An error in interpretation of double-reciprocal plots and Scatchard plots in studies of binding of fluorescent probes to proteins, and alternative proposals for determining binding parameters

Abstract

One of the objects of experiments in which a fluorochrome is added to suspensions of cell membranes is to determine the parameters n and K _D , the capacity of unit mass of protein to bind fluorochrome and the dissociation constant, respectively. Currently, these are estimated from Scatchard plots, construction of which first requires that observed fluorescence intensity be converted to moles of bound fluorochrome. This in turn is said to be possible by analysis of the intercept of a plot of reciprocal fluorescence intensity against reciprocal protein concentration. However, analysis of the classical mass action equilibrium equation, upon which the foregoing procedures are said to be based, reveals that the intercept of the double-reciprocal plot always underestimates the desired value. The error is formalized and shown to increase without bound with fluorochrome concentration. The error in turn leads to erroneous assessment of n and K _D . Alternative methods for calculating the desired parameters are proposed, based on direct plots of fluorescence intensity.