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.