A quick, easily integrated technique for testing enzyme substrate specificity
22 Apr 2009A new applications note from Paraytec describes how the ActiPix D-100 UV Area Imaging detector provides a novel technique for testing biocatalyst substrate specificity towards a mixture of UV active compounds using a continuous engagement electrophoretically mediated microanalysis (EMMA) assay method.
In the described application - a plug of the substrates was injected into a fused silica capillary column containing the background electrolyte and dissolved enzyme. Using a capillary electrophoresis instrument with the ActiPix D-100, the components are seen to be separated in the initial part of the run, avoiding competitive binding and inhibition problems that occur in standard enzyme assays.
Experimental data is shown that demonstrates how the EMMA assay using the ActiPix and a silica capillary with 3 loops uniquely enables multi-component substrate specificity analysis for the tyramine oxidase enzyme. The peaks corresponding to each of the separated components were measured at multiple time points along the looped capillary, providing intrinsic self-referencing and enabling ready identification of tyramine and 2-phenylethylamine as the only reactive components. The new method provides a quick, easily integrated technique for assessing enzyme-substrate specificity using a multi-compound mixture of substrates and had the potential to be broadly applicable to other UV active compounds. The method only requires minimal amounts of reagent, making it perfectly suited for evaluating enzyme specificity even if only nanolitre amounts of enzyme are available.
A copy of the Application Note (AN002) can be downloaded by following the company article webpage link on the right hand side of the screen.
The ActiPix D100 is the world's first quantitative UV area imaging system. The miniature detector contains a high resolution 1280x1024 Active Pixel sensor. Compact in design, the ActiPix D100 consists of a control box connected via a fibre optic cable and communications cable to a remote sensor head. The sensor head holds easily exchangeable, application-specific cartridges. The cartridges are used in conjunction with powerful software, enabling the end-user to easily switch between applications. The detector can be used as a 'plug and play' accessory linked in-line to single or multiple peripheral devices, such as syringe pumps, existing CE or nanoLC instrumentation. These can be further coupled to other devices such as mass spectrometers giving a greater depth of information. Detection is performed at a selected wavelength by means of interchangeable filters. The detector wavelength range is 190 to 1100 nm.
Related Products
Request Quote for All Products
ActiPix™ D100 UV Area Imaging System
Paraytec LtdNow available The Paraytec ActiPix D100 UV imaging detector is now available for purchase. This keenly awaited product is set to transform UV-Vis detection technology with its increased sensitivity, minute sample requirements and wide dynamic range - unlocking completely new applications for UV-Vis absorbance detection and greatly enhancing existing applications. Key Product Features - Miniature UV detector that can be placed in combination with existing lab hardware, e.g. LC-MS - Capillary-based detection, in-line or discrete allowing sub-microlitre sample volume - High spatial resolution - Five orders of magnitude linear dynamic range - Powerful active pixel sensor provides real-time images - Can be used down to 190 nm in the UV Description The Paraytec ActiPix D100 is a miniature capillary UV detector, designed for on-line monitoring at a single UV wavelength, particularly for use with the separation methods of liquid chromatography and capillary electrophoresis. The ActiPix D100 consists of a control box and a small remote sensor head containing a capillary cartridge. The remote head is designed for positioning exactly at your required detection point, and can be literally clipped in to an analysis stream. Its small size (approx 5x7x7cm) means that it is ideal for in-line detection with LC, nano-LC, LC-MS and CE. The miniature size allows the detector to be used as a ‘clip-on’ accessory linked to existing separations instrumentation and in line with a mass spectrometer. Detection is performed at a selected wavelength by means of exchangeable filters. Light is delivered via a fibre optic coupled light source. Readout of signals on the active pixel sensor and absorbance calculations are carried out at MHz rates, with all processing done in hardware on custom electronic assemblies. Processed data is output in real-time to computer using an industry-standard high-speed serial data link. Specifications - Wavelengths: Single wavelength filters selectable in range 190 – 1100 nm - Bandpass: 10 nm - Baseline noise in absorbance: 5 µAU - Linear dynamic range: 5 orders of magnitude: 5 µAU – 0.5 AU - Peptide limit of quantification: 100 ng / ml (190 nm) - Spatial resolution: 70 µm - Tolerance for eluents: Useable with all standard eluents (methanol with 1 M acetic acid etc.) over full range down to 190 nm - Sensor: CMOS Active Pixel Sensor - Size of detector: Approx 5x7x7 cm Features Uses fused silica capillary - Goes down to 190 nm in UV - Enables low volumes of sample to be analysed discretely or in a continuous stream Patented signal referencing technology - Highly sensitive - Quantitate peptide at 100 pg per µL at 190 nm Over five orders of magnitude linear dynamic range - 5 µAU – 0.5 AU - Reduces the need for dilution - Enables minor components and major components to be quantified simultaneously High spatial resolution - Can resolve peaks in complex mixtures, e.g. proteomics and µUPLC Real time monitoring of velocity - Enables integration with process control, e.g. high throughput fraction collection Miniature detector - Enables placement of one or more detectors in a process stream, e.g. high throughput purification, HPLC, MS, UPLC, monitoring of components in a column