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

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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 (~5 μL) 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 μm. 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 FTIR stopped flow system is presented, including a demonstration of the alkaline conformational transition of cytochrome c as an example.

Keywords: CYTOCHROME C; FOURIER TRANSFORM INFRARED SPECTROSCOPY; FT-IR SPECTROSCOPY; MICRO-SYSTEM TECHNOLOGY; STOPPED FLOW; TIME-RESOLVED SPECTROSCOPY; TRANSMISSION SPECTROSCOPY

Document Type: Research Article

DOI: http://dx.doi.org/10.1366/000370203322554581

Publication date: November 1, 2003

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