Capillary electrophoresis in the assay of the hydrolysis of glycine-containing peptides by a protease from Pseudomonas aeruginosa.
A rapid and simple capillary zone electrophoresis (CZE) method for measuring the activity of a minor protease from Pseudomonas aeruginosa is described. When glycine-containing oligopeptides were used as substrates, it was possible to separate and quantify substrate and products. Moreover oligopeptide hydrolysates were analysed by fast atom bombardment mass spectrometry providing the sites of splitting of the substrates. By comparison with CZE calibration curves constructed with reference peptides, the initial rates of hydrolysis were calculated. The method, validated for pentaglycine hydrolysis, was also used for the analysis of reaction mixtures and for monitoring the enzymic hydrolysis of various peptides in order to investigate enzyme specificity.
Vessillier
S
S
Laboratoire de Biochimie Analytique et de Synthèse Bioorganique, UFR Chimie-Biochimie, Université Lyon 1, Villeurbanne, France.
Bernillon
J
J
Saulnier
J
J
Wallach
J
J
D019075:Electrophoresis, Capillary; D010450:Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification; D005998:Glycine / Q000737:chemistry*; D006868:Hydrolysis; D007202:Indicators and Reagents; D009842:Oligopeptides / Q000737:chemistry*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D015203:Reproducibility of Results; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013379:Substrate Specificity
D007202:Indicators and Reagents; D009842:Oligopeptides; D010450:Endopeptidases; D005998:Glycine