|Edwards, Judson - Vince|
Submitted to: Peptide Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/25/2005
Publication Date: 7/1/2005
Citation: Edwards, J.V., Pierre, S.C., Bopp, A.F., Goynes, W.R. 2005. Detection of Human Neutrophil Elastase with Peptide-Bound Cross-Linked Ethoxylate Acrylate Resin Analogs. Peptide Research. 66:160-168.
Interpretive Summary: Chronic wounds are a major worldwide health problem. This research is directed at strengthening the role of cotton-based textiles and allied technologies in the growing market of high tech wound dressings. This paper demonstrates an important technology for development of an enzyme detection device that could be used in combination with a cotton-based, chronic wound dressing. High levels of elastase in the non-healing wound break down the recruitment of new tissue necessary for healing to take place. A detection device for assessment of elastase levels in the chronic wound could be used to determine when it is best to apply chronic wound dressings that sequester and inhibit elastase, and to monitor levels of elastase during wound dressing usage. The technology will benefit the consumer and enhance the managed efficacy of cotton wound dressings designed for chronic wound treatment.
Technical Abstract: As an approach to develop an elastase detection method in chronic wound fluid an assessment of the elastase-substrate adsorption at the solid-liquid interface was made. N-succinyl-Ala-Ala-Pro-Val-p-nitroanilide (suc-Ala-Ala-Pro-Val-pNA), a chromo genic human neutrophil elastase (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 substrate hydrolysis by HNE was obtained. The enzyme activities of chromogenic substrates bound to CLEAR resin were compared to activities of chromogenic substrates in solution. 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 are compared with the same two analogs in solution. An increase in visible wavelength absorbance was observed with increased resin weight and enzyme concentration. 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 gave an increased dose response with increasing enzyme units based on visible absorbance. The synthesis and assay of elastase substrates on CLEAR resin gives an understanding of substrate-enzyme adsorption and activity at the solid-liquid interface.