Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 125-134 |
| Number of pages | 10 |
| Journal | Journal of Chromatography A |
| Volume | 846 |
| Issue number | 1-2 |
| DOIs | |
| Publication status | Published - Jun 18 1999 |
Fingerprint
Keywords
- Cathepsins
- Elastases
- Enzymes
- Micellar electrokinetic chromatography
- Peptides
- Pseudomonas aeruginosa
ASJC Scopus subject areas
- Analytical Chemistry
Cite this
Simultaneous determination of Pseudomonas aeruginosa elastase, human leukocyte elastase and cathepsin G activities by micellar electrokinetic chromatography. / Viglio, S.; Luisetti, M.; Zanaboni, G.; Döring, G.; Worlitzsch, D.; Cetta, G.; Iadarola, P.
In: Journal of Chromatography A, Vol. 846, No. 1-2, 18.06.1999, p. 125-134.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Simultaneous determination of Pseudomonas aeruginosa elastase, human leukocyte elastase and cathepsin G activities by micellar electrokinetic chromatography
AU - Viglio, S.
AU - Luisetti, M.
AU - Zanaboni, G.
AU - Döring, G.
AU - Worlitzsch, D.
AU - Cetta, G.
AU - Iadarola, P.
PY - 1999/6/18
Y1 - 1999/6/18
N2 - 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.
AB - 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.
KW - Cathepsins
KW - Elastases
KW - Enzymes
KW - Micellar electrokinetic chromatography
KW - Peptides
KW - Pseudomonas aeruginosa
UR - http://www.scopus.com/inward/record.url?scp=0032781220&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0032781220&partnerID=8YFLogxK
U2 - 10.1016/S0021-9673(98)01056-5
DO - 10.1016/S0021-9673(98)01056-5
M3 - Article
C2 - 10420604
AN - SCOPUS:0032781220
VL - 846
SP - 125
EP - 134
JO - Journal of Chromatography A
JF - Journal of Chromatography A
SN - 0021-9673
IS - 1-2
ER -