An evaluation of NBD-phospholipids as substrates for the measurement of phospholipase and lipase activities

Abstract

Because most of the existing assays of phospholipase activity are quite laborious, the use of 1-acyl-2-[6-(7-nitro-1,3-benzoxadiazol-4-yl)amino]caproyl labeled phospholipids (NBD phospholipids) was investigated to determine whether they could be used as substrates in the routine assay of various phospholipases and lipases. NBD-labeled analogues of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidic acid were evaluated. There was about a 50-fold increase in fluorescence upon hydrolysis of the NBD hexanoic acid from the NBD phospholipid, confirming an earlier report. This change in fluorescence was constant over the normal physiological pH range (pH 5–9). Detergents and bovine serum albumin interfered with the assay in a concentration dependent manner. An increase in fluorescence and a concomitant increase in NBD hexanoic acid was detected with the two phospholipase A₂ enzymes. Although a change in fluorescence was detected with a phospholipase C, careful evaluation revealed that the rate of increase in fluorescence was not proportional to the rate of production of diacylglycerol product. Neither of the two phospholipase D enzymes which were tested were able to cause an increase in fluorescence when incubated with NBD phospholipids. A small increase in fluorescence was detected with each of the four lipases. Of the five NBD lipids tested, the highest rates of hydrolysis were consistently obtained with NBD-phosphatidylglycerol followed by NBD-phosphatidylcholine.