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Water-soluble macromolecular fluorogenic substrates for assaying proteinases: determination of pancreatic elastase
F Anjuère1, M Monsigny, R Mayer
1Département de Biochimie des Glycoconjugués et des Lectines Endogènes, CNRS, Orléans, France.
Abstract:
Water-soluble macromolecular fluorogenic substrates were synthesized in order to develop an easy specific proteinase assay. The validity of this method was tested with porcine pancreatic elastase by using its specific peptidic substrate Ala-Ala-Pro-Ala linked to a hydrosoluble polymer. The octapeptidic sequence FTC-epsilon Aca-Ala-Ala-Pro-Ala-Gly-Gly-Gly was linked to a water-soluble and neutral poly-L-lysine derivative. The aminocaproyl residue and the triglycyl sequence were added in order to improve the stability of the substrate, and the accessibility of the specific sequence Ala-Ala-Pro-Ala to elastase, respectively. The assay is based on the quantitative precipitation of the polymeric substrate in isopropanol while the released soluble fluorescent peptidic moiety is fluorometrically titrated in the supernatant.