Protease determination using an optimized alcohol enzyme electrode.
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 ethyl 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 degrees 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.
Bardeletti
G
G
Laboratoire de Biochimie Biotechnologie, Université Claude Bernard-Lyon, Villeurbanne, France.
Carillon
C
C
D000429:Alcohol Oxidoreductases / Q000737:chemistry*; D015374:Biosensing Techniques; D002138:Calibration; D002175:Candida / Q000201:enzymology; D010450:Endopeptidases / Q000032:analysis*; D004800:Enzymes, Immobilized; D000431:Ethanol / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000032:analysis / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D000432:Methanol / Q000378:metabolism; D013696:Temperature; D014357:Trypsin / Q000032:analysis
D004800:Enzymes, Immobilized; D000431:Ethanol; D006861:Hydrogen Peroxide; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D010450:Endopeptidases; D014357:Trypsin; D000432:Methanol