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 e ffect) is often used to map the dimensions of binding sites on a protein ta rget. The implicit assumption of steric hindrance is rarely confirmed with direct binding measurements, yet other mechanisms for cutoff exist. We stud ied 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 ex change and light scattering. BSA binds the n-alkanols specifically and, at 1 mM total concentration, is stabilized with increasing potency up to decan ol (C10), where a loss in stabilizing potency occurs. Cutoff in stabilizing potency is concentration-dependent and occurs at progressively longer n-al kanols at progressively lower total n-alkanol concentrations. Light scatter ing measurements of n-alkanol/BSA solutions show a smooth decline in bindin g stoichiometry with increasing chain length until C14-16, where it levels off at similar to 2:1 (alkanol:BSA). MGB does not bind the n-alkanols speci fically and is destabilized by them with increasing potency until C10, wher e a loss in destabilizing potency occurs. Like BSA, MGB demonstrates a conc entration-dependent cutoff point for the n-alkanols. Derivation of the numb er of methylenes bound at K-D and the free energy contribution per bound me thylene showed that no discontinuity existed to explain cutoff, rendering s teric 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 di scriminator of target relevance to general anesthesia.