REAL-TIME ENZYME-KINETICS MONITORED BY DUAL-COLOR FLUORESCENCE CROSS-CORRELATION SPECTROSCOPY

A method for sensitively monitoring enzyme kinetics and activities by using dual color fluorescence crosscorrelation spectroscopy is describ ed, This universal method enables the development of highly sensitive and precise assays for real-time kinetic analyses of any catalyzed cle avage or addition reaction, where a chemical linkage is formed or clea ved through an enzyme's action between two fluorophores that can be di scriminated spectrally, In this work a homogeneous assay with restrict ion endonuclease EcoRI and a 66-bp double-stranded DNA containing the GAATTC recognition site and fluorophores at each 5' end is described. The enzyme activity can be quantified down to the low picomolar range (>1.6 pM) where the rate constants are linearly dependent on the enzym e concentrations over two orders of magnitude. Furthermore, the reacti ons were monitored online at various initial substrate concentrations in the nanomolar range, and the reaction rates were clearly represente d by the Michaelis-Menten equation with a K-M of 14 +/- 1 nM and a k(c at) of 4.6 +/- 0.2 min(-1). In addition to kinetic studies and activit y determinations, it is proposed that enzyme assays based on the dual- color fluorescence cross-correlation spectroscopy will be very useful for high-throughput screening and evolutionary biotechnology.