Abstract
A new continuous fluorescence-displacement assay for enzymes that release long-chain fatty acids [Wilton (1990) Biochem. J. 266, 435-439] is described in detail for pig pancreatic triacylglycerol lipase. The assay involves the displacement of the highly fluorescent fatty acid probe 11-(dansylamino)undecanoic acid from rat liver fatty acid-binding protein by long-chain fatty acids released as a result of enzyme activity. The assay is surprisingly effective for triacylglycerol lipase, allowing the expression of full activity with low concentrations of substrates in the absence of detergents. The initial rate of decrease in fluorescence is linearly related to enzyme concentration, and activity can be detected in the assay down to concentrations of 10 pg of pure enzyme/ml. The assays demonstrated the quantitative conversion of limiting amounts of substrate into the monacylglycerol. This observation allowed the assay to be used to measure substrates such as triacylglycerols and particularly 1,2-diacylglycerols at concentrations down to about 0.1 microM. Because phospholipase C releases 1,2-diacylglycerols, the coupling of this enzyme to excess lipase allowed the measurement of pure phospholipase C from Bacillus cereus at concentrations down to about 2 ng/ml, and the initial rate of fall in fluorescence in the assay was linearly related to enzyme activity.
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