[3] Nuclear magnetic resonance spectroscopy to follow phospholipase kinetics and products

Abstract

Nuclear magnetic resonance (NMR) spectroscopy is a useful technique for monitoring the kinetics of phospholipase action and the physical state of substrates and products produced. High-resolution 1H, 13C, and 31P NMR studies have been used to characterize lipid structure and dynamics in micelles and small unilamellar vesicles. NMR spectroscopic methods can also be used to monitor hydrolysis of lipids in different aggregates by phospholipases. The NMR methodology provides additional information on substrate and product structure and dynamics. The major advantage of NMR methods is that it changes in the substrate physical state as a function of enzyme action can be monitored. Different nuclei are sensitive to different parameters, and the choice of nucleus observed depends on the nature of the aggregate system and the phospholipase reaction to be studied. 13C NMR using enriched phosphatidylcholine (PC) is excellent for assaying the ester bond specificity of a phospholipase and 31P NMR is a better choice for enzyme action on mixed phospholipids in micellar systems or for phospholipase C where one of the products is water soluble. NMR spectroscopy is useful for mammalian phospholipases if reasonable substrate system is chosen. With the right nucleus, buffers and detergents may be present however they do not hinder observation of the desired resonances.