ELASTIN-DERIVED PEPTIDE BINDING TO A HYDROPHOBIC DOMAIN ON NEUTROPHIL ELASTASE

To understand the contributions of binding of elastin to domains removed from the active site of neutrophil elastase, we isolated an elastin-derived peptide (EDP) fraction, which we have previously shown was tightly linked to neutrophil elastase after prolonged digestion of elastin but which can be released from the enzyme with hydroxylamine. Elastin from human aorta was incubated with human neutrophil elastase under conditions favoring proteolysis. Low molecular weight species, including free EDP, were separated from the protein fraction by a small centrifuged gel filtration column. The high molecular weight protein fraction was subjected directly to 0.5 M hydroxylamine. The reaction mixture was then fractionated on a phosphocellulose column using an ionic gradient. A fraction was collected that exhibited fluorescence with a peak at similar to 410 nm when excited at 320 nm, indicating the presence of desmosine acid (or) isodesmosine. A second peak with amidolytic activity towards methoxysuccinyl-ala-Ala-Pro-Val-p-nitroaniline (MeOSucAAPVpNa), but no fluorescence at 410 nm was also detected at the same elution volume where free elastase appeared. After removal of low molecular weight digestion products but prior to treatment with hydroxylamine, the putative elastase-EDP complex possessed no amidolytic activity towards MeOSucAAPVpNa. When the liberated EDP was added to elastase in an amidolytic assay, the EDP behaved as only a partial noncompetitive inhibitor (V-max/V-max similar or equal to 90%), but bound with high affinity to neutrophil elastase (K-i similar or equal to 29 nM), as detected by its ability to quench elastase endogenous fluorescence. The complete emission spectrum of the mixture of elastase and EDP obtained at excitation wavelengths specific for tryptophan and desmosine/ isodesmosine suggests that the EDP was in a hydrophobic environment which was close to at least one of the three tryptophan residues in the enzyme. Based on fluorescence energy transfer, we have estimated a distance between the elastase and EDP of similar to 10 +/- 3 Angstrom A (1 Angstrom A = 0.1 nm) during elastinolysis. This pattern of binding to a hydrophobic site on neutrophil elastase without competitive inhibition of amidolytic activity was consistent with the importance of hydrophobic interactions between neutrophil elastase and elastin within a region of the enzyme removed from the active site.