|Edwards, Judson - Vince|
Submitted to: National Organization for the Professional Advancement of Black Chemists and Chemical Engineers
Publication Type: Abstract Only
Publication Acceptance Date: 4/13/2003
Publication Date: N/A
Technical Abstract: An excessive concentration of elastase in chronic non-healing wounds has been shown to deleteriously degrade cytokine growth factors necessary for healing. Therefore, an approach to develop elastase detection in chronic wound fluid was investigated by using a selectively modified resin to interact with elastase. Specific chromogenic substrates such as N-succinyl-Ala-Ala-Pro-Val-para-nitroanilide (suc-Ala-Ala-Pro-Val-pNA) and suc-Ala-Ala-Pro-Ala-pNA were immobilized to glycine-cross-linked ethoxylate acrylate resins (Gly-CLEAR) by a carbodiimide reaction. The solid-liquid phase reaction of the selectively modified resins with elastase releases a chromophore (para-nitroaniline, pNA) to produce a colorimetric response. Several enzyme assays were prepared to determine the amount of para-nitroaniline released and establish the equilibrium conditions for solid-liquid phase interactions. The enzymatic activities of CLEAR-Gly-suc-Ala-Ala-Pro-Val-pNA and CLEAR-Gly-suc-Ala-Ala-Pro-Ala-pNA on the solid-phase resin were compared with suc-Ala-Ala-Pro-Val-pNA and suc-Ala-Ala-Pro-Ala-pNA in solution. The initial enzyme assays entailed reacting the substrate-resin (from 1 mg to 5 mg) with 1 unit of elastase. The release of pNA from the immobilized substrate was not enough to develop a colorimetric response; however, substrate-enzyme interactions were detected in absorbance readings. To verify that chromogenic substrates were present on the resin, the soluble active substrate analog was released from the resin through saponification where ethoxylate ester linkages between the resin polymer and substrate were hydrolyzed. The resin-released conjugate of the elastase substrate, which has an increased dose response with increasing enzyme units ranging from 2 to 6 units was supported by visible absorbance. Yet, increasing the amount of immobilized substrates on resin to ~20 mg had a response different than that of the soluble substrate for enzymatic assays. The elastase diffuses in the immobilized matrix that prevents the release of pNA in the supernatant. Therefore, protein analyses were investigated to show the partitioning of enzyme between the solid-liquid interfaces. By characterizing the enzyme-immobilized substrate interactions, this study forms the basis for a human neutrophil elastase detection method in chronic wound fluid.