PNP insertion at the head-group region of phospholipid bilayers, as revealed by 1H-NMR techniques

CLOP, E. M.; FRACETO, L.; DE PAULA, E.; PERILLO, M.A.

Montevideo Uruguay

Congreso; 6TH International Conference of bilogical phisics; 5TH Southern Cone Biophisical Congress, 36th Reunión Anual de la Sociedad Argentina de Biofísica; 2007

Sociedad Argentina de Biofísica

Para-nitrophenol (PNP) is one of the hydrolytic products of p-nitrophenylphosphate (PNPP), generally used as a substrate in kinetic studies of different phosphatases. In heterogeneous media, the rates of these reactions, when determined by absorbance of PNP, may be obscured by dielectric effects of the environment on PNP extinction coefficient values (note that Pm/w, PNP>0 and em¹ ew ). Moreover, em, being sensitive to the polarity of the environment, will depend on the depth of insertion of PNP within the membrane. In a previous work [1] we have demonstrated that, in non-polar homogeneous systems as well as in the presence of non-charged interfaces (EggPC MLVs and TX-100 micelles), the thermodynamic constant of the acid-base equilibrium of PNP increased with respect to a homogeneous water system. The calibration of these changes in terms of dielectric-constant of the environment indicated that dipolar interactions stabilized PNP within de polar head group regions of lipid water interfaces.The object of this work was to determine the PNP localization within lipid bilayers, through a higher resolution methodology based on a physical principle different from the one applied previously:  EPC SUVs (65 mM in 0.1 M carbonate buffer pH 10.2) were submitted to 1H-NMR analysis in the presence and in the absence of PNP (3:1 and 1.5:1 EPC:PNP molar ratios). Changes in de chemical shifts (CS) and longitudinal relaxation times (T1) of EPC hydrogens were determined. Intermolecular (PNP-EPC) dipolar interactions were investigated using ROESY experiments (100 and 300 ms) and two cross-peaks were detected between hydrogens from PNP and from EPC. The cross-peaks were assigned to hydrogens H1 and H2 of PNP with those from the choline lipid moiety. The short-range ring-current effect caused by the PNP aromatic ring on the CS of EPC gave us direct information about its preferential location within the polar head group membrane region. Accordingly, changes in longitudinal relaxations times of the lipid hydrogens disclosed details about the lipid dynamics in the presence of PNP. [1] Clop, EM and Perillo MA. SAB 2006.