| Assay Method Information | |
| | sPLA2V Activity Assay |
| Description: | Briefly, sn-2ester bond of the substrate 1,2-bis(heptanoylthio)-glycerophosphocholine was hydrolyzed by PLA2-V followed by the exposure of free thiols. These thiols activated the conversion of DTNB (5,5–dithio-bis-(2-nitrobenzoic acid) to 2-nitro-5-thiobenzoic acid, which was photometrically detected at 405 nm. After this, the assay proceeded in an aqueous buffer solution (pH 7.5) containing KCl (94 mM), CaCl2 (9 mM), Tris (24 mM), and Triton-X 100 (280 BM). Before the assay was started, substrate and PLA2-V was resuspended in assay buffer whereas DTNB was dissolved in an aqueous solution of Tris-HCl (pH 8) yielding an enzyme and DTNB concentrations of 100 ng/mL and 87 BM, respectively. 96-well micro titre plates containing substrate solution, DTNB, and the respective test substance were used to carry out assays at room temperature. |
| Affinity data for this assay | |
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