Steric hindrance is not required for n-alkanol cutoff in soluble proteins.

A loss of potency as one ascends a homologous series of compounds (cutoff effect) is often used to map the dimensions of binding sites on a protein target. The implicit assumption of steric hindrance is rarely confirmed with direct binding measurements, yet other mechanisms for cutoff exist. We studied the binding and effect of a series of n-alkanols up to hexadecanol (C16) on two model proteins, BSA and myoglobin (MGB), using hydrogen-tritium exchange and light scattering. BSA binds the n-alkanols specifically and, at 1 mM total concentration, is stabilized with increasing potency up to decanol (C10), where a loss in stabilizing potency occurs. Cutoff in stabilizing potency is concentration-dependent and occurs at progressively longer n-alkanols at progressively lower total n-alkanol concentrations. Light scattering measurements of n-alkanol/BSA solutions show a smooth decline in binding stoichiometry with increasing chain length until C14-16, where it levels off at approximately 2:1 (alkanol:BSA). MGB does not bind the n-alkanols specifically and is destabilized by them with increasing potency until C10, where a loss in destabilizing potency occurs. Like BSA, MGB demonstrates a concentration-dependent cutoff point for the n-alkanols. Derivation of the number of methylenes bound at K(D) and the free energy contribution per bound methylene showed that no discontinuity existed to explain cutoff, rendering steric hindrance unlikely. The data also allow an energetic explanation for the variance of the cutoff point in various reductionist systems. Finally, these results render cutoff an untenable approach for mapping binding site sterics in the absence of complementary binding measurements, and a poor discriminator of target relevance to general anesthesia.