Stopped Flow Apparatus for Time-Resolved Fourier Transform Infrared Difference Spectroscopy of Biological Macromolecules in ¹H₂O

Abstract

Stopped flow spectroscopy is an established technique for acquiring kinetic data on dynamic processes in chemical and biochemical reactions, and Fourier transform infrared (FT-IR) techniques can provide particularly rich structural information on biological macromolecules. However, it is a considerable challenge to design an FT-IR stopped flow system with an optical path length low enough for work with aqueous (1H2O) solutions. The system presented here is designed for minimal sample volumes (approximately 5 microL) and allows simultaneous FT-IR rapid-scan and VIS measurements. The system employs a micro-structured diffusional mixer to achieve effective mixing on the millisecond time scale under moderate flow and pressure conditions, allowing measurements in a cell path length of less than 10 microns. This makes it possible to record spectra in 1H2O solutions over a wide spectral range. The system layout is also designed for a combination of kinetic and static measurements, in particular to obtain detailed information on the faster spectral changes occurring during the system dead time. A detailed characterization of the FT-IR stopped flow system is presented, including a demonstration of the alkaline conformational transition of cytochrome c as an example.

Proteins in Action Monitored by Time‐Resolved FTIR Spectroscopy

“…This can be achieved in a stopped flow setup where the two components are mixed and transferred into the sample chamber. [32] Even faster mixing is possible using continuous flow systems. We have developed a micro mixing cell (Figure 2 C), which allows mixing of two components within the sub-millisecond time regime.…”

Using mid infrared technology as new method for the determination of the dwell time of salivary substitutes on three dimensional gingiva models

“…The washing solutions were directly analysed using an AquaSpec™ MIRA-LAB LHS-500analyser. AquaSpec™ spectroscopy is based on mid infrared spectroscopy in a spectral range of 3001–951 cm −1 [ 18 ]. Absorption spectra and PCA analysis were used for evaluation.…”

“…Mid infrared (MIR) spectroscopy is mainly used for the analysis of proteins and lipids [ 15 – 17 ]. However, the development of new flow-through cells with pathlength of <10 μm and small sample volumes optimized for the measurement of aqueous solutions [ 18 ] enables new applications as it is a fast method for the investigation of molecules in their native state in aqueous solutions very sensitively. With this method even traces of molecules can be detected.…”

Infrared Difference Spectroscopy as a Physical Tool to Study Biomolecules

“…Difference spectra can be calculated from spectra recorded before and after the illumination. Ligand release technique: Various ways are used to release the biomolecules of interest such as diffusion of substrates through a dialysis membrane (92)(93)(94), infrared variants of the stopped-flow and continuous-flow techniques (95,96). Voltage change: This method is used to initiate redox reactions of proteins by applying a potential step at the electrode in the electrochemical cell (97).…”