Nuclear magnetic resonance spin-lattice relaxation times (T(1)) measurements were performed in aqueous solutions of poly(ethylene glycol) (PEG) of 6000 Da molecular mass to study the dynamical relation between PEG and water molecules at different solute concentrations. (1)H-T(1) experiments were carried on at a low magnetic field in the time domain (20 MHz) and at a high field (400 MHz) to obtain spectral resolution. Two contributing components were identified in each proton system, PEG and water, presenting values of T(1) with very different orders of magnitude. The approximate matching between the shorter (1)H-T(1) values associated with water and PEG has lead us to conclude that there exists a network of interactions (hydrogen bonds) between the solute and the solvent, which results in the presence of an ordered and dehydrated structure of PEG folded or self-assembled in equilibrium with a more flexible monomer structure. Dynamic light scattering results were consistent with the formation of PEG aggregates, showing a mean size between 40 and 100 nm.

Functional consequences of constraining beta-Gal in bidimensional space were studied at defined molecular packing densities and constant topology. Langmuir-Blodgett films, LB15 and LB35 composed of dipalmitoyl phosphatidylcholine and K. lactis beta-Gal, were obtained by transferring Langmuir films (L) initially packed at 15 and 35 mN/m, respectively, to alkylated glasses. The beta-Gal-monolayer binding equilibrium, mainly the adsorption rate and affinity, depended on the initial monolayer's surface pressure (lower for higher pi i). At pi i = 15 and 35 mN/m, the surface excess (Gamma) followed downward parabolic and power-law tendencies, respectively, as a function of subphase protein concentration. Gamma values in L roughly reflected the protein surface density chemically determined in LBs (0-7.5 ng/mm2 at pi i = 0-35 mN/m and [beta-Gal] subphase = 0-100 microg/mL). The beta-Gal-catalyzed hydrolysis of o-nitrophenyl-galactopyranoside showed a Michaelian kinetics in solution as well as in LB15. KM, KM,LB15, Vmax, and Vmax,LB15 were 5.15 +/- 2.2 and 9.25 +/- 6 mM and 39.63 and 0.0096 +/- 0.0027 micromol/min/mg protein, respectively. The sigmoidal kinetics observed with LB35 was evaluated by Hill's model (K0.5 = 9.55 +/- 0.4 mM, Vmax,35 = 0.0021 micromol/min/mg protein, Hill coefficient n = 9) and Savageau's fractal model (fractal constant K f = 9.84 mM; reaction order for the substrate gs = 9.06 and for the enzyme ge = 0.62). Fractal reaction orders would reflect the fractal organization of the environment, demonstrated by AFM images, more than the molecularity of the reaction. Particular dynamics of the protein-lipid structural coupling in each molecular packing condition would have led to the different kinetic responses.

No 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.