|Edwards, Judson - Vince|
Submitted to: American Chemical Society Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 2/1/2003
Publication Date: 3/24/2003
Citation: Pierre, S.C., Edwards, J.V., Bopp, A.F., Goynes, W.R. 2003. Detection of Human Neutrophil Elastase with Selectively Modified Cross-Linked Ethoxylate Acrylate Resins [abstract]. American Chemical Society Abstracts. 225(1-2):CELL 66.
Technical Abstract: An approach to develop elastase detection in chronic wound fluid was investigated by solid-liquid phase interaction where the enzymatic substrate is immobilized to the solid phase. The substrates of interest are N-succinyl-Ala-Ala-Pro-Val-p -nitroanilide (suc-Ala-Ala-Pro-Val-pNA) and suc-Ala-Ala-Pro-Ala-pNA. These substrates were immobilized to glycine-cross-linked ethoxylate acrylate resins (Gly-CLEAR) by a carbodiimide reaction. Several enzyme assays were prepared to determine the amount of para-nitroaniline released and establish the equilibrium 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 interacting the substrate-resin ranging from 1 mg to 5 mg with 1 unit of elastase. The release of pNA, a chromophore, 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 broken. 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, enzymatic assays for immobilized substrates on resin at ~20 mg had a response different than that of the soluble substrate. The elastase diffuses into the immobilized substrate matrix where colorimetric response was not observed in the supernatant. Therefore, protein analyses were performed 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.