A new langmuir-schaefer-based method developed for catalytic studies of acetylcholinesterase in planar films of erythrocyte membranes

Autores
Felsztyna, Iván; Perillo, Maria Angelica; Clop, Eduardo Matias
Año de publicación
2017
Idioma
español castellano
Tipo de recurso
documento de conferencia
Estado
versión publicada
Descripción
Previously we reported that the catalytic activity of bovine erythrocyte acetylcholinesterase (BEA) located in Langmuir-Blodgett films (LB) of bovine erythrocyte membranes (BEM), LBBEA, depended on the curvature and packing of the molecular environment. Moreover, the specific activity of LBBEA was much lower than that of BEA in suspensions of BEM vesicles (SBEA). So, the present work was aimed at maximizing the specific activity of BEA recovered from the transfer of a Langmuir film (LF) from the air-aqueous interface to alkylated solid surfaces and improving the precision of the enzymatic assays. Three main changes were introduced to the previously assayed method. a) Phosphate saline buffer (PBS), pH 7.4 was used instead of H2O as the subphase over which was spread the BEM to form the LF, assuming that this composition, closer to physiological conditions, would be more effective than water in preserving the BEA protein structure/activity and the LF organization. b) BEA in LF films (LFBEA) was transferred from air-PBS interface to hydrophobic flat surfaces by the LangmuirSchaefer technique (LS) to obtain LSBEA samples. c) A new device was designed to allow performing the whole enzymatic activity assay using a unique LS film as well as the reading of the absorbance values in the same container. The LF of BEM at the air-PBS interface, compared with LF formed over H2O, showed surface pressure vs area (π-A) isotherms more expanded at low π, more compressible, with a bi-dimensional transition at lower π and lower minimal A. The surface potential reached 250 mV at the collapse point in both conditions (H2O and PBS). The specific activity resulted SBEA>>LSBEA>LBBEA. The use of PBS in the subphase and the transfer of LF at π=35mN/m instead of 10 mN/m improved the recovery of specific activity in LSBEA and LBBEA. The homogeneity of BEA distribution in LSBEA samples highly improved the precision of the kinetic parameters determined in different molecular packing conditions.
Fil: Felsztyna, Iván. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina
Fil: Perillo, Maria Angelica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina
Fil: Clop, Eduardo Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina
V Reunión Científica del IIByT (CONICET-UNC)
Córdoba capital
Argentina
Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones Biológicas y Tecnológicas
Universidad Nacional de Córdoba
Materia
Langmuir-Schafer
Langmuir-Blodgett
Acetylcholinesterase
Enzyme kinetics
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/245573
Acceder
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network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling A new langmuir-schaefer-based method developed for catalytic studies of acetylcholinesterase in planar films of erythrocyte membranesFelsztyna, IvánPerillo, Maria AngelicaClop, Eduardo MatiasLangmuir-SchaferLangmuir-BlodgettAcetylcholinesteraseEnzyme kineticshttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Previously we reported that the catalytic activity of bovine erythrocyte acetylcholinesterase (BEA) located in Langmuir-Blodgett films (LB) of bovine erythrocyte membranes (BEM), LBBEA, depended on the curvature and packing of the molecular environment. Moreover, the specific activity of LBBEA was much lower than that of BEA in suspensions of BEM vesicles (SBEA). So, the present work was aimed at maximizing the specific activity of BEA recovered from the transfer of a Langmuir film (LF) from the air-aqueous interface to alkylated solid surfaces and improving the precision of the enzymatic assays. Three main changes were introduced to the previously assayed method. a) Phosphate saline buffer (PBS), pH 7.4 was used instead of H2O as the subphase over which was spread the BEM to form the LF, assuming that this composition, closer to physiological conditions, would be more effective than water in preserving the BEA protein structure/activity and the LF organization. b) BEA in LF films (LFBEA) was transferred from air-PBS interface to hydrophobic flat surfaces by the LangmuirSchaefer technique (LS) to obtain LSBEA samples. c) A new device was designed to allow performing the whole enzymatic activity assay using a unique LS film as well as the reading of the absorbance values in the same container. The LF of BEM at the air-PBS interface, compared with LF formed over H2O, showed surface pressure vs area (π-A) isotherms more expanded at low π, more compressible, with a bi-dimensional transition at lower π and lower minimal A. The surface potential reached 250 mV at the collapse point in both conditions (H2O and PBS). The specific activity resulted SBEA>>LSBEA>LBBEA. The use of PBS in the subphase and the transfer of LF at π=35mN/m instead of 10 mN/m improved the recovery of specific activity in LSBEA and LBBEA. The homogeneity of BEA distribution in LSBEA samples highly improved the precision of the kinetic parameters determined in different molecular packing conditions.Fil: Felsztyna, Iván. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; ArgentinaFil: Perillo, Maria Angelica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; ArgentinaFil: Clop, Eduardo Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; ArgentinaV Reunión Científica del IIByT (CONICET-UNC)Córdoba capitalArgentinaConsejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones Biológicas y TecnológicasUniversidad Nacional de CórdobaConsejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones Biológicas y TecnológicasPerillo, Maria AngelicaGarcia, Daniel Asmed2017info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/conferenceObjectReuniónBookhttp://purl.org/coar/resource_type/c_5794info:ar-repo/semantics/documentoDeConferenciaapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/245573A new langmuir-schaefer-based method developed for catalytic studies of acetylcholinesterase in planar films of erythrocyte membranes; V Reunión Científica del IIByT (CONICET-UNC); Córdoba capital; Argentina; 2017; 20-20CONICET DigitalCONICETspainfo:eu-repo/semantics/altIdentifier/url/https://www.iibyt.conicet.unc.edu.ar/reuniones-cientificas-anuales/Nacionalinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T09:48:17Zoai:ri.conicet.gov.ar:11336/245573instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-03 09:48:17.495CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv A new langmuir-schaefer-based method developed for catalytic studies of acetylcholinesterase in planar films of erythrocyte membranes
title A new langmuir-schaefer-based method developed for catalytic studies of acetylcholinesterase in planar films of erythrocyte membranes
spellingShingle A new langmuir-schaefer-based method developed for catalytic studies of acetylcholinesterase in planar films of erythrocyte membranes
Felsztyna, Iván
Langmuir-Schafer
Langmuir-Blodgett
Acetylcholinesterase
Enzyme kinetics
title_short A new langmuir-schaefer-based method developed for catalytic studies of acetylcholinesterase in planar films of erythrocyte membranes
title_full A new langmuir-schaefer-based method developed for catalytic studies of acetylcholinesterase in planar films of erythrocyte membranes
title_fullStr A new langmuir-schaefer-based method developed for catalytic studies of acetylcholinesterase in planar films of erythrocyte membranes
title_full_unstemmed A new langmuir-schaefer-based method developed for catalytic studies of acetylcholinesterase in planar films of erythrocyte membranes
title_sort A new langmuir-schaefer-based method developed for catalytic studies of acetylcholinesterase in planar films of erythrocyte membranes
dc.creator.none.fl_str_mv Felsztyna, Iván
Perillo, Maria Angelica
Clop, Eduardo Matias
author Felsztyna, Iván
author_facet Felsztyna, Iván
Perillo, Maria Angelica
Clop, Eduardo Matias
author_role author
author2 Perillo, Maria Angelica
Clop, Eduardo Matias
author2_role author
author
dc.contributor.none.fl_str_mv Perillo, Maria Angelica
Garcia, Daniel Asmed
dc.subject.none.fl_str_mv Langmuir-Schafer
Langmuir-Blodgett
Acetylcholinesterase
Enzyme kinetics
topic Langmuir-Schafer
Langmuir-Blodgett
Acetylcholinesterase
Enzyme kinetics
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Previously we reported that the catalytic activity of bovine erythrocyte acetylcholinesterase (BEA) located in Langmuir-Blodgett films (LB) of bovine erythrocyte membranes (BEM), LBBEA, depended on the curvature and packing of the molecular environment. Moreover, the specific activity of LBBEA was much lower than that of BEA in suspensions of BEM vesicles (SBEA). So, the present work was aimed at maximizing the specific activity of BEA recovered from the transfer of a Langmuir film (LF) from the air-aqueous interface to alkylated solid surfaces and improving the precision of the enzymatic assays. Three main changes were introduced to the previously assayed method. a) Phosphate saline buffer (PBS), pH 7.4 was used instead of H2O as the subphase over which was spread the BEM to form the LF, assuming that this composition, closer to physiological conditions, would be more effective than water in preserving the BEA protein structure/activity and the LF organization. b) BEA in LF films (LFBEA) was transferred from air-PBS interface to hydrophobic flat surfaces by the LangmuirSchaefer technique (LS) to obtain LSBEA samples. c) A new device was designed to allow performing the whole enzymatic activity assay using a unique LS film as well as the reading of the absorbance values in the same container. The LF of BEM at the air-PBS interface, compared with LF formed over H2O, showed surface pressure vs area (π-A) isotherms more expanded at low π, more compressible, with a bi-dimensional transition at lower π and lower minimal A. The surface potential reached 250 mV at the collapse point in both conditions (H2O and PBS). The specific activity resulted SBEA>>LSBEA>LBBEA. The use of PBS in the subphase and the transfer of LF at π=35mN/m instead of 10 mN/m improved the recovery of specific activity in LSBEA and LBBEA. The homogeneity of BEA distribution in LSBEA samples highly improved the precision of the kinetic parameters determined in different molecular packing conditions.
Fil: Felsztyna, Iván. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina
Fil: Perillo, Maria Angelica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina
Fil: Clop, Eduardo Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina
V Reunión Científica del IIByT (CONICET-UNC)
Córdoba capital
Argentina
Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones Biológicas y Tecnológicas
Universidad Nacional de Córdoba
description Previously we reported that the catalytic activity of bovine erythrocyte acetylcholinesterase (BEA) located in Langmuir-Blodgett films (LB) of bovine erythrocyte membranes (BEM), LBBEA, depended on the curvature and packing of the molecular environment. Moreover, the specific activity of LBBEA was much lower than that of BEA in suspensions of BEM vesicles (SBEA). So, the present work was aimed at maximizing the specific activity of BEA recovered from the transfer of a Langmuir film (LF) from the air-aqueous interface to alkylated solid surfaces and improving the precision of the enzymatic assays. Three main changes were introduced to the previously assayed method. a) Phosphate saline buffer (PBS), pH 7.4 was used instead of H2O as the subphase over which was spread the BEM to form the LF, assuming that this composition, closer to physiological conditions, would be more effective than water in preserving the BEA protein structure/activity and the LF organization. b) BEA in LF films (LFBEA) was transferred from air-PBS interface to hydrophobic flat surfaces by the LangmuirSchaefer technique (LS) to obtain LSBEA samples. c) A new device was designed to allow performing the whole enzymatic activity assay using a unique LS film as well as the reading of the absorbance values in the same container. The LF of BEM at the air-PBS interface, compared with LF formed over H2O, showed surface pressure vs area (π-A) isotherms more expanded at low π, more compressible, with a bi-dimensional transition at lower π and lower minimal A. The surface potential reached 250 mV at the collapse point in both conditions (H2O and PBS). The specific activity resulted SBEA>>LSBEA>LBBEA. The use of PBS in the subphase and the transfer of LF at π=35mN/m instead of 10 mN/m improved the recovery of specific activity in LSBEA and LBBEA. The homogeneity of BEA distribution in LSBEA samples highly improved the precision of the kinetic parameters determined in different molecular packing conditions.
publishDate 2017
dc.date.none.fl_str_mv 2017
dc.type.none.fl_str_mv info:eu-repo/semantics/publishedVersion
info:eu-repo/semantics/conferenceObject
Reunión
Book
http://purl.org/coar/resource_type/c_5794
info:ar-repo/semantics/documentoDeConferencia
status_str publishedVersion
format conferenceObject
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/245573
A new langmuir-schaefer-based method developed for catalytic studies of acetylcholinesterase in planar films of erythrocyte membranes; V Reunión Científica del IIByT (CONICET-UNC); Córdoba capital; Argentina; 2017; 20-20
CONICET Digital
CONICET
url http://hdl.handle.net/11336/245573
identifier_str_mv A new langmuir-schaefer-based method developed for catalytic studies of acetylcholinesterase in planar films of erythrocyte membranes; V Reunión Científica del IIByT (CONICET-UNC); Córdoba capital; Argentina; 2017; 20-20
CONICET Digital
CONICET
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dc.publisher.none.fl_str_mv Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones Biológicas y Tecnológicas
publisher.none.fl_str_mv Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones Biológicas y Tecnológicas
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