Langmuir Films from Human Placental Membranes: Preparation, Rheology, Transfer to Alkylated Glasses, and Sigmoidal Kinetics of Alkaline Phosphatase in the Resultant Langmuir-Blodgett Film

Abstract

In the present study, we studied the activity of human placental alkaline phosphatase (PLAP) constraint in a planar surface in controlled molecular packing conditions. For the first time, Langmuir films (LFs) were prepared by the spreading of purified placental membranes (PPM) on the air-water interface and their stability and rheological properties were studied. LFs exhibited a collapse pressure pi(C) = 48 mN/m, hysteresis during the compression-decompression cycle (C-D), indicating a plastic deformation, and a compressibility modulus (K) compatible with liquid-expanded phases. A phase transition point appeared at pi(T) = 28 mN/m and, following successive C-D, it moved toward lower surface areas and higher K, suggesting the lost of some non-PLAP proteins as components of vesicles that might protrude from the monolayer (confirmed by combining lipid/protein molar ratio analysis, PAGE-SDS and V(max)). LFs were transferred at 35 mN/m to alkylated glasses to obtain Langmuir-Blodgett films (LB(35)) the stability of which was confirmed by AFM. The kinetics of p-nitrophenyl phosphate (pNPP) hydrolysis at 37 degrees C catalyzed by PPM was Michaelian and exhibited the thermostability at 60 degrees C typical of PLAP. In LB(35), PLAP exhibited a sigmoidal kinetics which resembled the behavior of the partially metalated enzyme but might become from a cross-talk between protein and membrane structures.