An improved spectrophotometric phospholipase A2 assay using 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine as substrate and lipoxygenase as coupled enzyme

• Published in: Applied biological chemistry Vol. 56; no. 4; pp. 369 - 376
• Main Authors: Soccio, Mario, Trono, Daniela, Laus, Maura N., Pastore, Donato
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01.08.2013; Springer Nature B.V; 한국응용생명화학회
• Subjects: Absorbance; Applied Microbiology; Assaying; Biological Techniques; Bioorganic Chemistry; Chemistry; Chemistry and Materials Science; Enzymatic activity; Enzyme activity; Enzymes; Ethanol; Lecithin; Lipoxygenase; Liquid oxygen; LOX-1 protein; Lysophosphatidylcholine; Original Article; Phosphatidylcholine; Phosphatidylethanolamine; Phospholipase; Phospholipase A2; Phospholipids; Reaction kinetics; Reaction products; Soybeans; Spectrophotometry; Substrates; Venom; 농학; spectrophotometric assay; lipoxygenase; enzymatic assay; honey bee venom phospholipase A; phosphatidylcholine
• ISSN: 1738-2203; 2468-0834; 2234-344X; 2468-0842
• DOI: 10.1007/s13765-013-3018-8

An improved spectrophotometric assay of phospholipase A 2 (PLA 2 ) activity based on the coupled PLA 2 /lipoxygenase (LOX) reactions using 1-palmitoyl-2-linoleoyl- sn -glycero-3-phosphatidylcholine (PC LIN ) as substrate is reported. The PLA 2 -mediated release of free linoleate is continuously monitored by following the absorbance increase at 234 nm caused by its conversion into the conjugated diene hydroperoxide catalyzed by the coupled soybean LOX-1 reaction. The new protocol includes the use of Tween 20 (3 μL/μmol phospholipid) as surfactant and of ethanol (15 μL/mL reaction mixture), that ensure clearness of reaction mixture and linear increase of absorbance in the course of reaction. This method was tested on a purified secretory PLA 2 from honey bee venom (HBV-PLA 2 ). The enzyme did not discriminate among PC LIN , phosphatidylcholine, and phosphatidylethanolamine, but showed the highest rate using 1,2-dilinoleoyl-sn-glycero-3-phosphatidylcholine (PC DILIN ). Nevertheless, the use of PC DILIN is not recommended, as it may induce an overestimation of enzyme activity, because not only the free linoleate, but also the reaction product 1-linoleoyl-lysophosphatidylcholine, are known to be oxidized by LOX. HBV-PLA 2 showed maximal activity at pH 9.0, hyperbolic kinetics (K m , 74.2±2.9 μM; V max , 827±7 μmol/min/mg protein) and competitive inhibition (K i about 5 μM) by palmityl trifluoromethyl ketone, a classical PLA 2 inhibitor. Interestingly, the HBV-PLA 2 /soybean LOX-1 coupled reactions also allow an accurate assay of PC LIN concentration. In the whole, these results demonstrate that this improved PLA 2 /LOX assay allows a very accurate, simple, and rapid measurement of enzyme activity and substrate concentration.