Figure 2.

(A) The Ca²⁺-affinity of CR56 was determined by rate dialysis [Ca²⁺]_bound/[CR56] versus the free Ca²⁺ concentration. The concentration of CR56 was 15 μM throughout the analysis. (B) Scatchard analysis of the data shown in panel A. The solid line represents the best fit by least-squares analysis as described in the materials and methods section. The fact that the experimental points are located on a straight line indicates absence of cooperativity between the two Ca²⁺ binding sites. Strong positive cooperativity would result in upward curvature, while strong negative cooperativity would result in downward curvature of the Scatchard plot. The average affinity is determined to, K_D = 10.6 μM assuming two identical sites (n = 2.2).