Stopped-Flow Application Notes

SX 20 Enzymes Teaser

Determination of Enzyme Kinetics Using Stopped-Flow Spectroscopy

This study describes the use of an SX20 spectrometer to measure the presteady state kinetics of a well-studied enzymatic reaction: hydrolysis of p-Nitrophenyl acetate catalysed by α-chymotrypsin. Kinetic parameters such as rate constants and the Michaelis-Menten constant are evaluated.

Since it was first studied over 50 years ago, extensive research has established the detailed mechanism of this reaction.

SX20 FP Teaser

Stopped-Flow Fluorescence Polarisation/Anisotropy

Stopped-flow fluorescence polarisation/anisotropy is a highly useful technique for obtaining valuable kinetic information about biomolecular interactions and structural changes of biomolecules. An example from the literature illustrates how the SX20 Fluorescence Polarisation accessory is used to gain insights into the kinetics of the interaction between a helicase and its RNA binding partner.

SX20 FRET Teaser

Stopped-Flow Fluorescence (Förster) Resonance Energy Transfer (FRET)

Stopped-flow FRET is a useful technique for studying many types of biomolecular interactions. The method provides kinetic information regarding distance changes occurring on the millisecond timescale. This application note gives an introduction to the technique along with a review of some application experiments from the scientific literature.

SX20 PDA Teaser

High Sensitivity Direct Mount Photodiode Array for the SX20

The directly mounted photodiode array accessory ensures highly efficient light collection for stopped-flow absorbance measurements. The improved design ensures that ultimate sensitivity is achieved even at levels that maybe needed to study species that exhibit photobleaching. As an example of the many applications of this accessory, this application note demonstrates its use to measure the kinetics of an inorganic ligand exchange reaction.

SF3 Teaser

Shining Light on Non-Chemical Steps in Protein Catalysis

Stopped-flow experiments using Applied Photophysics SX20 and Chirascan V100 SF.3 systems allow monitoring of different spectral properties including fluorescence, absorbance, and even far-and near-UV CD.With these tools. This study outlines the transition between fully folded and locally unfolded states of Tsa1, in its sulfenic acid form CP-SOH, is not in rapid equilibrium as previously hypothesised, but is almost irreversible (kFF~0s-1)and in direct competition with the hyperoxidation step.

Sulfonamides Teaser

Determination of Species-Specific Rate Constants of the Reaction Between Ferrate(VI) and Sulfonamide Antibiotics

Applied Photophysics SX20 stopped-flow spectrometer can be used to study the species-specific rate constants of the reactions between FeVI and SAs. Values of k at pH 7.0 and 8.0 were used in this study, to learn the half-lives of the elimination of SAs by FeVI. For the practical application of this work, if FeVI concentration is in excess at pH 8.0 and 25.0 °C then the removal of SAs would be short and in the range of 12.52 – 361.20 s depending on the SA species. Due to the dependence of this oxidation reaction on FeVI concentration and pH, application conditions could be fine tuned to majorly reduce time spent in the removal of SAs

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