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

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

San Miguél deTucumán

Congreso; III Latin American Federation of Biophysical Societies (LAFeBS) IX IberoAmerican Congress of Biophysics XLV Reunion Anual SAB 2016; 2016

Sociedad Argentina de Biofísica(SAB)-Latin American Federation of Biophysical Societies (LAFeBS)

Previously we reported that the catalytic activity of bovine erythrocyte acetylcholinesterase (BEA) located in Langmuir-Blodgett films (LB) ofbovine erythrocyte membranes (BEM), LBBEA, depended on the curvature and on the 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 atmaximizing the specific activity of BEA recovered from the transfer ofa Langmuir film (LF) from the air-aqueous interface to alkylated solidsurfaces and improving the precision of the enzymatic assays. Threemain changes were introduced to the previously assayed method. a)Phosphate saline buffer (PBS), pH 7.4, was used instead of H2O as thesubphase over which was spread the BEM to form the LF, assuming thatthis 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 was transferred from air-PBS interface to hydrophobic flat surfaces by the Langmuir-Schaefer technique(LS) to obtain LSBEA samples. c) A new device was designed to allowperforming the whole enzymatic activity assay using a unique LS filmas 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 overH2O, showed surface pressure vs area (π-A) isotherms more expandedat low π, more compressible, with a bi-dimensional transition at lowerπ and lower minimal A. The surface potential reached 250 mV at thecollapse point in both conditions (H2O and PBS). The specific activityresulted SBEA»LSBEA>LBBEA. The use of PBS in the subphase andthe transfer of LF at π=35mN/m instead of 10 mN/m improved the recovery of specific activity in LSBEA and LBBEA. The homogeneity ofBEA distribution in LSBEA samples highly improved the precision ofthe kinetic parameters determined in different molecular packing conditions.