Ca2l to bovine a-lactalbumin in the absence and presence of EGTA

Literature values for the Kd for Ca2 + in bovine a-lactalbumin range over 3 orders of magnitude. There is a difference between two results obtained with EGTA as a metalion 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 has been 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 yM, which was determined fluorimetrically, gave a Kd for Ca2 + of 1.3 + 0.1 nm. When oc-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 two methods are so similar as to indicate that the affinity for Ca2 + was unaffected by the presence of EGTA.