Quantitative Analysis of Ca2+-Binding by Flow Dialysis

  • Michio Yazawa
Part of the Methods in Molecular Biology™ book series (MIMB, volume 173)


Ca2+-binding to proteins can be measured directly by equilibrium dialysis (1,2), the standard method for the direct measurement of the binding of small ligand molecules by macromolecules. In this method, a semipermeable cellulose bag containing a solution of macromolecules is immersed in the buffer solution containing ligand molecules and is incubated to attain both the chemical and diffusion equilibrium. The method can be improved with the use of two small thin chambers separated by the cellulose membrane, which may reduce the incubation time required to achieve diffusion equilibrium (microdialysis) (3). Ligand molecules are usually labeled with the radioactive isotopes for quantitative determinations, and ligand molecules bound to the macromolecule in the equilibrium state are determined directly from the difference between the free concentration in the dialysate and the total concentration in the protein solution. Binding of ligand to the protein molecule can be calculated from the known value of the protein concentration, and the ligand bindings at several free concentrations of the ligand are determined from independent experiments to yield a ligand binding curve from which the maximum number of ligand binding and the equilibrium constants are estimated. In this method, the ligand binding equilibrium, which is usually obtained within less than a second, has to be assessed after attainment of the diffusion equilibrium of ligands across the membrane, which usually takes a much longer time - on the order of several hours.


Protein Solution Free Ligand Lower Chamber Dialysis Membrane Ligand Molecule 
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.


  1. 1.
    Potter, J. D. and Gergely, J. (1975) The calcium and magnesium binding sites on troponin and their role in the regulation of myofibrillar adenosine triphosphatase. J. Biol. Chem. 250, 4628–4633.PubMedGoogle Scholar
  2. 2.
    Crouch, T. H. and Klee, C. B. (1980) Positive cooperative binding of calcium to bovine brain calmodulin. Biochemistry 19, 3692–3698.PubMedCrossRefGoogle Scholar
  3. 3.
    Teraoka, H. and Nierhaus, K. H. (1979) Measurement of the binding of antibiotics to ribosomal particles by means of equilibrium dialysis. Methods Enzymol. 59, 862–866.PubMedCrossRefGoogle Scholar
  4. 4.
    Colowick, S. P. and Womack, F. C. (1969) Binding of diffusible molecules by mac-romolecules: rapid measurement by rate of dialysis. J. Biol. Chem. 244, 774–777.PubMedGoogle Scholar
  5. 5.
    Womack, F. C. and Colowick, S. P. (1973) Rapid measurement of binding of ligands by rate of dialysis. Methods Enzymol. 27, 464–471.PubMedCrossRefGoogle Scholar
  6. 6.
    Haiech, J., Klee, C. B., and Demaille, J. G. (1981) Effects of cations on affinity of calmodulin for calcium: ordered binding of calcium ions allows the specific activation of calmodulin-stimulated enzymes. Biochemistry 20, 3890–3897.PubMedCrossRefGoogle Scholar
  7. 7.
    Minowa, O. and Yagi, K. (1984) Calcium binding to tryptic fragments of calmodulin. J.Biochem. 56, 1175–1182.Google Scholar
  8. 8.
    Yazawa, M., Ikura, M., Hikichi, K., Luan, Y., and Yagi, K. (1987) Communication between two globular domains of calmodulin in the presence of mastoparan or caldesmon fragment. J. Biol. Chem. 262, 10,951–10,954.PubMedGoogle Scholar
  9. 9.
    Porumb, T. (1994) Determination of calcium-binding constants by flow dialysis. Anal. Biochem. 220, 227–237.PubMedCrossRefGoogle Scholar
  10. 10.
    Stemmer, P. M. and Klee, C. (1994) Dual calcium ion regulation of calcineurin by calmodulin and calcineurin B. Biochemistry 33, 6859–6866.PubMedCrossRefGoogle Scholar
  11. 11.
    Yazawa, M., Vorherr, T., James, P., Carafoli, E., and Yagi, K. (1992) Binding of calcium by calmodulin: influence of the calmodulin binding domain of the plasma membrane calcium pump. Biochemistry 31, 3172–3176.CrossRefGoogle Scholar
  12. 12.
    Starovasnik, M. A., Davis, T. N., and Klevit, R. E. (1993) Similarities and differences between yeast and vertebrate calmodulin: an examination of the calcium binding and structural properties of calmodulin from the yeast Saccharomyces cerevisiae. Biochemistry 32, 3261–3270.PubMedCrossRefGoogle Scholar
  13. 13.
    Yazawa, M., Sakuma, M., and Yagi, K. (1980) Calmodulins frommuscles of marine invertebrates, scallop and sea anemone. J. Biochem. 87, 1313–1320.PubMedGoogle Scholar
  14. 14.
    Feldmann, K. (1978) New devices for flow dialysis and ultrafiltration for the study of protein-ligand interactions. Anal. Biochem. 38, 225–235.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Michio Yazawa
    • 1
  1. 1.Division of Chemistry, Graduate School of Science, Hokkaido UniversitySapporoJapan

Personalised recommendations