Reaction monitoring of enzyme-catalyzed ester cleavage by time-resolved fluorescence and electrospray mass spectrometry: method development and comparison

Abstract

Two complementary methods for reaction monitoring of the esterase-catalyzed cleavage of bis(2-pyridylmethyl)(2-acetoxyphenyl)amine are developed and compared. While enzyme-amplified lanthanide luminescence (EALL) allows for the time-resolved fluorescence determination of the intrinsically non-fluorescent product, both substrate and product of the enzymatic reaction may be determined simultaneously by electrospray mass spectrometry (ESI-MS). Excitation wavelength for the Tb(III) complex of the reaction product is 297 nm and emission was detected at 545 nm, which is the characteristic emission wavelength of the terbium(III) ion. In contrast to other EALL techniques, the presented method allows for the direct monitoring of an enzymatic conversion without any further sample preparation (e.g., rebuffering). For the mass spectrometric measurements the mass traces were set to m/z=306, 328, 348, and 370 for the protonated ester, the resulting phenol and their sodium adducts, respectively.