Letter pubs.acs.org/JPCL
Evaluation of pH at Charged Lipid/Water Interfaces by HeterodyneDetected Electronic Sum Frequency Generation Achintya Kundu,† Shoichi Yamaguchi,† and Tahei Tahara*,†,‡ †
Molecular Spectroscopy Laboratory, RIKEN, Wako, Saitama 351-0198, Japan Ultrafast Spectroscopy Research Team, RIKEN Center for Advanced Photonics (RAP), Wako, Saitama 351-0198, Japan
‡
S Supporting Information *
ABSTRACT: Although the interface pH at a biological membrane is important for biological processes at the membrane, there has been no systematic study to evaluate it. We apply novel interface-selective nonlinear spectroscopy to the evaluation of the pH at model biological membranes (lipid/water interfaces). It is clearly shown that the pH at the charged lipid/water interfaces is substantially deviated from the bulk pH. The pH at the lipid/water interface is higher than that in the bulk when the head group of the lipid is positively charged, whereas the pH at the lipid/water interface is lower when the lipid has a negatively charged head group. SECTION: Surfaces, Interfaces, Porous Materials, and Catalysis
A
equilibrium of the pH indicator. We used two lipids having oppositely charged head groups but the same acyl chain. A cationic lipid is 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), and an anionic lipid is 1,2-dipalmitoyl-sn-glycero-3phosphoglycerol (DPPG), as shown in Figure 1b. The lipid/ water interfaces were formed with the lipids adsorbed at the air/water interface, where the hydrophobic acyl chains of the lipid point upward to the air and their hydrophilic charged head groups point downward to bulk water. The interface density of the lipids was approximately 2 molecules nm−2, and the lipid monolayers were in the liquid condensed phase (the surface pressures at the DPTAP/water and DPPG/water interfaces were 40 ± 3 and 25 ± 3 mN m−1, respectively). The pH indicator was coadsorbed at the lipid/water interfaces with its alkyl chain aligned with the lipid acyl chains and its chromophore facing interfacial water. The density ratio of the lipid to the pH indicator was 10. The Π−A (surface pressure− area) curves of the lipid monolayers with and without the pH indicator were nearly identical, which means that the structure of the lipid/water interfaces is not influenced by the pH indicator. The pH of the bulk aqueous phase was controlled by sodium hydroxide and hydrochloric acid. The ionic strength of the bulk solution at different bulk pHs was kept at 0.4 ± 0.01 mol dm−3 by adding sodium chloride. The interfacial electronic χ(2) (second-order nonlinear optical susceptibility) spectra of the pH indicator were measured with HD-ESFG. (The detail of the measurement is given in the Supporting Information.)
biological membrane separates the cytoplasm and cellular organelles from the exterior of the cell.1,2 The interface pH at the biological membrane3−5 is crucial for many biochemical processes such as ion transport, adsorption of molecules, orientation of the membrane proteins, and binding of therapeutic peptides and drugs. Therefore, it is essentially important to know the interface pH at the biological membrane. However, only a few experimental6−8 and theoretical 9−11 studies have been reported so far. In experimental studies,7,8 the interface pH or interface potential at the biological membrane was estimated using fluorescence spectroscopy, but an ambiguity remains due to the acid−base equilibrium in the excited state. In theoretical studies,9−11 the interface pH or interface potential at the biological membrane was calculated, but the obtained results were far from the experimental values.6−8 In this regards, the interface pH at the biological membrane has not yet been understood well. To evaluate the interface pH and compare it with the bulk pH, it is desirable to apply a common method to the interface and the bulk. One of the most reliable methods to determine the bulk pH is UV−visible absorption spectrometry using a pH indicator. Because interface-selective electronic spectroscopy has recently become possible by heterodyne-detected electronic sum frequency generation (HD-ESFG),12−18 we are now able to carry out quantitative pH spectrometry for interfaces.12,18 In this Letter, we evaluate the interface pH of model biological membranes (lipid/water interfaces) by pH spectrometry using HD-ESFG. To investigate the pH at lipid/water interfaces, we chose a surface-active pH indicator, 4-heptadecyl-7-hydroxycoumarin, which was used to determine the pH at the air/water interface in our recent study.18 Figure 1a shows the acid−base © XXXX American Chemical Society
Received: February 3, 2014 Accepted: February 6, 2014
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feature, and it can be interpreted in the same way as UV−visible absorption spectra under the present two-photon resonant and one-photon nonresonant conditions. (Note that the Im χ(2) signal appears with a negative sign, reflecting the absolute orientation of the chromophore.13,19) In the Im χ(2) spectra, a peak at 377 nm is observed at bulk pH 8.4, and it is assigned to the conjugate base (A−). (The peak position was determined by the fitting analysis. See the Supporting Information for details.) With lowering of the bulk pH from 8.4 to 4.6, the intensity of the Im χ(2) signal due to A− gradually decreases, owing to the shift in the acid−base equilibrium. At the bulk pH lower than 6.0, the contribution of the acid (HA) is predominant at wavelengths shorter than 350 nm. The imaginary and real parts of the χ(2) spectra exhibit isosbestic points approximately at 350 and 390 nm, respectively, which assures the reliability of the measurements. Figure 3a and b shows the interface-selective electronic χ(2) spectra of the pH indicator at the negatively charged DPPG/
Figure 1. (a) Acid−base equilibrium of a surface-active pH indicator, 4-heptadecyl-7-hydroxycoumarin. (b) Chemical structures of a cationic lipid (DPTAP) and an anionic lipid (DPPG).
Figure 2a and b shows the interface-selective electronic χ(2) spectra of the pH indicator at the positively charged DPTAP/ water interface. The horizontal axis is the wavelength corresponding to the sum frequency ω1 + ω2, and the vertical axis stands for the imaginary and real parts of χ(2). The imaginary part of χ(2) (Im χ(2)) exhibits an absorptive spectral
Figure 3. (a) Imaginary and (b) real parts of the χ(2) spectra of the pH indicator at the DPPG/water interface. Black, pink, green, blue, and red lines represent spectra obtained at bulk pH 8.6, 9.1, 10.2, 10.6, and 11.9, respectively.
water interface. Also in this case, a peak due to A− is observed at 366 nm in the Im χ(2) spectrum at bulk pH 11.9. (See the Supporting Information for details.) With lowering of the bulk pH from 11.9 to 8.6, the signal due to A− decreases, and the contribution of HA is predominant in the wavelength region shorter than 350 nm at the bulk pH lower than 10.6. In this spectral change, the imaginary and real parts of the χ(2) spectra exhibit isosbestic points at around 345 and 380 nm, respectively. Compared with the positively charged DPTAP/ water interface, the population of A− is much less at the negatively charged DPPG/water interface at the same bulk pH. This indicates that the interface pH is substantially different between the two interfaces. We note that the pH indicator is always adsorbed at the lipid/water interfaces, and hence, the observed pH dependence of the χ(2) spectra is solely ascribed to
Figure 2. (a) Imaginary and (b) real parts of the χ(2) spectra of the pH indicator at the DPTAP/water interface. Black, pink, green, blue, and red lines represent spectra obtained at bulk pH 4.6, 5.9, 7.2, 7.7, and 8.4, respectively. 763
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= [A−] is realized at bulk pH 10.7. Therefore, the pKa of the pH indicator at the DPPG/water interface is represented as 10.7 + Δ. This Δ is the pH difference between the DPPG/water interface and the bulk. We note that the acid−base equilibrium of this pH indicator was examined at various positively and negatively charged micelles interfaces, and apparent pKa values at those interfaces have been reported.20,21 The bulk pH values at [HA] = [A−] (i.e., apparent pKa) obtained in this study for the DPTAP and DPPG interfaces are in good agreement with the values reported for positively and negatively charged micelle interfaces, respectively.20,21 Because the pKa value of the pH indicator is represented with the bulk pH and the pH difference between the bulk and the interface (Δ), the interface pH can be obtained if we can obtain the pKa value of the indicator at each interface. The pKa of the acid−base equilibrium is proportional to the standard reaction Gibbs energy, which is given by the standard chemical potentials of the reactants and products. Because the standard chemical potential of a chemical species is governed by the relative dielectric constant of the surrounding medium (ε), the pKa can be given as a function of ε.12,18,20,21 In fact, Fernández and Fromherz experimentally determined a relation between ε and the pKa of the pH indicator, which is depicted in Figure 5a.21 This curve shows that the pKa increases as ε lowers. This is readily understood because the neutral HA is stabilized in low ε media, compared to the charged A−. As the relation between the pKa and ε is given, the pKa is obtainable from ε. The pH indicator used in this study is a solvatochromic dye, so that its electronic transition energy is sensitive to ε of the surroundings. Drummond and Grieser have experimentally examined a relation between the absorption peak wavelength of A− and ε and obtained the curve shown in Figure 5b.20 The curve shows that the peak wavelength (λmax) becomes longer as the ε value is lowered because the basic form of the pH indicator shows negative solvatochromism. The transition energy may also be affected by the charges of the lipid head groups. Therefore, we examined the ionic strength dependence of the absorption spectrum in the bulk and also estimated the peak wavelength shift induced by the Stark effect using a simple model (see the Supporting Information for details). They showed that the effect of the charge on the transition energy is negligible (
