Comparison of Fluorescence Lifetime and Fluorescence Intensity Readouts using a Homogeneous Protease-assay

Introduction

In this work two measurement parameters are compared, the fluorescence intensity (FI) and fluorescence lifetime (FLT), which could be acquired efficiently in a multi parameter readout from one and the same measurement.

The biochemical reaction being investigated consists of a fluorescent labelled enzyme-substrate (case in as a biopolymer carrying BODIPY FL), which is cleaved by the protease a-chymotrypsin.

In the beginning of the assay the fluorescence signal is internally quenched as a result of energy transfer interactions among the BODIPY labels. The continuous fragmentation of the biopolymer backbone in consequence of enzymatic degradation leads to a rise of the fluorescence signal while the quenching effects decrease. In the assay this is expressed in an increase of both, intensity and lifetime.