Nature of the individual Ca2+ binding sites in Ca2+-regenerated bacteriorhodopsin

The binding constants, K1 and K2, and the number of Ca2+ ions in each of the two high affinity sites of Ca2+-regenerated bacteriorhodopsin (bR) are determined potentiometrically at different pH values in the range of pH 3.5–4.5 by using the Scatchard plot method. From the pH dependence of K1 and K2, it was found that two hydrogen ions are released for each Ca2+ bound to each of the two high affinity sites. Furthermore, we have measured by a direct spectroscopic method the association constant, Ks, for the binding of Ca2+ to deionized bR, which is responsible for producing the blue to purple color change. Comparing the value of Ks and its pH dependence with those of K1 and K2 showed that the site corresponding to Ks is to be identified with that of K2. This is in agreement with the conclusion reached previously, using a different approach, which showed that it is the second Ca2+ that causes the blue to purple color change.Our studies also show that in addition to the two distinct high affinity sites, there are about four to six sites with lower binding constants. These are attributed to the nonspecific binding in bR