title: A facile assay to monitor secretory phospholipase A₂ using 8-anilino-1-naphthalenesulfonic acid.
abstract: Secretory phospholipases A2 (sPLA2s) 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 sPLA2 inhibition. Thus, it demands the need for screening of potential leads for sPLA2 inhibition. To date, sPLA2 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-1-naphthalenesulfonic acid (ANS) as interfacial hydrophobic probe. The modified assay required a 10ng concentration of sPLA2. 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 sPLA2 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 sPLA2 catalysis are well documented and validated in this study.
authors.affiliation: Department of Biotechnology, Sri Jayachamarajendra College of Engineering, Mysore 570006, India.
authors.forename: Hamse K
authors.initials: HK
authors.lastname: Vivek
authors.affiliation: Department of Biotechnology, Sri Jayachamarajendra College of Engineering, Mysore 570006, India.
authors.forename: Supritha G
authors.initials: SG
authors.lastname: Swamy
authors.affiliation: Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570006, India.
authors.forename: Babu S
authors.initials: BS
authors.lastname: Priya
authors.affiliation: Department of Pharmacology, National University of Singapore, Singapore 117597, Republic of Singapore.
authors.forename: Gautam
authors.initials: G
authors.lastname: Sethi
authors.affiliation: Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570006, India.
authors.forename: Kanchugarakoppal S
authors.initials: KS
authors.lastname: Rangappa
authors.affiliation: Department of Biotechnology, Sri Jayachamarajendra College of Engineering, Mysore 570006, India. Electronic address: nanju_chem@yahoo.com.
authors.forename: S Nanjunda
authors.initials: SN
authors.lastname: Swamy
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002138:Calibration; D003840:Deoxycholic Acid / Q000737:chemistry; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry / Q000378:metabolism; D017815:Elapidae; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008823:Micelles; D017830:Octoxynol / Q000737:chemistry; D054497:Phospholipases A2, Secretory / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011794:Quercetin / Q000494:pharmacology; D017840:Daboia; D013050:Spectrometry, Fluorescence
chemical_list: C515594:8-anilino-1-naphthalenesulfonic acid; D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D008823:Micelles; D003840:Deoxycholic Acid; D017830:Octoxynol; D011794:Quercetin; D054497:Phospholipases A2, Secretory; D002118:Calcium; D004134:Dimyristoylphosphatidylcholine
keywords: DMPC; Fluorescence property of ANS in response to sPLA(2); LPC; Triton X-100