Testing Enzyme Substrate Specificity

A 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 three 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 enzymesubstrate 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.