High-affinity binding of Ca2+ to bovine α-lactalbumin in the absence and presence of EGTA

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Abstract
Literature values for the Kd for Ca2+ in bovine .alpha.-lactalbumin range over 3 orders of magnitude. There is a difference between 2 results obtained with EGTA [ethylene glycol bis(.beta.-aminoethyl ether)-N,N,N'',N''-tetraacetic acid] as a metal-ion buffer, partly because different values for the Kd of Ca2+-EGTA were used in the calculations, and a much wider difference between results obtained in the presence and absence of EGTA, which was attributed to an interaction between EGTA and the protein. Titrations in a flow-dialysis cell showed that Mn2+ competed with Ca2+ for the high-affinity site on the protein, and the results, combined with a Kd for Mn2+ of 2.1 .+-. 0.1 .mu.M, which was determined fluorimetrically, gave a Kd for Ca2+ of 1.3 .+-. 0.1 nM. When .alpha.-lactalbumin containing 45Ca2+ was titrated with EGTA in a flow-dialysis cell, and widely accepted metal-chelation data for EGTA were used in the calculations, a Kd for Ca2+ of 1.10 .+-. 0.03 nM was obtained. The results from the 2 methods are so similar as to indicate that the affinity for Ca2+ was unaffected by the presence of EGTA.

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