LIMITATIONS INHERENT IN DELTAPH METHOD OF DETETERMINING BINDING ISOTHERMS OF BOVINE SERUM ALBUMIN

Binding isotherms, determined by the ΔpH method of Scatchard and Black, of a large variety of aliphatic and aromatic anions, containing up to 14 carbon atoms, have been compared with isotherms obtained with the same substances by equilibrium dialysis and in two instances by measurement of potentials across permselective membranes. With symmetric nondeformable ions, e.g., aromatic anions, the ΔpH method gives results in good agreement with those determined by dialysis. With aliphatic long-chain anions, the amount of binding and the binding constants are systematically underestimated by factors which increase with the affinity. In these comparisons, due regard has been taken of (a) the effects of the necessary differences in the amounts of competing electrolyte present when each of the three methods is used, and (b) need to correct for the lack of buffering capacity in very dilute solutions of unbuffered protein. Two methods for evaluation of an empirical electrostatic factor, ω, from the binding measurements in 0.001 M NaCl are discussed. Explanation of the much lower ω computed from that calculated by application of Debye-Hückel theory is sought in terms of applicability of the Linderstrom-Lang model to noncovalently bound long-chain ions with hydrophobic tails, and of differences between the effective charge Z of the macroion and the charge computed stoichiometrically. It is shown that as a practical matter the ΔpH method cannot be applied to any ligand of high affinity (K > 105) because of the large effect in this method of small errors in determining concentration or protein molecular weight. © 1969, American Chemical Society. All rights reserved.