using pulsed-field gradient (PFG)-31P nuclear magnetic resonance spectroscopy in solutions containing 2.7-5.0 mM hemoglobin (Hb) and a range of DPG concentrations. The dependence of the measured values of D on the fraction of the total DPG in the sample that is bound to Hb enabled the estimation of the dissociation constants (Kd) of complexes of DPG with carbonmonoxygenated, oxygenated, and deoxygenated Hb; the values of Kd (mM), measured at 25 degrees C, pH 6.9 and in 100 mM bis Tris/50 mM KCl, were 1.98 +/- 0.26, 1.8 +/- 0.5 and 0.39 +/- 0.26, respectively. In intact erythrocytes the apparent diffusion coefficient, Dapp, of DPG was larger in oxygenated and carbonmonoxygenated cells (6.17 +/- 0.20 x 10(-11) m2s-1) than in deoxygenated cells (4.10 +/- 0.23 x 10(-11) m2s-1). Changes in intracellular DPG concentration (5-55 mM) in erythrocytes, brought about by incubation in a medium containing inosine and pyruvate, did not result in significant changes in the value of Dapp; this result supports the hypothesis that DPG binds to other sites in the erythrocyte. Monte Carlo simulations of diffusion in biconcave discs were used to test the adequacy of the values of Kd estimated in solution to describe the binding of DPG to Hb in oxygenated and deoxygenated erythrocytes. The results of the simulations implied that the value of Kd estimated for deoxygenated Hb-DPG was greater than expected from the experiments involving intact erythrocytes. This difference is surmised to be at least partly due to the difficulty of measuring D at low-ligand concentrations. Notwithstanding this shortcoming, the PFG method appears to be suitable for probing interactions between macromolecules and ligands when the Kd is in the millimolar range. It is one of the few techniques available in which these interactions can be studied in intact cells. In addition, the Monte Carlo simulations of the diffusion experiments highlighted important differences between theory and experiment relating to the nature of molecular motion inside the cells.

Full text

PDF

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arnone A. X-ray diffraction study of binding of 2,3-diphosphoglycerate to human deoxyhaemoglobin. Nature. 1972 May 19;237(5351):146–149. doi: 10.1038/237146a0. [DOI] [PubMed] [Google Scholar]
  2. Beck J. S. Relations between membrane monolayers in some red cell shape transformations. J Theor Biol. 1978 Dec 21;75(4):487–501. doi: 10.1016/0022-5193(78)90358-2. [DOI] [PubMed] [Google Scholar]
  3. Benesch R., Benesch R. E. Intracellular organic phosphates as regulators of oxygen release by haemoglobin. Nature. 1969 Feb 15;221(5181):618–622. doi: 10.1038/221618a0. [DOI] [PubMed] [Google Scholar]
  4. Berger H., Jänig G. R., Gerber G., Ruckpaul K., Rapoport S. M. Interaction of haemoglobin with ions. Interactions among magnesium, adenosine 5'-triphosphate, 2,3-bisphosphoglycerate, and oxygenated and deoxygenated human haemoglobin under simulated intracellular conditions. Eur J Biochem. 1973 Oct 18;38(3):553–562. doi: 10.1111/j.1432-1033.1973.tb03090.x. [DOI] [PubMed] [Google Scholar]
  5. Beutler E., Mathai C. K. A comparison of normal red cell ATP levels as measured by the firefly system and the hexokinase system. Blood. 1967 Sep;30(3):311–320. [PubMed] [Google Scholar]
  6. Bock J. L., Wenz B., Gupta R. K. Changes in intracellular Mg adenosine triphosphate and ionized Mg2+ during blood storage: detection by 31P nuclear magnetic resonance spectroscopy. Blood. 1985 Jun;65(6):1526–1530. [PubMed] [Google Scholar]
  7. Chasis J. A., Mohandas N. Erythrocyte membrane deformability and stability: two distinct membrane properties that are independently regulated by skeletal protein associations. J Cell Biol. 1986 Aug;103(2):343–350. doi: 10.1083/jcb.103.2.343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cohen C. M., Foley S. F. Biochemical characterization of complex formation by human erythrocyte spectrin, protein 4.1, and actin. Biochemistry. 1984 Dec 4;23(25):6091–6098. doi: 10.1021/bi00320a029. [DOI] [PubMed] [Google Scholar]
  9. Fabry M. E., San George R. C. Effect of magnetic susceptibility on nuclear magnetic resonance signals arising from red cells: a warning. Biochemistry. 1983 Aug 16;22(17):4119–4125. doi: 10.1021/bi00286a020. [DOI] [PubMed] [Google Scholar]
  10. Fossel E. T., Solomon A. K. Modulation of 2,3-diphosphoglycerate 31P-NMR resonance positions by red cell membrane shape. Biochim Biophys Acta. 1976 Jun 17;436(2):505–511. doi: 10.1016/0005-2736(76)90212-1. [DOI] [PubMed] [Google Scholar]
  11. Garby L., De Verdier C. H. Affinity of human hemoglobin A to 2,3--diphosphoglycerate. Effect of hemoglobin concentration and of pH. Scand J Clin Lab Invest. 1971 Jun;27(4):345–350. doi: 10.3109/00365517109080229. [DOI] [PubMed] [Google Scholar]
  12. Gerber G., Berger H., Jänig G. R., Rapoport S. M. Interaction of haemoglobin with ions. Quantitative description of the state of magnesium, adenosine 5'-triphosphate, 2,3-bisphosphoglycerate, and human haemoglobin under simulated intracellular conditions. Eur J Biochem. 1973 Oct 18;38(3):563–571. doi: 10.1111/j.1432-1033.1973.tb03091.x. [DOI] [PubMed] [Google Scholar]
  13. Gupta R. K., Benovic J. L., Rose Z. B. Location of the allosteric site for 2,3-bisphosphoglycerate on human oxy- and deoxyhemoglobin as observed by magnetic resonance spectroscopy. J Biol Chem. 1979 Sep 10;254(17):8250–8255. [PubMed] [Google Scholar]
  14. HERRING W. B., LEAVELL B. S., PAIXO L. M., YOE J. H. Trace metals in human plasma and red blood cells. A study of magnesium, chromium, nickel, copper and zinc. I. Obser- vations of normal subjects. Am J Clin Nutr. 1960 Nov-Dec;8:846–854. doi: 10.1093/ajcn/6.2.846. [DOI] [PubMed] [Google Scholar]
  15. Hamasaki N., Rose Z. B. The binding of phosphorylated red cell metabolites to human hemoglobin A. J Biol Chem. 1974 Dec 25;249(24):7896–7901. [PubMed] [Google Scholar]
  16. Haner R. L., Schleich T. Measurement of translational motion by pulse-gradient spin-echo nuclear magnetic resonance. Methods Enzymol. 1989;176:418–446. doi: 10.1016/0076-6879(89)76023-7. [DOI] [PubMed] [Google Scholar]
  17. Higashi T., Yamagishi A., Takeuchi T., Kawaguchi N., Sagawa S., Onishi S., Date M. Orientation of erythrocytes in a strong static magnetic field. Blood. 1993 Aug 15;82(4):1328–1334. [PubMed] [Google Scholar]
  18. Labotka R. J., Schwab C. M. A dialysis cell for nuclear magnetic resonance spectroscopic measurement of protein-small molecule binding. Anal Biochem. 1990 Dec;191(2):376–383. doi: 10.1016/0003-2697(90)90235-2. [DOI] [PubMed] [Google Scholar]
  19. Marshall W. E., Costello A. J., Henderson T. O., Omachi A. Organic phosphate binding to hemoglobin in intact human erythrocytes determined by 31P nuclear magnetic resonance spectroscopy. Biochim Biophys Acta. 1977 Feb 22;490(2):290–300. doi: 10.1016/0005-2795(77)90004-6. [DOI] [PubMed] [Google Scholar]
  20. Moriyama R., Lombardo C. R., Workman R. F., Low P. S. Regulation of linkages between the erythrocyte membrane and its skeleton by 2,3-diphosphoglycerate. J Biol Chem. 1993 May 25;268(15):10990–10996. [PubMed] [Google Scholar]
  21. Petersen A., Kristensen S. R., Jacobsen J. P., Hørder M. 31P-NMR measurements of ATP, ADP, 2,3-diphosphoglycerate and Mg2+ in human erythrocytes. Biochim Biophys Acta. 1990 Aug 17;1035(2):169–174. doi: 10.1016/0304-4165(90)90112-a. [DOI] [PubMed] [Google Scholar]
  22. Russu I. M., Wu S. S., Bupp K. A., Ho N. T., Ho C. 1H and 31P nuclear magnetic resonance investigation of the interaction between 2,3-diphosphoglycerate and human normal adult hemoglobin. Biochemistry. 1990 Apr 17;29(15):3785–3792. doi: 10.1021/bi00467a027. [DOI] [PubMed] [Google Scholar]
  23. Schindler M., Koppel D. E., Sheetz M. P. Modulation of membrane protein lateral mobility by polyphosphates and polyamines. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1457–1461. doi: 10.1073/pnas.77.3.1457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sheetz M. P., Casaly J. 2,3-Diphosphoglycerate and ATP dissociate erythrocyte membrane skeletons. J Biol Chem. 1980 Oct 25;255(20):9955–9960. [PubMed] [Google Scholar]
  25. Simpson L. O. The effects of saline solutions on red cell shape: a scanning-electron- microscope-based study. Br J Haematol. 1993 Dec;85(4):832–834. doi: 10.1111/j.1365-2141.1993.tb03237.x. [DOI] [PubMed] [Google Scholar]
  26. Stewart I. M., Chapman B. E., Kirk K., Kuchel P. W., Lovric V. A., Raftos J. E. Intracellular pH in stored erythrocytes. Refinement and further characterisation of the 31P-NMR methylphosphonate procedure. Biochim Biophys Acta. 1986 Jan 23;885(1):23–33. doi: 10.1016/0167-4889(86)90034-0. [DOI] [PubMed] [Google Scholar]
  27. Van Kampen E. J., Zijlstra W. G. Determination of hemoglobin and its derivatives. Adv Clin Chem. 1965;8:141–187. doi: 10.1016/s0065-2423(08)60414-x. [DOI] [PubMed] [Google Scholar]
  28. van KAMPEN E., ZIJLSTRA W. G. Standardization of hemoglobinometry. II. The hemiglobincyanide method. Clin Chim Acta. 1961 Jul;6:538–544. doi: 10.1016/0009-8981(61)90145-0. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES