Insulin binding monitored by fluorescence correlation spectroscopy.
OBJECTIVE
The characteristics of insulin binding to its receptors have been extensively studied by the radioligand binding assay. We used fluorescence correlation spectroscopy to determine the distribution of diffusion times and further novel data on the kinetics of insulin's binding to its receptor.


METHODS
Cultured human renal tubular cells (HRTC) were incubated with tetramethyl rhodamine labelled insulin (Rh-Ins) for 60 min. Fluorescence intensity fluctuations and autocorrelation functions for Rh-Ins, free in the incubation medium and bound to the cell membrane, were studied at single-molecule 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 tauD1 = 0.8 ms, tauD2 = 20 ms) and corresponding weight fractions of y1 = 0.43 and y2 = 0.42. Specificity of the binding was shown by the dislocation of bound Rh-Ins when excess unlabelled insulin was added. Scatchard analysis showed a nonlinear plot, revealing two binding processes with different affinities (Kass approximately 2 x 10(10) M(-1) and approximately 1 x 10(9) M(-1), respectively).


CONCLUSIONS
The fluorescence correlation spectroscopy results show two classes of binding sites with different affinities for insulin, or interactions between receptor sites consistent with negative cooperativity. This conclusion is in agreement with studies of insulin binding using radioligand binding assays. Because of its high sensitivity (single molecule detection), FCS, provides additional data allowing a more precise evaluation of the kinetics of ligand-receptor interactions at low expression levels in living cells.
Zhong
Z H
ZH
Department of Surgical Sciences, Karolinska Hospital, Stockholm, Sweden.
Pramanik
A
A
Ekberg
K
K
Jansson
O T
OT
Jörnvall
H
H
Wahren
J
J
Rigler
R
R
D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D004058:Diffusion; D005456:Fluorescent Dyes; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D007684:Kidney Tubules; D007700:Kinetics; D007834:Lasers; D011972:Receptor, Insulin / Q000378:metabolism; D012235:Rhodamines; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013816:Thermodynamics
D005456:Fluorescent Dyes; D007328:Insulin; D012235:Rhodamines; D011972:Receptor, Insulin