Real-time spectrophotometric microplate assay for galactolipase activity using synthetic or natural galactolipids in...

Galactolipases catalyze the hydrolysis of galactolipids, a major class of membrane lipids in photosynthetic organisms and an important dietary source of polyunsaturated fatty acids. Quantitative determination of galactolipase activity remains analytically challenging because galactolipids are amphiphilic molecules and catalytic rates strongly depend on interfacial organization within lipid aggregates. Here we describe a real-time spectrophotometric microplate assay for galactolipase activity based on synthetic medium chain monogalactosyldiacylglycerol (C8-MGDG) and digalactosyldiacylglycerol (C8-DGDG) organized in mixed micelles with bile salts. Enzymatic hydrolysis was monitored through proton release associated with fatty acid ionization and detected using pH sensitive chromogenic indicators producing linear responses across pH values from 6.0 to 9.2. The assay displayed a well-defined linear working range, with proportional increases in reaction rate between 5.75 and 46 ng of enzyme per well. Using guinea pig pancreatic lipase-related protein 2 (GPLRP2) as a galactolipase reference standard, optimum specific activities of 1206 U mg^-1 toward MGDG and 1508 U mg^-1 toward DGDG were obtained at pH 8.2. The method discriminated against enzymes with galactolipase activity from lipases that do not hydrolyze galactolipids. The assay also enabled detection of galactolipase activity on galactolipids obtained from spinach leaves. At pH 8.2, increasing amounts of GPLRP2 produced proportional increases in activity over the tested range of 11.25-90 ng of enzyme per well, confirming that the assay maintains a linear response when applied to complex natural lipid mixtures. This method provides a versatile analytical platform for galactolipase characterization, comparative enzymatic studies, inhibitor screening, and activity profiling in biological samples.