LACTOSE REPRESSOR-OPERATOR DNA INTERACTIONS - KINETIC-ANALYSIS BY A SURFACE-PLASMON RESONANCE BIOSENSOR

Lactose repressor binding to operator DNA and subsequent dissociation of the complex was monitored continuously by a biosensor, measuring surface plasmon resonance. In this analysis a synthetic, double-stranded oligonucleotide containing the operator site was immobilized on the sensor surface and repressor protein was passed over the surface. The formation of the repressor-operator complex was specific and could be inhibited by isopropyl-β-D-thiogalactopyranoside inducer. From the association curve, the apparent kass was determined to be 1.8 × 106 M-1 s-1. Dissociation of the complex was, for the first time for the lac repressor, determined as an uncatalyzed reaction and the kdiss was determined to be 3.4 × 10-4 s-1. As a reference, the repressor-operator interaction was analyzed by electrophoretic mobility shift assay under similar reaction conditions. With this method the equilibrium binding constant was calculated to be 2.4(±0.2) × 108 M-1. The corresponding value calculated from biosensor data was 5.1 × 109 M-1. © 1993 Academic Press, Inc.