partition-based method for the rapid assay of phospholipases a using short chain fluorescent phospholipid analogs

ALOISE MM,; GIMENEZ G,; FLORIN-CHRISTENSEN M,; ISOLA ELD,; FLORIN-CHRISTENSEN J,

Villa Carlos Paz, Córdoba,

Otro; XXXVIII Reunión de la Sociedad Argentina de Bioquímica y Biología Molecular.; 2002

Sociedad Argentina de Bioquímica y Biología Molecular.

Direct quantitation of phospholipase A1 and A2 activity by end point assays requires separation of the reaction products from the phospholipid substrate. We here describe a fast method for this purpose that eliminates chromatographic steps, using C6-NBD phospholipid analogues. Because C6-NBD-FA and C6-NBD-lysophospholipids are highly polar, a simple partition in the system chloroform-methanol-0.1 M ammonium hydroxide   (1:1:0.9 v/v) results in the separation of the parental phospholipids from either type of reaction products in less than 30 seconds. Thus, quantitation of fluorescence in the upper phase provides a direct measurement of the amount of hydrolysis. Since the corresponding fluorescent diacylglycerol and phosphatidic acid partition in the lower phase, this method also discriminates phospholipases A from phospholipases C and D. Thus, this method provides a rapid, selective and economical assay suitable for large amounts of samples. the use of this novel assay is exemplified using trypanosomal phospholipase A1 recently descrived in our laboratory.