A facile assay to monitor secretory phospholipase A(2) using 8-anilino-1-naphthalenesulfonic acid

作者：Vivek Hamse K; Swamy Supritha G; Priya Babu S; Sethi Gautam; Rangappa Kanchugarakoppal S; Swamy S Nanjunda^*

来源：Analytical Biochemistry, 2014, 461: 27-35.

DOI：10.1016/j.ab.2014.05.024

• Fluorescence property of ANS in response to sPLA(2)
• Triton X-100
• LPC
• DMPC

Secretory phospholipases A(2) (sPLA(2)s) are present in snake venoms, serum, and biological fluids of patients with various inflammatory, autoimmune and allergic disorders. Lipid mediators in the inflammatory processes have potential value for controlling phospholipid metabolism through sPLA(2) inhibition. Thus, it demands the need for screening of potential leads for sPLA(2) inhibition. To date, sPLA(2) activity has been assayed using expensive radioactive or chromogenic substrates, thereby limiting a large number of assays. In this study, a simple and sensitive NanoDrop assay was developed using non-fluorogenic and non-chromogenic phospholipid substrate 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 8-anilino-l-naphthalenesulfonic acid (ANS) as interfacial hydrophobic probe. The modified assay required a long concentration of sPLA(2). ANS, as a strong anion, binds predominantly to cationic group of choline head of DMPC through ion pair formation, imparting hydrophobicity and lipophilicity and resulting in an increase in fluorescence. Triton X-100 imparts correct geometrical space during sPLA(2) catalyzing DMPC, releasing lysophospholipid and acidic myristoyl acid, which in turn alters the hydrophobic environment prevailing around ANS-DMPC, which leads to weakening of the electrostatic ion pair interaction between DMPC and ANS ensuing decrease in fluorescence. These characteristic fluorescence changes between DMPC and ANS in response to sPLA(2) catalysis are well documented and validated in this study.

• 出版日期2014-9-15