Quantitative analyses of the interaction between calcium ions and human serum albumin

We examined the suitability of nine organic buffers for studying calcium binding to albumin by equilibrium dialysis. Results obtained with defatted human serum albumin showed that 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid were superior. Scatchard analysis of the experimental data from an extensive study performed in HEPES at pH 7.4 and 20 °C revealed (putting n(i) = 1, i = 1-4) k₁ = 367 L/mol, k₂ = 314 L/mol, k₃ = 291 L/mol, and k₄ = 179 L/mol. The very weak binding was characterized as n₅ = 10 and k₅ = 40 L/mol. The results were also analyzed in terms of stoichiometric association constants. The constants K₁ and K₂ were calculated to be 1513 and 647 L/mol, respectively, whereas the other constants were considered undeterminable. pH studies showed that in the interval 6.8-7.4, binding was not influenced by changes in acidity. Increasing pH to above the physiological value resulted in increased binding. At pH 8.0, k₁ was increased almost fourfold, whereas k₂ and k₃ were approximately doubled. These findings indicate that the neutral to basic transition is important for the calcium-binding properties of albumin. The transition is a reversible, gradual conformational change of the protein at pH 6-9.