Dual-Channel Stopped-Flow Apparatus for Simultaneous Fluorescence, Anisotropy, and FRET Kinetic Data Acquisition for Binary and Ternary Biological Complexes.

The Stopped-Flow apparatus (SF) tracks molecular events by mixing the reactants in sub-millisecond regimes. The reaction of intrinsically or extrinsically labeled biomolecules can be monitored by recording the fluorescence, F ( t ), anisotropy, r ( t ), polarization, p ( t ), or FRET, F ( t ) FRET , traces at nanomolar concentrations. These kinetic measurements are critical to elucidate reaction mechanisms, structural information, and even thermodynamics. In a single detector SF, or L-configuration, the r ( t ), p ( t ), and F ( t ) traces are acquired by switching the orientation of the emission polarizer to collect the IVV and IVH signals however it requires two-shot experiments. In a two-detector SF, or T-configuration, these traces are collected in a single-shot experiment, but it increases the apparatus' complexity and price. Herein, we present a single-detector dual-channel SF to obtain the F ( t ) and r ( t ) traces simultaneously, in which a photo-elastic modulator oscillates by 90° the excitation light plane at a 50 kHz frequency, and the emission signal is processed by a set of electronic filters that split it into the r ( t ) and F ( t ) analog signals that are digitized and stored into separated spreadsheets by a custom-tailored instrument control software. We evaluated the association kinetics of binary and ternary biological complexes acquired with our dual-channel SF and the traditional methods; such as a single polarizer at the magic angle to acquire F ( t ), a set of polarizers to track F ( t ), and r ( t ), and by energy transfer quenching, F ( t ) FRET . Our dual-channel SF economized labeled material and yielded rate constants in excellent agreement with the traditional methods.