Figure 5. Binding of calcium or zinc ions to phosphorylated CCK₈ enhances subsequent ferric ion binding.

(A) The change in free [Ca²⁺] during addition of aliquots of calcium chloride to 44.6 μM CCK₈ (▼),15.2 μM CCK₈PO₄ (▲) or 35.6 μM CCK₈SO₄ (◆) in 10 mM Na⁺ PIPES, pH 6.5, 100mM NaCl, 0.005% Tween 20, 0.4% DMSO, 293 K was measured with a calcium-selective electrode as described in Experimental. Points are means of at least three separate experiments; solid and dashed lines represent the best fits with the program BioEqs to one-site and two-site models, respectively. The CCK₈PO₄ data was better fitted by a two-site than a one-site model. Binding of Ca²⁺ to the first CCK₈PO₄ site (K_d = 46 μM) was tighter than binding to CCK₈ (K_d = 83 μM), which in turn was at least 10-fold tighter than binding to CCK₈SO₄ (K_d = 870 μM). (B,C) Addition of aliquots of calcium chloride (B, ▲) or zinc sulphate (C,■) to 40 μM CCK₈PO₄ in the same buffer did not result in any increase in the absorption at 275 nm up to a molar ratio of 5. Subsequent addition of aliquots of ferric chloride to the solutions containing calcium (△) or zinc (□) resulted in a greater increase in absorption than that seen when ferric ions were added to CCK₈PO₄ in the absence of calcium or zinc ions (●). Points are means of at least three separate experiments; bars represent the SEM. The solid lines represent the best fit to a two-site ordered model with the program BioEqs, with the K_d and A₂₇₅ values for ferric ions only given in Table 1. The data for ferric ions in the presence of calcium ions was simulated (dashed line) as described in Experimental with the equation for a competitive two-site ordered model and the K_d and A₂₇₅ values given in Figure 6.