COMPARISON BETWEEN THE BINDING OF CA2+ AND MG2+ TO THE 2 HIGH-AFFINITY SITES OF BACTERIORHODOPSIN

Bacteriorhodopsin contains Ca2+ and Mg2+ ions whose removal inhibits its proton pump function. The binding constants of Ca2+ to the high-affinity sites were determined by the use of a calcium ion specific electrode. The unavailability of magnesium ion specific electrode prevented a similar determination for Mg2+. Th, binding constant of Mg2+ to the binding site of highest affinity is determined by using a calcium ion selective electrode to measure the concentration of free Ca2+ in competition with Mg2+ for the binding. The binding constant of Mg2+ to the second high affinity site is determined spectrally. The two high-affinity binding constants for Mg2+ are compared with those obtained for Ca2+ in the absence and the presence of Mg2+. The fact that the presence of low concentration of one metal ion does not affect the binding constant of the other metal ion to the other binding site supports the assumption of the independence of the two high-affinity sites of one another. The difference in the observed values of the binding constants of the two high-affinity sites for Ca2+ and Mg2+ is qualitatively discussed in terms of the enthalpy and entropy changes in the binding equilibrium.