In vitro characterization of an in situ microdialysis sampling assay for elastase activity detection.
A microdialysis sampling method has been developed to detect the in vitro presence of a proteolytic enzyme, porcine elastase, external to a microdialysis probe. Elastase converts the substrate, succinyl(Ala)(3)-p-nitroanilide (suc(Ala)(3)-p-NA), to p-nitroaniline (p-NA). The substrate, suc(Ala)(3)-p-NA, was locally delivered through the microdialysis probe to external solutions containing different elastase activities (0.025-0.5 units/mL). The product, p-NA, was recovered back into the probe. Dialysates containing both suc(Ala)(3)-p-NA and p-NA were quantified using HPLC-UV. Different microdialysis suc(Ala)(3)-p-NA extraction efficiencies (EE) were observed among different elastase-containing solutions (buffer and 0.3% agar solutions). The p-NA concentrations recovered back into the microdialysis probe correlated with the elastase activity external to the microdialysis probe. The greatest fraction of p-NA recovered as compared to substrate lost occurred with the highest flow rate used (5.0 microL/min). However, the highest concentrations of p-NA recovered occurred at the lowest flow rates. This method may allow for microdialysis sampling to be used as a means to study localized enzyme activity.
Steuerwald
Amy J
AJ
Department of Chemistry and Chemical Biology, Cogswell Laboratories, Rensselaer Polytechnic Institute, Troy, NY 12180-3590, USA.
Villeneuve
Joshua D
JD
Sun
Li
L
Stenken
Julie A
JA
D000362:Agar; D000814:Aniline Compounds / Q000737:chemistry / Q000378:metabolism; D002021:Buffers; D002851:Chromatography, High Pressure Liquid; D007202:Indicators and Reagents; D007700:Kinetics; D017551:Microdialysis; D009842:Oligopeptides / Q000737:chemistry; D010196:Pancreatic Elastase / Q000032:analysis* / Q000378:metabolism*; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet
D000814:Aniline Compounds; D002021:Buffers; D007202:Indicators and Reagents; D009842:Oligopeptides; C008625:nitroaniline; C015516:succinyl-trialanine-4-nitroanilide; D000362:Agar; D010196:Pancreatic Elastase