It has been hypothesized that nonspecific reversible binding of cytosk
eletal proteins to lipids in cells may guide their binding to integral
membrane anchor proteins. In a model system, we measured desorption r
ates k(off) (off-rates) of the erythrocyte cytoskeletal proteins spect
rin and protein 4.1 labeled with carboxyfluorescein (CF), at two diffe
rent compositions of planar phospholipid membranes (supported on glass
), using the total internal reflection/fluorescence recovery after pho
tobleaching (TIR/FRAP) technique. The lipid membranes consisted of eit
her pure phosphatidylcholine (PC) or a 3:1 mixture of PC with phosphat
idylserine (PS). In general, the off-rates were not single exponential
s and were fit to a combination of fast, slow, and irreversible fracti
ons, reported both separately and as a weighted average, By a variatio
n of TIR/FRAP, we also measured equilibrium affinities (the ratio of s
urface-bound to bulk protein concentration) and thereby calculated on-
rates, k(on) The average off-rate of CF-4.1 from PC/PS (similar to 0,0
08/s) is much slower than that from pure PC (similar to 1.7/s), Despit
e the consequent increase in equilibrium affinity at PC/PS, the on-rat
e at PC/PS is also substantially decreased (by a factor of 40) relativ
e to that at pure PC. The simultaneous presence of (unlabeled) spectri
n tends to substantially decrease the on-rate (and the affinity) of CF
-4.1 at both membrane types, Similar experiments for CF-spectrin alone
showed much less sensitivity to membrane type and generally faster of
f-rates than those exhibited by CF-4,1, However, when mixed with (unla
beled) 4,1, both the on-rate and off-rate of CF-spectrin decreased dra
stically at PC/PS (but not PC), leading to a somewhat increased affini
ty, Clearly, changes in affinity often involve countervailing changes
in both on-rates and off-rates, In many of these studies, the effect o
f varying ionic strength and bulk concentrations was examined; it appe
ars that the binding is an electrostatic attraction and is far from sa
turation at the concentrations employed, These results and the techniq
ues implemented carry general implications for understanding the funct
ional role of nonspecific protein binding to cellular lipid membranes.