Parinaric acids as probes of binding domains in neutrophil elastase.

Human neutrophil elastase has an extended hydrophobic substrate binding site which serves as a target for a number of hydrophobic inhibitors. We show here that the parinaric acids, fluorescent-conjugated tetraenoic fatty acids of plant origin, are inhibitors of neutrophil elastase. cis-Parinaric acid (cis-PA) interacts with the enzyme in two inhibitory modes. The high affinity interaction (Ki = 55 +/- 6 nM) results in partial noncompetitive inhibition of amidolytic activity, with 82% residual activity. A lower affinity interaction with cis-PA (Ki = 4 +/- 1 microM) results in competitive inhibition. trans-PA also acts as a high affinity partial noncompetitive inhibitor of elastase with a Ki equal to that for cis-PA but has no low affinity competitive inhibitory action. The endogenous fluorescence from the 3 tryptophan residues in elastase is partially quenched on binding cis- or trans-PA. Dependence of quenching of tryptophan fluorescence on PA concentration is consistent with binding to a single site with an apparent Kd of 26 +/- 3 nM, which may be equivalent to the high affinity partial noncompetitive inhibitory binding mode. Analysis of quenching according to the modified Forster theory of energy transfer developed by Snyder and Freire (Snyder, B., and Freire, E. (1982) Biophys. J. 40, 137-148) leads to an estimate of apparent closest indole-PA distance of 13 +/- 3 A. Fluorescence of either cis- or trans-PA is apparently unperturbed upon binding in the high affinity mode to elastase, but at micromolar cis-PA concentrations, binding to elastase results in a blue shift and 20% increase in intensity of PA emission, suggesting that the lower affinity competitive inhibitory binding mode of binding to elastase provides a hydrophobic environment for cis-PA.