Solute accessibility to N epsilon-fluorescein isothiocyanate-lysine-23 cobra alpha-toxin bound to the acetylcholine receptor. A consideration of the effect of rotational diffusion and orientation constraints on fluorescence quenching.

To obtain information on the disposition of alpha-toxin when bound to the acetylcholine receptor (AChR), we evaluated the accessibility of solutes to fluorescein isothiocyanate (FITC) conjugated to alpha-toxin (siamensis 3) at lysine 23 (FITC-toxin) by measuring the rate constants for iodide quenching of the fluorescence of fluorescein free in solution and FITC-toxin free in solution and bound to AChR. Relative to the free fluorescein, we observed a 55% reduction in the quenching rate constant for the unbound FITC-toxin and 80% reduction for the AChR-bound FITC-toxin. It is tempting to interpret a decrease in the quenching rate constant as due to an increase in the masking of the labeling fluorophore, which in our case would then be indicative of masking of fluorescein conjugated to the free toxin and masking of FITC-toxin, in the region of lysine 23, when bound to AChR. However, elementary considerations indicate that the quenching rate depends not only on geometrical masking factors but also on the translational and rotational mobilities of the labeled molecules as well as orientational constraints. To evaluate these effects we have established quantitative relations between the rate of fluorescence quenching, the degree of masking of fluorophore, translational and rotational rates, and orientational constraints of the labeled macromolecules, using recent formulations for the rate of reaction between asymmetric molecules (Shoup et al., 1981, Biophys. J., 36:619-714). These relations predict that the decrease in quenching constant observed for the labeled FITC-toxin as well as the AChR-bound FITC-toxin is largely due to differences in translational and rotational rates and orientational constraints and not to significant increases in geometrical masking. Our theoretical formulation shows that the quenching rate can be decreased by a factor of 2-5 merely by immobilizing a fluorophore on the surface of a large protein without any significant increase in geometrical masking.