ON THE NEED TO CONSIDER KINETIC AS WELL AS THERMODYNAMIC CONSEQUENCESOF THE PARKING PROBLEM IN QUANTITATIVE STUDIES OF NONSPECIFIC-BINDINGBETWEEN PROTEINS AND LINEAR POLYMER-CHAINS

Attention is drawn to a need for caution in the thermodynamic characte rization of nonspecific binding of a large ligand to a linear acceptor such as a polynucleotide or a polysaccharide-because of the potential for misidentification of a transient (pseudoequilibrium) state as tru e equilibrium. The time course of equilibrium attainment during the bi nding of a large ligand to nonspecific three-residue sequences of a li near acceptor lattice has been simulated, either by numerical integrat ion of the system of ordinary differential equations or by a Monte Car lo procedure, to identify the circumstances under which the kinetics o f elimination of suboptimal ligand attachment (called the parking prob lem) create such difficulties. These simulations have demonstrated tha t the potential for the existence of a transient plateau in the time c ourse of equilibrium attainment increases greatly (i) with increasing extent of acceptor saturation (i.e., with increasing ligand concentrat ion), (ii) with increasing magnitude of the binding constant, and (iii ) with increasing length of the acceptor lattice. Because the capacity of the polymer lattice for ligand is most readily determined under co nditions conducive to essentially stoichiometric interaction, the para meter so obtained is thus likely to reflect the transient (irreversibl e) rather than equilibrium binding capacity. A procedure is described for evaluating the equilibrium capacity from that irreversible paramet er; and illustrated by application to published results [M. Nesheim, M .N. Blackburn, C.M. Lawler, K.G. Mann, J. Biol. Chem. 261 (1986) 3214- 3221] for the stoichiometric titration of heparin with thrombin. (C) 1 998 Elsevier Science B.V. All rights reserved.