title: 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.
authors.affiliation: Micro-biolytics GmbH, Georges Koehler Allee 102, D-79110 Freiburg, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Masuch
authors.forename: David A
authors.initials: DA
authors.lastname: Moss
mesh_terms: D001704:Biopolymers / Q000032:analysis / Q000737:chemistry; D045304:Cytochromes c / Q000032:analysis* / Q000737:chemistry*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D017022:Flow Injection Analysis / Q000295:instrumentation* / Q000379:methods; D046911:Macromolecular Substances; D044085:Microfluidics / Q000295:instrumentation* / Q000379:methods; D009038:Motion; D011487:Protein Conformation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017550:Spectroscopy, Fourier Transform Infrared / Q000295:instrumentation* / Q000379:methods; D014867:Water / Q000737:chemistry*
chemical_list: D001704:Biopolymers; D046911:Macromolecular Substances; D014867:Water; D045304:Cytochromes c