A continuous spectrophotometric assay for phospholipase A(2) activity.
This paper describes a simple continuous spectrophotometric method for assaying phospholipase A(2) (PLA(2)) activity. The procedure is based on a coupled enzymatic assay, using dilinoleoyl phosphatidylcholine as phospholipase substrate and lipoxygenase as coupling enzyme. The linoleic acid released by phospholipase was oxidized by lipoxygenase and then phospholipase activity was followed spectrophotometrically by measuring the increase in absorbance at 234 nm due to the formation of the corresponding hydroperoxide from the linoleic acid. The optimal assay concentrations of hog pancreatic phospholipase A(2) and lipoxygenase were established. PLA(2) activity varied with pH, reaching its optimal value at pH 8.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3mM. Phospholipid hydrolysis followed classical Michaelis-Menten kinetics (V(m)=1.8 microM/min, K(m)=4.5 microM, V(m)/K(m)=0.4 min(-1)). This assay also allows PLA(2) inhibitors, such as p-bromophenacyl bromide or dehydroabietylamine acetate, to be studied. This method was proved to be specific since there was no activity in the absence of phospholipase A(2). It also has the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase A(2).
Jiménez
M
M
Departamento de Bioquímica y Biología Molecular A, Universidad de Murcia, Espinardo 30071, Murcia, Spain. mja@um.es
Cabanes
J
J
Gandía-Herrero
F
F
Escribano
J
J
García-Carmona
F
F
Pérez-Gilabert
M
M
D003840:Deoxycholic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010741:Phospholipases A / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013053:Spectrophotometry / Q000379:methods*
D003840:Deoxycholic Acid; D010741:Phospholipases A