Thursday, May 9, 2013

Applications Thursday: Monitoring transglutaminase activity with a fluorescence assay

Transglutaminases are group of enzymes that are especially important in protein cross-linking that is crucial for tissue homeostasis. Regulation of these enzymes appears to be necessary as human diseases have been linked to either the lack of or excessive cross-linking.

Structure of quenched substrate

In BMG LABTECH application note 234 entitled 'Real-time fluorescence assay for monitoring transglutaminase activity' we describe an assay which is amenable to high throughput analysis to screen for regulators and inhibitors of transglutaminase activity. The application exploits the fact that in the presence of excess cross-linked substrate transglutaminases catalyze isopeptide bond hydrolysis. A quenched fluorescent probe that mimics a crosslinked transglutaminase reaction product is used; such that when transglutaminase is active, hydrolysis of the isopeptide bond leads to release of the quencher and an increase in light emission, which can be detected in a BMG microplate reader. For this application note the FLUOstar OPTIMA and Omega were used.

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