Detection and quantification of leucyl arninopeptidase after native electrophoresis using leucine-p-nitroanilide
Abstract
A general method for detecting leucyl aminopeptidase activity after native polyacrylamide gel electrophoresis (PAGE) in situ is described. The method is based on diazotization of p-nitroaniline, liberated in the polyacrylamide gel by leucyl aminopeptidase action on leucine-p-nitroanilide (LpNA) and subsequent coupling with a chromogen, 1-naphthylamine, until a pink azo dye product at the position of enzyme activity is obtained. A possible use of this technique for leucyl aminopeptidase detection and quantification is indicated. This method was found to be reproducible with the coefficient of variation below 15% for a 32-fold range, while the colored area of enzyme activity was in linear dependence to enzyme activity. Applications of this method with some other aminoacyl-p-nitroanilides and for detection of kidney bean leucyl aminopeptidase isoforms are demonstrated.
Keywords:
isoenzymes / leucyl aminopeptidase / Phaseolus vulgaris / p-nitroanilide / zymogramSource:
Electrophoresis, 2005, 26, 12, 2476-2480Publisher:
- Wiley-V C H Verlag Gmbh, Weinheim
DOI: 10.1002/elps.200500047
ISSN: 0173-0835
PubMed: 15920779
WoS: 000230239900022
Scopus: 2-s2.0-21644465014
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IHTMTY - JOUR AU - Božić, Nataša AU - Vujčić, Zoran PY - 2005 UR - https://cer.ihtm.bg.ac.rs/handle/123456789/4670 AB - A general method for detecting leucyl aminopeptidase activity after native polyacrylamide gel electrophoresis (PAGE) in situ is described. The method is based on diazotization of p-nitroaniline, liberated in the polyacrylamide gel by leucyl aminopeptidase action on leucine-p-nitroanilide (LpNA) and subsequent coupling with a chromogen, 1-naphthylamine, until a pink azo dye product at the position of enzyme activity is obtained. A possible use of this technique for leucyl aminopeptidase detection and quantification is indicated. This method was found to be reproducible with the coefficient of variation below 15% for a 32-fold range, while the colored area of enzyme activity was in linear dependence to enzyme activity. Applications of this method with some other aminoacyl-p-nitroanilides and for detection of kidney bean leucyl aminopeptidase isoforms are demonstrated. PB - Wiley-V C H Verlag Gmbh, Weinheim T2 - Electrophoresis T1 - Detection and quantification of leucyl arninopeptidase after native electrophoresis using leucine-p-nitroanilide VL - 26 IS - 12 SP - 2476 EP - 2480 DO - 10.1002/elps.200500047 ER -
@article{ author = "Božić, Nataša and Vujčić, Zoran", year = "2005", abstract = "A general method for detecting leucyl aminopeptidase activity after native polyacrylamide gel electrophoresis (PAGE) in situ is described. The method is based on diazotization of p-nitroaniline, liberated in the polyacrylamide gel by leucyl aminopeptidase action on leucine-p-nitroanilide (LpNA) and subsequent coupling with a chromogen, 1-naphthylamine, until a pink azo dye product at the position of enzyme activity is obtained. A possible use of this technique for leucyl aminopeptidase detection and quantification is indicated. This method was found to be reproducible with the coefficient of variation below 15% for a 32-fold range, while the colored area of enzyme activity was in linear dependence to enzyme activity. Applications of this method with some other aminoacyl-p-nitroanilides and for detection of kidney bean leucyl aminopeptidase isoforms are demonstrated.", publisher = "Wiley-V C H Verlag Gmbh, Weinheim", journal = "Electrophoresis", title = "Detection and quantification of leucyl arninopeptidase after native electrophoresis using leucine-p-nitroanilide", volume = "26", number = "12", pages = "2476-2480", doi = "10.1002/elps.200500047" }
Božić, N.,& Vujčić, Z.. (2005). Detection and quantification of leucyl arninopeptidase after native electrophoresis using leucine-p-nitroanilide. in Electrophoresis Wiley-V C H Verlag Gmbh, Weinheim., 26(12), 2476-2480. https://doi.org/10.1002/elps.200500047
Božić N, Vujčić Z. Detection and quantification of leucyl arninopeptidase after native electrophoresis using leucine-p-nitroanilide. in Electrophoresis. 2005;26(12):2476-2480. doi:10.1002/elps.200500047 .
Božić, Nataša, Vujčić, Zoran, "Detection and quantification of leucyl arninopeptidase after native electrophoresis using leucine-p-nitroanilide" in Electrophoresis, 26, no. 12 (2005):2476-2480, https://doi.org/10.1002/elps.200500047 . .