Determining the Substrate Permeability through the Bilayer of Large Unilamellar Vesicles of DOPC. A Kinetic Study.

LUNA, M. A.; J J SILBER; L E SERENO; CORREA N.M*; MOYANO F.

RSC-Advances

ROYAL SOC CHEMISTRY

Año: 2016 vol. 6 p. 62594 - 62601

In this work we determine the permeability of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)vesicles in the presence of different cholesterol (Cho) contents, by using the enzymatic hydrolysis of Nbenzoyl-L-tyrosine p-nitroanilide (Bz-Try-pNA) catalyzed by a-chymotrypsin (a-CT). The reaction wasfirst studied in homogeneous media in a 4% p/p ethanol?water mixture and then in DOPC vesicles atdifferent Cho content. In homogenous media, ethanol helps to solubilize the substrate, which is almostinsoluble in water and therefore increases the effective concentrations. In DOPC vesicles, ethanol doesnot destroy the bilayer. In both cases, the enzymatic hydrolysis can be followed by UV-visiblespectroscopy. The hydrolysis of Bz-Try-pNA catalyzed by a-CT follows the Michaelis?Mentenmechanism and the kinetic parameters: kcat, KM and kcat/KM were evaluated in both systems at the samesolvent mixture compositions. To obtain the kinetic parameters and the permeability of the reactant inDOPC : Cho vesicles, we use a simple mathematical model and dynamic light scattering (DLS)measurements. The results show that the hydrolysis reaction takes place in the water entrapped in theinterior of the DOPC vesicles and, that the enzyme encapsulated inside the vesicles, despite thesignificant differences in the permeability values of Bz-Try-pNA, has similar catalytic effectsindependently on the Cho composition used.