In vitro characterization of an in situ microdialysis sampling assay for elastase activity detection.

ABSTRACT

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.