Assay of snake venom phospholipase A2 using scattering mode of a spectrofluorimeter

When aggregated micelles of phospholipids; are hydrolysed by phospholipase A(2) (PLA(2)) in an aqueous dispersion, scattering from the solution is decreased. Hydrolysis of dimyristoyl phosphatidylcholine by PLA(2) from Russell's viper venom has been investigated using a spectrofluorimeter at 650 urn. The rate of decrease in scattering was linearly dependent with venom concentration, while the initial lag at the onset was inversely related to it. Similar dependency was observed with substrate concentration. The reaction was inhibited with venom preincubated with antivenom or withdrawal of Ca+2 by EGTA. Gas-liquid chromatography of the product showed liberation of myristic acid. The amount of fatty acid released by 1 mg of venom was found to be 3470 and 3680 nmol/min, using scattering mode and pH-stat titrimetric assay respectively, that indicated a good correlation between them. The sensitivity of detection by the scattering mode was double that of the titrimetric assay.