Association Kinetics of Gene Product Fragments Derived from E. coli 1-Galactosidase

1 Association Kinetics of Gene Product Fragments derived from E. coli (1-Galactosidase 

Fluorescence correlation spectroscopy (FCS) was used to elucidate the association kinetics of lacZ fragments derived from E. coli (3-galactosidase. This example demonstrates the power of FCS to provide mechanistic information by allowing the interaction to be observed at the molecular level. In general, it demonstrates how FCS may be used as a powerful tool for kinetic measurements in the life sciences. 

The [3-galactosidase analog consists of two subunits a large dimeric polypeptide (200 kDa), denoted enzyme acceptor (EA)2, and a small polypeptide (20 kDa), denoted enzyme donor (ED). (EA)2 and ED are enzymatically inactive but spontaneously associate to give enzymatically active tetramers. In CEDIA assays, [45,46] the hapten or analyte is covalently linked to the ED, and an analyte-specific antibody is used to inhibit the assembly of the enzymatically active tetramers. Analyte in a patient s serum competes with the analyte in the analyte-ED conjugate for antibody, modulating the amount of active B- 

A detailed study of association was performed in order to elucidate the reaction mechanism prior to enzymatic conversion of the substrate. The data give strong evidence for a fast pre-equilibrium state followed by a slower association into a stable complex. De- 

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