Ca(2+) and Na(+) binding to high affinity sites of calcium-containing proteins measured by capillary electrophoresis.
A method for the determination of stability constants of metal ion-protein binding, based on capillary electrophoresis, is presented. It utilizes the change in electrophoretic mobility of the protein upon binding of a metal ion. Taking advantage of edta(4-) as a controller of the free Ca(2+) concentration, a [Ca(2+)](free) as low as 10(-9) M has been attained in the solutions. We have found this method very useful for measuring binding of Ca(2+) to proteins, where the stability constant is in the range 10(5)-10(8) M(-1). The stability constants for the binding of Ca(2+) to proteinase K and bovine alpha-lactalbumin has by this method been measured at an ionic strength of 0.1 M, pH(c) 7.40 and 25 degrees C. For proteinase K a constant of 10(7.4) M(-1) is found, and for alpha-lactalbumin the constant has been found to be 10(9.2) M(-1). The structural stability of both proteins are found to be affected by the presence of Na(+) in the buffer solutions. From this observation, association constants for binding of Na(+) to the Ca(2+) sites have been calculated to 10(2.4) M(-1) for proteinase K and 10(3.5) M(-1) for alpha-lactalbumin. Less than 50 microg have been used of each protein in this study, an obvious advantage over other methods.
Rasmussen
Bodil W
BW
Department of Chemistry, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
Bjerrum
Morten J
MJ
D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D004492:Edetic Acid / Q000737:chemistry; D019075:Electrophoresis, Capillary; D019286:Endopeptidase K / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry; D009994:Osmolar Concentration; D011485:Protein Binding; D012964:Sodium / Q000737:chemistry* / Q000378:metabolism
D001059:Apoproteins; D007768:Lactalbumin; D004492:Edetic Acid; D012964:Sodium; D019286:Endopeptidase K; D008274:Magnesium; D002118:Calcium