Detection of human neutrophil elastase with peptide-bound cross-linked ethoxylate acrylate resin analogs

An assessment of elastase-substrate kinetics and adsorption at the solid-liquid interface of peptide-bound resin was made in an approach to the solid-phase detection of human neutrophil elastase (HNE), which is found in high concentration in chronic wound fluid. N-succinyl-alanine-alanine-proline-valine-p-nitroanilide (suc-Ala-Ala-Pro-Val-pNA), a chromogenic HNE substrate, was attached to glycine-cross-linked ethoxylate acrylate resins (Gly-CLEAR) by a carbodiimide reaction. To assess the enzyme-substrate reaction in a two-phase system, the kinetic profile of resin-bound peptide substrate hydrolysis by HNE was obtained. A glycine and di-glycine spacer was placed between the resin polymer and substrate to assess the steric and spatial requirements of resin to substrate with enzyme hydrolysis. The enzymatic activities of suc-Ala-Ala-Pro-Val-pNA and suc-Ala-Ala-Pro-Ala-pNA on the solid-phase resin were compared with similar analogs in solution. An increase in visible wavelength absorbance was observed with increasing amounts of substrate-resin and enzyme concentration. Enzyme hydrolysis of the resin-bound substrate was also demonstrated on a polypropylene surface, which was employed for visible absorbance of released chromophore. A soluble active substrate analog was released from the resin through saponification of the ethoxylate ester linkages in the resin polymer. The resin-released conjugate of the HNE substrate demonstrated an increased dose response with increasing enzyme concentration. The synthesis and assay of elastase substrates bound to CLEAR resin gives an understanding of substrate-elastase adsorption and activity at the resin's solid-liquid interface for HNE detection with a solid-phase peptide.