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Applied Biochemistry and Biotechnology

, Volume 43, Issue 3, pp 177–188 | Cite as

Protease determination using an optimized alcohol enzyme electrode

  • G. Bardeletti
  • C. Carillon
Article

Abstract

A new method for the determination of protease activities is described. In this large family, trypsin is used as a protease model that cleaves the enthyl or methyl ester of artificial substrates producing ethanol or methanol. Alcohol is detected using an alcohol oxidase enzyme electrode. The H2O2 production that occurs is measured amperometrically. At 30°C, in a 0.1M phosphate buffer, pH 7.5, the enzyme electrode response for ethanol was calibrated at 3.10−6–3.10−3 M and for methanol from 3.10−7 to 4.10−4 M in the cell measurement. Trypsin levels as determined by the proposed method and by a conventional spectrophotometric method are in good agreement when using the same measurement conditions. A detection limit of 10 U·L−1 and a linear calibration curve of 10–100,000 U·L−1 in the sample were obtained. Measuring time for the required trypsin solution concentration was from 4 min (for the most dilute samples) to 1 min (for the most concentrate samples). In a typical experiment, protease measurements did not inactivate the alcohol oxidase on the probe, nor did a more classical use for alcohol detection. The procedure developed could permit any protease estimation on the condition that they hydrolyze ester bonds from synthetic substrate.

Index Entries

Amperometric enzyme electrode ethanol methanol protease activity trypsin 

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Copyright information

© Humana Press Inc. 1993

Authors and Affiliations

  • G. Bardeletti
    • 1
  • C. Carillon
    • 1
  1. 1.Laboratoire de Biochimie Biotechnologie, EP19 CNRS, Bat 303Université Claude Bernard-Lyon 1Villeurbanne CedexFrance

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