title: A constant area monolayer method to assess optimal lipid packing for lipolysis tested with several secreted phospholipase A2.
abstract: We present an analysis of lipid monolayer hydrolysis at a constant area to assess the optimal lateral surface pressure value (Πopt) and thus, the surface packing density of the lipid, at which the activity of a given lipolytic enzyme is maximal. This isochoric method consists of a measurement of the decrease down to zero of the Πopt of phospholipid substrate monolayer due to continuous hydrolysis using only one reaction compartment. We performed the comparison of both approaches using several commercially available and literature-evaluated sPLA2s. Also, we characterized for the first time the profile of hydrolysis of DLPC monolayers catalyzed by a sPLA2 from Streptomyces violaceoruber and isoenzymes purified from Bothrops diporus venom. One of these viper venom enzymes is a new isoenzyme, partially sequenced by a mass spectrometry approach. We also included the basic myotoxin sPLA2-III from Bothrops asper. Results obtained with the isochoric method and the standard isobaric one produced quite similar values of Πopt, validating the proposal. In addition, we propose a new classification parameter, a lipolytic ratio of hydrolysis at two lateral pressures, 20 mN·m(-1) and 10 mN·m(-1), termed here as LR20/10 index. This index differentiates quite well "high surface pressure" from "low surface pressure" sPLA2s and, by extension; it can be used as a functional criterion for the quality of a certain enzyme. Also, this index could be added to the grouping systematic criteria for the superfamily proposed for phospholipase A2.
authors.affiliation: Centro de Investigaciones en Química Biológica de Córdoba, CIQUIBIC, Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina.
authors.forename: Pablo J
authors.initials: PJ
authors.lastname: Yunes Quartino
authors.affiliation: Institut Pasteur de Montevideo, Instituto de Investigaciones Biológicas Clemente Estable, Unidad de Bioquímica y Proteómica Analíticas, Uruguay.
authors.forename: Madelón
authors.initials: M
authors.lastname: Portela
authors.affiliation: Institut Pasteur de Montevideo, Instituto de Investigaciones Biológicas Clemente Estable, Unidad de Bioquímica y Proteómica Analíticas, Uruguay.
authors.forename: Analía
authors.initials: A
authors.lastname: Lima
authors.affiliation: Institut Pasteur de Montevideo, Instituto de Investigaciones Biológicas Clemente Estable, Unidad de Bioquímica y Proteómica Analíticas, Uruguay.
authors.forename: Rosario
authors.initials: R
authors.lastname: Durán
authors.affiliation: Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.
authors.forename: Bruno
authors.initials: B
authors.lastname: Lomonte
authors.affiliation: Centro de Investigaciones en Química Biológica de Córdoba, CIQUIBIC, Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina. Electronic address: gfidelio@mail.fcq.unc.edu.ar.
authors.forename: Gerardo Daniel
authors.initials: GD
authors.lastname: Fidelio
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000379:methods*; D003198:Computer Simulation; D004789:Enzyme Activation; D008066:Lipolysis; D008563:Membrane Lipids / Q000737:chemistry*; D008956:Models, Chemical; D054467:Phospholipases A2 / Q000032:analysis / Q000737:chemistry*; D053835:Unilamellar Liposomes / Q000737:chemistry*
chemical_list: D008563:Membrane Lipids; D053835:Unilamellar Liposomes; D054467:Phospholipases A2
keywords: Bothrops asper myotoxin; Bothrops diporus sPLA(2); Isochoric method; Lipid monolayer; Phospholipase A(2); Surface pressure