A Sensitive and Continuous Fluorometric Activity Assay Using a Natural Substrate

Abstract

Calpain activity can be assayed by a variety of methods which almost invariably rely on measuring the liberation of trichloroacetic acid (TCA)-soluble peptides from a protein substrate, usually casein. The soluble peptides are then measured either by their absorption at 280 nm, or by their radioactivity if the casein was radioactively labeled. The major difficulty in this type of assay is that the small peptides need to be separated from partially digested or undigested casein, which makes the assays tedious, and precludes continuous monitoring of activity. A continuous assay is possible with fluorescent peptide substrates (1), but these are relatively poor substrates for calpain so that such an assay is insensitive. A continuous and sensitive assay, suitable therefore also for kinetic studies, has been developed, using MAP2-DTAF as substrate (5-[4,6-dichlorotriazin-2-yl amino])-fluorescein[DTAF]-labeledmicrotubule-associated protein 2 [MAP2] (2). The assay depends upon the observation that release of small peptides carrying only one fluorescent label, by digestion of a protein labeled with many fluorescent groups, is accompanied by a significant increase in fluorescence of the solution, without any need for separation. Detailed consideration of the conditions and kinetics of the assay is given in (3), but the assay is most useful in the range of 10–180 ng of calpain, and is therefore 10²–10³-fold more sensitive than the commonly used calpain assays.