Electrophoretically mediated microanalysis assay for sirtuin enzymes

Abstract

An electrophoretically mediated microanalysis (EMMA) assay for the human sirtuin SIRT1 has been developed using 9-fluorenylmethoxycarbonyl (Fmoc)-labeled peptides, i.e. Fmoc-KK(Ac)-NH(2), Fmoc-KK(Ac)L-NH(2) and Fmoc-RHKK(Ac)-NH(2), as substrates. The partial filling mode was applied due to the incompatibility between the incubation buffer, pH 8.0, and the BGE that had a pH of 2.7 or 2.3 depending on the analytes. Incubation and subsequent analyte separation were carried out in a 37/30 cm, 50 μm id fused-silica capillary at 37°C. An injection sequence of incubation buffer, enzyme, substrate, enzyme and incubation buffer was selected because the electrophoretic mobility of SIRT1 was not known. The assay was optimized with regard to the length of the injected plugs, the mixing voltage and mixing time as well as the activity (concentration) of SIRT1. The EMMA assay was subsequently applied to the determination of the Michaelis-Menten constants, K(m), and the maximum velocity, V(max), as well as the determination of the inhibitory constants, IC(50), of inhibitors. Data obtained with the in-capillary assay were in accordance with the literature data or an offline SIRT1 assay.