J. Biol. Chem., Vol. 260, Issue 6, 3451-3455, 03, 1985
The effect of ligand heterogeneity on the Scatchard plot. Particular relevance to lipoprotein binding analysis
CM Mendel, V Licko and JP Kane
Computer simulations of equilibrium binding studies of a mixture of two
labeled ligands binding competitively to a single class of identical and
independent sites (receptors) were performed to investigate how ligand
heterogeneity affects the observed data in such studies. The simulated data
are presented in Scatchard plots. Ligand heterogeneity was generally found
to be indistinguishable from the case of a homogeneous ligand when usual
experimental conditions applied (that is, Scatchard plots of the data were
straight lines). Some factors that increased the probability of recognizing
heterogeneity in the system were identified, however. These are 1) a large
difference between the dissociation constants of the two ligands, 2) a high
concentration of receptors relative to the dissociation constant of the
higher-affinity ligand, 3) a high concentration of the lower-affinity
ligand relative to that of the higher-affinity ligand, 4) a high specific
activity of the lower-affinity ligand relative to that of the
higher-affinity ligand, and 5) lack of experimental error. When ligand
heterogeneity (under certain conditions) did cause curvilinearity in the
Scatchard plot, the curve formed was always concave-downwards. Thus, ligand
heterogeneity may occasionally mimic positive cooperativity, but never
mimics negative cooperativity or multiple classes of binding sites.
Implications of these findings for equilibrium binding studies involving
lipoproteins (which are generally isolated as heterogeneous mixtures of
particles) are discussed in detail. These findings are also relevant to
equilibrium binding studies using ligands which are mixtures of
stereoisomers or which contain chemical or radiochemical impurities.
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