Electrophoretic Methods for Study of Hemoglobins

  • Henri Wajcman
Part of the Methods in Molecular Biology™ book series (MIMM, volume 82)

Abstract

Electrophoresis, a technique consisting of the migration of electrically charged molecules under an applied electric field, occupies one of the most important places in the history of the study of hemoglobin (Hb). HbS, the first abnormal Hb described, was discovered in 1949 by Pauling et al. (1), using moving boundary electrophoresis. Later, Hb variants were detected by zone electrophoresis on paper, starch gel, or cellulose acetate (2,3). With the exception of cellulose acetate electrophoresis, which is still used in some laboratories, these procedures have been replaced by isoelectric focusing (IEF) (4). In IEF, a pH gradient is established by carrier ampholytes subjected to an electric current. The Hb molecule migrates across this gradient until it reaches the position where its net charge is zero (isoelectric point [pI]). It then concentrates into a sharp band.

Keywords

Cellulose Acetate Amino Acid Exchange Electrophoretic Method Globin Chain Carrier Ampholyte 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Pauling, L., Itano, H. A., Singer, S. J., and Wells, I. C. (1949) Sickle cell anemia, a molecular disease. Science 110, 543.PubMedCrossRefGoogle Scholar
  2. 2.
    Huisman, T. H. J. and Jonxis, J. H. P. (1977) The Hemoglobinopathies Techniques of Identification, Clinical and Biochemical Analysis, vol. 6, Marcel Dekker, New York.Google Scholar
  3. 3.
    Huisman, T. H. J. (1986) Introduction and review of standard methodology for the detection of hemoglobin abnormalities, in The Hemoglobinopathies, Methods in Hematology, vol. 15 (Huisman, T. H. J., ed.), Churchill Livingstone, Edinburgh.Google Scholar
  4. 4.
    Righetti, P. G., Gianaza, E., Bianchi-Bosisio, A., and Cossu, G. (1986) Conventional isoelectric focusing and immobilized pH gradients for hemoglobin separation and identification, in The Hemoglobinopathies, Methods in Hematology, vol. 15 (Huisman, T. H. J., ed.), Churchill Livingstone, Edinburgh, p. 47.Google Scholar
  5. 5.
    Barwick, R. C. and Schneider, R. G. (1980–1981) The computer-assisted differentiation of hemoglobin variants, in Human Hemoglobins and Hemoglobinopathies: A Review to 1981. Texas Reports on Biology and Medicine, vol 40. (Schneider, R. G., Charache, S., and Schroeder, W. A., eds.), University of Texas Medical Branch, Galveston, TX, pp. 143–156.Google Scholar
  6. 6.
    Basset, P., Beuzard, Y., Garrel, M. C., and Rosa, J. (1978) Isoelectric focusing of human hemoglobin: its application to screening, to the characterization of 70 variants, and to the study of modified fractions of normal hemoglobins. Blood 51, 971–982.PubMedGoogle Scholar
  7. 7.
    Righetti, P. G. (1983) Isoelectric Focusing: Theory, Methodology and Applications, Elsevier, Amsterdam.Google Scholar
  8. 8.
    Bjellqvist, B., Ek, K., Righetti, P. G., Gianazza, E., Gorg, A., Westermeier, R., and Postel, W. (1982) Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications. J. Biochem. Biophys. Methods 6, 317–339.PubMedCrossRefGoogle Scholar
  9. 9.
    Righetti, P. G., Gianazza, E., Bianchi-Bosisio, A., Wajcman, H., and Cossu, G. (1989) Electrophoretically silent hemoglobin mutants as revealed by isoelectric focusing in immobilized pH gradients. Electrophoresis 10, 595–599PubMedCrossRefGoogle Scholar
  10. 10.
    Cossu, G., Manca, M., Pirastru, M. G., Bullitta, R., Bianchi-Bosisio, A., Gianazza, E., and Righetti, P. G. (1982) Neonatal screening of β-thalassemias by thin layer isoelectric focusing. Am. J. Hematol. 13, 149–157.PubMedCrossRefGoogle Scholar
  11. 11.
    Drysdale, J. W., Righetti, P., and Bunn, H. F. (1971) The separation of human and animal hemoglobins by isoelectric focusing in polyacrylamide gel. Biochim. Biophys. Acta. 229, 42–50.PubMedGoogle Scholar
  12. 12.
    Schneider, R. G. and Barwick, R. C. (1982) Hemoglobin mobility in citrate agar electrophoresis: its relationship to anion binding. Hemoglobin 6, 199–208.PubMedCrossRefGoogle Scholar
  13. 13.
    Alter, B. P., Goff, S. C., Efremov, G. D., Gravely, M. E., and Huisman, T. H. J. (1980) Globin chain electrophoresing: a new approach to the determination of the G/A ratio in fetal haemoglobin and to the studies of globin synthesis Br. J. Haematol. 44, 527–534.PubMedCrossRefGoogle Scholar
  14. 14.
    Radola, B. J. (1973) Analytical and preparative isoelectric focusing in gel-stabilized layers. N Y Acad. Sci. 209, 127–143.CrossRefGoogle Scholar
  15. 15.
    Westermeier, R. (1997) Electrophoresis in Practice, 2nd ed., VCH Verlagsgesellschaft, Weinheim, Germany.Google Scholar
  16. 16.
    Harano, T., Ueda, S., Harano, K., and Shibata, S. (1980) Improved method for quantitation of biosynthesized human globin chains in reticulocytes by use of urea cellulose acetate membrane electrophoresis. Proc. Jap. Acad. 56(B), 230–234.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2003

Authors and Affiliations

  • Henri Wajcman
    • 1
  1. 1.INSERM U468Hôpital Henri MondorCreteilFrance

Personalised recommendations