Insulin binding monitored by fluorescence correlation spectroscopy

Aim/hypothesis. The characteristics of insulin binding to its receptors hav e been extensively studied by the radioligand binding assay. We used fluore scence correlation spectroscopy to determine the distribution of diffusion times and further novel data on the kinetics of insulin's binding to its re ceptor. Methods. Cultured human renal tubular cells (HRTC) were incubated with tetr amethyl rhodamine labelled insulin (Rh-Ins) for 60 min. Fluorescence intens ity fluctuations and autocorrelation functions for Rh-Ins, free in the incu bation medium and bound to the cell membrane, were studied at single-molecu le detection sensitivity in a 0.2 fL confocal volume. Results. Measurements at the cell membrane revealed Rh-Ins binding with at least two diffusion components (diffusion times tau (D1) = 0.8 ms, tau (D2) = 20 ms) and corresponding weight fractions of y(1) = 0.43 and y(2) = 0.42 . Specificity of the binding was shown by the dislocation of bound Rh-Ins w hen excess unlabelled insulin was added. Scatchard analysis showed a nonlin ear plot, revealing two binding processes with different affinities (K-ass similar to 2 (.)10(10) M-1 and similar to 1 (.) 10(9) M-1, respectively). Conclusion/ interpretation. The fluorescence correlation spectroscopy resul ts show two classes of binding sites with different affinities for insulin, or interactions between receptor sites consistent with negative cooperativ ity. This conclusion is in agreement with studies of insulin binding using radioligand binding assays. Because of its high sensitivity (single molecul e detection), FCS, provides additional data allowing a more precise evaluat ion of the kinetics of ligand-receptor interactions at low expression level s in living cells.