Simultaneous determination of Pseudomonas aeruginosa elastase, human leukocyte elastase and cathepsin G activities by micellar electrokinetic chromatography.
Micellar electrokinetic chromatography (MEKC) is a new method for analysing proteolytic activities simultaneously present in incubation mixtures. Here we demonstrate that MEKC differentiates between the enzymatic activities of Pseudomonas aeruginosa elastase (PsE) and human leukocyte elastase (HLE) or cathepsin G (Cat G) in assays using the chromogenic peptide substrates Suc-Ala-Ala-Ala-NA or Suc-Ala-Ala-Pro-Phe-NA, respectively (where Suc = succinyl and NA = 4-nitroaniline/u-nitroanilide). When PsE and Cat G were incubated at equimolar ratio with Suc-Ala-Ala-Pro-Phe-NA, the PsE-specific cleavage products PheNA and Suc-Ala-Ala-Pro were detected whereas inhibition of the metalloproteinase PsE with EDTA resulted in detection of NA and Suc-Ala-Ala-Pro-Phe only. Similarly, when PsE and HLE were incubated at equimolar ratio with Suc-Ala-Ala-Ala-NA, the PsE-specific cleavage products Suc-Ala and Ala-Ala-NA were detected whereas at an PsE-HLE ratio 1:50, both the PsE-specific and the HLE-specific cleavage products NA and Suc-Ala-Ala-Ala were separated. MEKC also allowed determination of the kinetic constants for the interactions of PsE, Cat G and HLE with the substrates considered.
Viglio
S
S
Dipartimento di Biochimica A. Castellani, Università di Pavia, Italy.
Luisetti
M
M
Zanaboni
G
G
Döring
G
G
Worlitzsch
D
D
Cetta
G
G
Iadarola
P
P
D000595:Amino Acid Sequence; D056649:Cathepsin G; D002403:Cathepsins / Q000737:chemistry / Q000378:metabolism*; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods*; D006801:Humans; D007700:Kinetics; D019272:Leukocyte Elastase / Q000737:chemistry / Q000378:metabolism*; D010196:Pancreatic Elastase / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D012697:Serine Endopeptidases; D013379:Substrate Specificity
D010446:Peptide Fragments; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase