Detection and quantification of leucyl aminopeptidase after native electrophoresis using leucine-p-nitroanilide.
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.
Bozić
Natasa
N
Center of Chemistry, Institute of Chemistry, Technology and Metallurgy, Serbia and Montenegro. n.bozic@eunet.yu
Vujcić
Zoran
Z
D000813:Anilides / Q000378:metabolism*; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007527:Isoenzymes / Q000032:analysis / Q000302:isolation & purification; D007668:Kidney / Q000201:enzymology; D007931:Leucyl Aminopeptidase / Q000032:analysis* / Q000302:isolation & purification; D027805:Phaseolus / Q000201:enzymology; D015203:Reproducibility of Results; D012639:Seeds / Q000201:enzymology; D013552:Swine
D000813:Anilides; D007527:Isoenzymes; C001536:1-leucine-4-nitroanilide; D007931:Leucyl Aminopeptidase