THE EFFECT OF PROTEIN-CONCENTRATION ON ION-BINDING

The concentration of protein in a solution has been found to have a si gnificant effect on ion binding affinity, It is well known that an inc rease in ionic strength of the solvent medium by addition of salt modu lates the ion-binding affinity of a charged protein due to electrostat ic screening. In recent Monte Carlo simulations, a similar screening h as been detected to arise from an increase in the concentration of the protein itself, Experimental results are presented here that verify t he theoretical predictions; high concentrations of the negatively char ged proteins calbindin D-9k and calmodulin are found to reduce their a ffinity for divalent cations, The Ca2+-binding constant of the C-termi nal site in the Asn-56 --> Ata mutant of calbindin D-9k has been measu red at seven different protein concentrations ranging from 27 mu M to 7.35 mM by using H-1 NMR, A 94% reduction in affinity is observed when going from the lowest to the highest protein concentration, For calmo dulin, we have measured the average Mg2+-binding constant of sites I a nd II at 0.325, 1.08, and 3.25 mM protein and find a 13-fold differenc e between the two extremes. Monte Carlo calculations have been perform ed for the two cases described above to provide a direct comparison of the experimental and simulated effects of protein concentration on me tal ion affinities, The overall agreement between theory and experimen t is good. The results have important implications for all biological systems involving interactions between charged species.