Direct determination of phospholipase D activity by infrared spectroscopy

To determine phospholipase D (PLD) activity, an infrared spectroscopy assay was developed, based on the phosphate vibrational mode of phospholipids such as dimyristoylphophatidylcholine (DMPC), lysophosphatidylglycerol (lysoPG), dipalmitoylphosphatidylethanolamine (DPPE), and lysophosphatidylserine (lysoPS). The phosphate bands served to monitor the hydrolysis rates of phospholipids with PLD. The measurements could be performed within less than 20 min with 10 μl of buffer containing 2 to 40 mM DMPC and 10 to 200 ng of Streptomyces chromofuscus PLD (corresponding to 350–7000 pmol of DMPC hydrolyzed per minute). The limit of sensitivity was approximately 10 ng of PLD at 100 mM Tris–HCl (pH 8.0) with 10 mM Ca2+ and 2.5 mg ml−1 Triton X-100. Reproducible specific activity of PLD (35 ± 5 nmol of hydrolyzed DMPC min−1 μg−1 PLD) measured by the infrared assay remained stable over 50 to 200 ng of PLD and over 5 to 40 mM DMPC. The feasibility of this assay to determine the hydrolysis rate of other phospholipids such as lysoPG, DPPE, and lysoPS was confirmed. The IC50 of cobalt (800 ± 200 μM), a known S. chromofuscus PLD inhibitor, was measured by means of the infrared assay, demonstrating that this assay can be used to screen PLD activity and/or the specificity of its inhibitors.