Abstract
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 ofn-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 totaln-alkanol concentrations. Light scattering measurements ofn-alkanol/BSA solutions show a smooth decline in binding stoichiometry with increasing chain length until C14–16, where it levels off at ∼2∶1 (alkanol∶BSA). MGB does not bind then-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.
Footnotes
- Received January 14, 1999.
- Accepted May 11, 1999.
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Send reprint requests to: R. G. Eckenhoff, M.D., Department of Anesthesia, 772 Dulles Building, Hospital of the University of Pennsylvania, University of Pennsylvania Health System, 3400 Spruce St., Philadelphia, PA 19104-4283. E-mail:reckenho{at}mail.med.upenn.edu.
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This work was supported by National Institute of General Medical Sciences Grants 51595 and 55876.
- The American Society for Pharmacology and Experimental Therapeutics
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