A NEW LANGMUIR-SCHAEFER-BASED METHOD DEVELOPED FOR CATALYTIC STUDIES OF ACETYLCHOLINESTERASE IN PLANAR FILMS OF ERYTHROCYTE MEMBRANES

CLOP, E. M.; PERILLO, M. A.; FELSZTYNA, I.

Córdoba capital

Encuentro; V Reunión Científica del IIByT (CONICET-UNC); 2017

CONICET

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, thespecific 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 recoveredfrom the transfer of a Langmuir film (LF) from the air-aqueous interface to alkylated solidsurfaces and improving the precision of the enzymatic assays. Three main changes wereintroduced to the previously assayed method. a) Phosphate saline buffer (PBS), pH 7.4 wasused instead of H2O as the subphase over which was spread the BEM to form the LF, assumingthat this composition, closer to physiological conditions, would be more effective than water inpreserving 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 LangmuirSchaefertechnique (LS) to obtain LSBEA samples. c) A new device was designed to allowperforming the whole enzymatic activity assay using a unique LS film as well as the reading ofthe 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 moreexpanded at low π, more compressible, with a bi-dimensional transition at lower π and lowerminimal A. The surface potential reached 250 mV at the collapse point in both conditions (H2Oand PBS). The specific activity resulted SBEA>>LSBEA>LBBEA. The use of PBS in thesubphase and the transfer of LF at π=35mN/m instead of 10 mN/m improved the recovery ofspecific activity in LSBEA and LBBEA. The homogeneity of BEA distribution in LSBEAsamples highly improved the precision of the kinetic parameters determined in differentmolecular packing conditions.