Localization of para-nitrophenol within the lipid bilayer

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

Rosario, Santa Fe, Argentina

Congreso; XXXV Annual Meeting of the Argentinean Biophysical Society; 2006

Sociedad Argentina de Biofísica

Para-nitro phenol (PNP) is one of the hydrolysis products of p-nitrophenylphosphate, a compound generally used as a substrate in enzyme kinetic studies of many phostatases. The rates of these reactions are frequently determined by measurements of the absorbance of the product formed. If the membrane-water partition coefficient of the product is non-zero, as may be expected for PNP (Poctanol-water, PNP>0) [1], the magnitude determined for the reaction rate may be affected in the presence of membrane-water interfaces due to the departure of the value of the extinction coefficient of the product within the membrane phase from its value in water (em-ew¹0) [2]. Moreover, em, being sensitive to the polarity of the environment, will depend on the localization of PNP within the membrane depth.The objective of the present study was to estimate the localization of p-nitrophenol (PNP) within model membranes to contribute to the understanding of enzyme kinetics in heterogeneous systems. We studied the acid-base equilibrium of PNP in the presence and in the absence of two non-charged interfaces, PC MLVs and Triton X-100 (TX100), and compared these values with those obtained in solutions of dioxane of known dielectric constants [3]. The DpKTX100 of PNP (pKTX100 - pKwater = 8.56?7.07=+1.49) was interpolated in a curve of DpK vs the values of dielectric constant of the solutions of 20-70 % P/V dioxane used as homogeneus systems of known polarity. The dielectric constant (DC) value DC=28 obtained suggested that PNP could penetrate deeply within the polar head group region of the micelle. In the presence of LUVs of PC the value DpKPC=1.059 (DC=41) reflected that the PNP acid-base equilibrium was taking place within the outer polar head group region of the membrane. The fact that DpKPC < DpKTX100 suggested a contribution of an electrostatic potential (+25.1 mV) that stabilized the charged dissociated species of PNP at the lipid-water interface. Our results support the hypothesis that PNP (a weak hydrophobic substance) concentrates in the membrane with respect to the aqueous phase through hydrophobic and dipolar interactions with phospholipids polar head groups. This information should be taken into account in the analysis of interfacial kinetics of reactions where PNP is obtained as a product.References[1] Schwarzenbach R.P., Stieri R., Folsom B.R. and Zeyer J., Environ.Sci.Technol. 1988, 22, 83-92.[2] Sanchez J.M., Ciklic I. and Perillo M.A., Biophys.Chem. 2005, 118, 69-77.[3] García D.A. and Perillo M.A., Biochim.Biophys.Acta, 1997, 1324,76-84.