Protease determination using an optimized alcohol enzyme electrode

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.