Localization of protonated and deprotonated species of p-nitrophenol within model membranes

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

Maresias

Workshop; 3rd International Workshop on Spectroscopy for Biology; 2010

LOCALIZATION OF PROTONATED AND DEPROTONATED SPECIES OFp-NITROPHENOL WITHIN MODEL MEMBRANESEduardo M. Clop1, Leonardo F. Fraceto2, Eneida de Paula3 and María A. Perillo11Biofísica-Química, Depto.-Química. FCEFyN, Univ.Nac. de Córdoba, Argentina. 2 Depto-Bioquímica,Universidade Estadual Paulista Julio de Mesquita Filho, Sorocaba, SP, Brasil. 3 Depto Bioquímica, IB,UNICAMP, Brasil. mclop@efn.uncor.edup-Nitrophenol (PNP) is one of the hydrolytic products of p-nitrophenyl phosphate, the syntheticsubstrate of many phosphatases (Pase). The partitioning of PNP within the membrane may alter the kinetics of Pase catalyzed reactions by affecting the product local concentration. Here we estimated the membrane localization of PNP, firstly by studying its acid-base equilibrium in two non-charged interfaces, EggPC vesicles (LUVs), and Triton X-100 micelles, as well as in several dioxane solutions of known dielectric constants. These results evidenced that the PNP acid-base equilibrium was taking place within the membrane?s polar head group region.We then applied 1H-NMR to evaluate the interaction of protonated (PNPH pH=5) anddeprotonated (PNP- pH=10.2) PNP in EPC small unilamellar vesicles (SUVs). Changes in chemical shifts (CS) and longitudinal relaxation times (T1) from EPC hydrogens were determined.Intermolecular dipolar interactions were investigated using ROESY experiments (100 and 300 ms) and two cross-peaks were detected between hydrogens from PNP- and EPC. The CS of EPC hydrogens in the presence of PNP gave us direct information about its preferential location within the polar head group region of the membrane. Moreover the PNPH seemed to be preferentially located deeper than PNP- as it was expected due to the lack of a net charge. Accordingly, changes in longitudinal relaxation times of the lipid hydrogens disclosed details about the lipid dynamics in the presence of PNP.Acknowledgements: Conicet, Foncyt, CAPES-SPU, LNLS, and FAPESP