Analysis of calcium binding to alpha-lactalbumin using a fluorescent calcium indicator.
A sensitive and rapid assay of Ca2+ binding to proteins was developed, based on the competition of Ca2+ binding to the protein of interest and fluo-3, a fluorescent Ca2+ indicator. Ca2+ binding to fluo-3 and bovine alpha-lactalbumin was analyzed at ten different pH values and a range of Na+ and K+ concentrations. We demonstrate that the binding constants of alpha-lactalbumin, determined by means of the competition assay and using intrinsic protein fluorescence, are the same within experimental error. The dissociation constant of the alpha-lactalbumin--Ca2+ complex in 50 mM Hepes containing 150 mM Na+ at pH 7.4 and 25 degrees C, was found to be 123 +/- 2 nM and 103 +/- 43 nM when determined by the competition assay and intrinsic protein fluorescence, respectively. Binding of Ca2+ to alpha-lactalbumin did not depend on pH in the range 6.6-8.4 and was differently affected by Na+ and K+. EDTA-agarose, a chelating chromatography material, was synthesized and used to remove Ca2+ from buffer and protein solutions. The total concentration of Ca2+ in 50 mM Hepes, containing 150 mM Na+ at pH 7.4, was lowered to 119 +/- 13 nM and the number of Ca2+ bound/molecule alpha-lactalbumin was lowered to 0.069 +/- 0.006. No interaction between fluo-3 and alpha-lactalbumin could be discerned from spectral analysis and fluorescence anisotropy measurements.
Eberhard
M
M
Department of Research, Kantonsspital, Basel, Switzerland.
Erne
P
P
D000814:Aniline Compounds; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D005456:Fluorescent Dyes; D007700:Kinetics; D007768:Lactalbumin / Q000378:metabolism*; D008433:Mathematics; D008962:Models, Theoretical; D011485:Protein Binding; D012964:Sodium / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D014966:Xanthenes
D000814:Aniline Compounds; D005456:Fluorescent Dyes; D014966:Xanthenes; C059715:Fluo-3; D007768:Lactalbumin; D012964:Sodium; D002118:Calcium