[Method for determining concentration of fluorescent probes binding to biological structures]

Fluorescent probes are used to study the structure and functions of proteins, biological membranes, lipoproteins, nucleic acids, nucleoproteins etc. The binding centers of these biological objects that interact with probes are usually rather heterogeneous. It is often impossible to describe the interaction of the probe with these centers in terms of simple Langmuir isotherms. Moreover, these centers can affect each other; as they are occupied with probe molecules, the properties of both free and probe-occupied centers change. The fluorescence quantum yield of the probe in different centers is not the same and can change due to their reciprocal influences. As a result, It is often impossible to determine even the number of probe molecules bound to these centers. In this paper we describe a method for determining the number of probe molecules bound to a biological object without regard to object heterogeneity and mutual influence of the centers. We abandoned the earlier accepted practice of calculating the number of bound molecules of the probe from the intensity of its fluorescence. Instead, the fluorescence of the probe is used only to compare solutions with different concentrations of probes and centers to achieve an equal occupancy of the centers with probe molecules. This makes it possible to measure the amount of the bound probe irrespective of the heterogeneity of the binding centers even in the presence of mutual influence of the centers.