Interaction of l-Phenylalanine with Lipid Monolayers at AirâWater

Article pubs.acs.org/JPCC

Cite This: J. Phys. Chem. C XXXX, XXX, XXX−XXX

Interaction of L‑Phenylalanine with Lipid Monolayers at Air−Water Interface at Different pHs: Sum-Frequency Generation Spectroscopy and Surface Pressure Studies Ankur Saha, Sumana SenGupta, Awadhesh Kumar,* and Prakash D. Naik Radiation & Photochemistry Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India Homi Bhabha National Institute (HBNI), Anushaktinagar, Mumbai 400 094, India S Supporting Information *

ABSTRACT: We employed vibrational sum-frequency generation (VSFG) spectroscopy to obtain molecular level understanding of interaction of L-phenylalanine (Phe) with lipid monolayers of zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at the air−water interface. The measured VSFG spectra in the CH stretch region due to the lipid and Phe, and the OH stretch region due to interfacial water molecules were analyzed. These results in combination with surface pressure studies reveal that the Phe molecules at acidic pH of 5.6 intercalate into DPPC monolayers, and replace some interfacial water molecules. Consequently, there is a decrease in the VSFG intensity in the OH stretch region in the Phe subphase, and a concomitant increase in the surface pressure of the DPPC monolayer. The exclusion of the water molecules is controlled by both the bulk concentration of Phe, and the surface concentration of DPPC. In contrast, at the neutral pH of 7.3 there is an increase in the VSFG intensity due to the interfacial water molecules in the Phe subphase, and a decrease in the surface pressure of the DPPC monolayer. A decrease in the surface pressure, implying an increase in the surface tension toward values of pure water, suggests condensation of lipids to some extent and exposing water regions in the surface. At the neutral pH, the Phe molecules interact mostly with the headgroup of the lipid monolayer without affecting directly the hydrophobic interaction with the tail region. At both the pHs, the microscopic order of the hydrocarbon chains of the DPPC molecules is observed to increase with increased VSFG intensity due to the terminal CH3 group, and decreased intensity due to the CH2 group. The interaction of DPPC monolayer with Phe is compared to that with sodium dodecyl sulfate (SDS), and the hydrophobic interactions between the side chains in the latter are found to be relatively much stronger. disorders.13,14 Such an interaction between Phe and DPPC monolayer has been investigated1,8 using Langmuir trough method, Brewster angle microscopy, confocal microscopy, and even molecular dynamics simulation. These studies1,8 report a change in the structure, and other surface properties of the monolayer, such as interfacial tension and morphology. Since an intermolecular interaction depends on the molecular states of interacting molecules, and the Phe molecules are known to exist in different states at different pH,15,16 it is expected that interaction of DPPC with Phe subphase at different pH can differ. Although Phe molecules are mostly in the zwitterionic state both in the ultrapure water of acidic pH, and at the physiological pH of ∼7.3, they are reported to still interact differently with DPPC monolayer in these pH conditions.8 It is shown that the Phe molecules at pH 7.3 do not penetrate the interface, but these molecules at pH 5.0 do penetrate the

1. INTRODUCTION A phospholipid monolayer at the air−water interface, particularly phosphatidylcholines (PC), is an excellent model for a cell membrane and a lung surfactant, and hence has attracted attention for decades.1 Basic understanding on the structure, orientation, and conformation of phospholipids as a monolayer, and the hydrogen bonding network of water molecules with the monolayer at the air−water interface has been reported.2 Since various biologically active molecules affect significantly the structural and biophysical properties of lipid monolayers, their interactions with monolayers are interesting topic of investigation. Several research groups have studied effects of surfactants,3−7 phenylalanine,1,8 drugs,9,10 and other chemicals11,12 on lipid monolayers. Since molecular interactions have weak strengths and long-range characters, their determination is challenging, and hence these studies are pursued both experimentally and theoretically. Studies on interaction of Phe with phospholipids are important, because this amino acid is associated with several diseases, such as Alzheimer’s disease, type II diabetes, and prion © XXXX American Chemical Society

Received: November 8, 2017 Revised: January 31, 2018 Published: February 6, 2018 A

DOI: 10.1021/acs.jpcc.7b11049 J. Phys. Chem. C XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry C Scheme 1. Chemical Structures of DPPC, Phe, and SDS

interface.1,8 Molecular dynamics simulation predicts that the penetration of the interface becomes deeper for the neutral Phe molecules.1 Since different states of L-phenylalanine intercalate to a different extent into a DPPC monolayer at the air−water interface, these authors1,8 demonstrated difference in the surface pressure, and ordering of the DPPC monolayer at different pH. Our interest was to understand these changes at the molecular level, using vibrational sum-frequency generation (VSFG) spectroscopy. Recent reviews on theory and applications of VSFG in general,17 and on Langmuir monolayers in particular18 provide details on the theoretical background and various applications of VSFG. We have employed this spectroscopy to investigate interaction of amino acid phenylalanine (Phe) with the zwitterionic phospholipid 1,2-dipalmitoyl-rac-glycero-3-phosphocholine (DPPC) monolayer, and compared the effects of interactions of Phe and SDS with DPPC. We have investigated the effects of these interactions on the molecular orientation of interfacial water molecules as well. Our VSFG and surface pressure studies support the recent work on interaction of Phe with DPPC, and provide additional information on vibrational spectral features at the air−water interface. Present work directly probed the consequence of interaction on the hydrophobic tails of DPPC molecules, and also on the interfacial water molecules. Chemical structures of DPPC, Phe, and SDS are given in Scheme 1. VSFG studies along with surface pressure measurements, at the acidic pH of ∼5.6 and at the physiological pH of ∼7.3, have provided insights into the nature of interaction between Phe and DPPC monolayer. We have kept the surface area constant, and altered the number of molecules per unit surface area by changing the number of molecules. This is in contrast to earlier studies using surface pressure−area (π−A) isotherm, wherein the number of molecules in the monolayer is kept constant, and the surface area is changed by compression to alter the number of molecules per unit surface area.

χr(2) (ωIR ) =

q

⎤ ⎥ ⎢⎣ (ωIR − ωq) + i Γq ⎥⎦ Aq

(2)

magnitude, damping constant, and resonant frequency, respectively, for a vibrational mode q. These parameters can be obtained from the measured VSFG spectra after fitting these to eqs 1 and 2. We have employed the relative values of Assp (in the ssp polarization combination) or Appp for the CH3 stretch for quantification. A particular polarization combination is denoted based on the polarization states of the SFG, visible, and IR beams in the sequence, for example ssp polarization implies that both SFG and visible beams have the s state, and the IR beam has p state. The s and p states refer to two polarization states, in which the light has an electric field polarized perpendicular and parallel to the plane of incidence, respectively.

3. EXPERIMENTAL SECTION 3.1. Vibrational Sum-Frequency Generation. VSFG spectroscopy is a nonlinear process involving the interaction of two input beams of fixed visible (generally 800 or 532 nm) and tunable infrared (IR beam, 2.3 to 10.0 μm) wavelengths to generate the signal beam at a frequency, which is the sum of the frequencies of the two input beams. The input IR and visible laser beams, after being focused partially, are aligned to propagate at incidence angles of respectively 55° and 60°, with respect to the surface normal, to overlap at the interface both spatially and temporally. To measure the VSFG spectra of the interfacial molecules, the IR wavenumber is tuned across the CH and OH stretching vibrational resonances in the region of 2800 to 3000 cm−1 and 3000 to 3600 cm−1, respectively. The measurements in the CH region are carried out in ssp and ppp polarization schemes, whereas for the OH region only ssp polarization was used. Although we have described the VSFG setup in our earlier publications,7,23−25 here essential details are mentioned in brief. A commercial Ekspla laser system (repetition rate =10 Hz and the pulse width =30 ps) was employed as a VSFG spectrometer with the spectral resolution of about 2 cm−1 for the CH vibrational bands. We used the visible beam at 532 nm, which is generated by frequency doubling of the fundamental output of a Nd:YAG laser (PL2241B, Ekspla, Lithuania). The tunable IR beam is generated in a difference frequency generator (DFG) with silver thiogallate (AgGaS2) crystal by mixing the idler output of an optical parametric generator (OPG) with the fundamental output of the Nd:YAG laser (1064 nm). The OPG (PG401, Ekspla), with a lithium triborate (LiB3O5) nonlinear crystal, was pumped by the third harmonic beam of the Nd:YAG laser (355 nm) to generate the idler output. Polarized VSFG spectra were

2. THEORETICAL BACKGROUND The SFG process is understood well theoretically, and detailed description is available in the literature.19−22 Briefly, the SFG intensity, ISFG, is found to depend on the absolute square of the second-order nonlinear susceptibility, χ(2), which has contribu(2) tion from both nonresonant (χ(2) nr ) and resonant terms (χr ), and expressed in eq 1, ISFG(ωSFG) ∝ |χ (2) (ωIR )|2 ∝ |χnr(2) + χr(2) (ωIR )|2

⎡

∑⎢

(1)

The eq 2 defines the resonant part, in which the symbols Aq, Γq, and ωq stand for the B


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longer present at the interface due to the presence of the monolayers at high number densities in our experiments. 4.1. VSFG Spectra of DPPC Monolayer at the Air− Water Interface. The VSFG spectra of the DPPC monolayer with the ssp polarization were measured in the CH stretching region with its varying surface density (5.3 × 1013 to 3.2 × 1014 molecules/cm2). At the lowest surface density of DPPC (5.3 × 1013 molecules/cm2), the signal could not be observed in the CH stretching region. With increased surface density of DPPC, primarily three usual vibrational bands could be observed at 2850, 2878, and 2941 cm−1, due to the CH2 symmetric stretch, νs(CH2), the CH3 symmetric stretch, νs(CH3) and CH3 Fermi resonance, νFR(CH3), respectively (shown in Figure S1 of Supporting Information). The Fermi resonance band is due to interaction of the bending overtone with symmetric stretching mode of the methyl group. The peak at 2941 cm−1 in the ssp spectra has also some contribution from CH3 asymmetric stretch, νas(CH3), which is mostly observed with the ppp polarization at 2967 cm−1. The observation of 2850 cm−1 band suggests presence of gauche defect in the alkyl chains of DPPC. With increasing surface concentration of DPPC three main effects are observed on VSFG spectra. First, overall VSFG intensities increase with increasing surface concentration of DPPC. However, the relative intensity of the νs(CH3) band to the νFR(CH3) increases with the DPPC concentration. The increase in the relative intensity of the νs(CH3) band with increasing surface concentration of DPPC may be because of the local aggregation of the methyl groups,28 which are responsible for the large enhancement of the van der Waals interaction. The concentration dependence of the relative intensity can also be explained based on the relative importance of the intermolecular and intramolecular interactions. At low surface concentration the intramolecular interaction dominates leading to relatively greater intensity of the νFR(CH3) band, because the Fermi resonance is defined as an intramolecular coupling.28,29 The relative importance of the intermolecular and intramolecular interactions will depend on the surface concentration of the DPPC molecules. With increasing surface concentration, intermolecular interaction dominates leading to increasing relative intensity of νs(CH3) band to the νFR(CH3) band. Second, the relative intensities of the 2850 to 2878 cm−1 stretching band drastically reduced, with a shift of the band peak position to lower wavenumbers (2850 to 2842 cm−1) with increased DPPC surface density. The shift of the νs(CH2) band at 2850 cm−1 to lower wavenumber, and its reduced relative intensity indicate that the acyl chains are conformationally ordered. Thus, these results suggest that the alkyl chains of DPPC acquire reduced gauche defects having increased trans configuration with increasing concentration.30−32 These effects of changed conformation, and chain ordering on increased concentration have been observed in the alkyl or acyl chains of several lipids,2,12,18,28 and surfactants,23,33,34 due to hydrophobic chain−chain interactions. Third, one additional weak band is seen at ∼2902 cm−1 at increased surface density of DPPC. This band is assigned to a combination of the νFR(CH2) band of the acyl group, and the νs(CH2) band of the phosphocholine headgroups,2,3,35 with some possible contribution from the CH2 symmetric stretch of the phospholipid glycerol moiety. This weak band could be observed because of greater DPPC surface concentration. 4.2. VSFG Spectra of DPPC Monolayer with Phe Subphase. The VSFG spectra of a DPPC monolayer at the

measured after allowing about 30 min for equilibration of the DPPC monolayer. The VSFG spectra were measured by averaging each spectral data point for 60 shots, and normalizing by IR and visible beam energies. Normalization was a simple operation of dividing the VSFG intensity by simultaneously measured IR and visible beam energies at each wavenumber. We have measured VSFG spectra in both the CH, and OH stretching regions for different DPPC surface concentration (1.3 × 1014 to 3.6 × 1014 molecules/cm2) with varying Phe bulk concentration (0 to 12 mM). We employed a standard spectral fitting procedure19,25,26 for the measured spectra using eqs 1 and 2 to quantitatively investigate the concentration dependence of the VSFG intensity. 3.2. Surface Pressure−Time (π−t) Adsorption Kinetics. The surface pressure of a Langmuir monolayer of DPPC was measured at the water subphase and the Phe solution subphase at room temperature of ∼297 K, employing a platinum Wilhelmy plate microbalance with an accuracy of ±0.02 mN/m, to understand interaction of Phe with the DPPC monolayer. We measured a change in the surface pressure by spreading a known volume of DPPC in CHCl3 solution at either the water or the Phe solution (known bulk concentration) subphase in Teflon troughs using a Hamilton syringe. A change in the surface pressure of DPPC at the Phe subphase with respect to the water subphase was recorded on equilibration of the system at the various concentrations of Phe and DPPC in both the acidic (pH ∼ 5.6) and neutral (∼7.3) pH conditions. 3.3. Materials and Sample Preparations. DPPC and Phe, both with purity ≥99%, were procured from SigmaAldrich, whereas chloroform with purity ≥99% was purchased from Thomas Baker (Mumbai). All these chemicals were used without further purification. A stock solution (∼100 μM) of DPPC in chloroform was used for a maximum of 2 days, and stored in a refrigerator even between uses. All the Phe solutions were prepared in ultrapure Millipore water of pH = 5.56, resistivity 18.2 MΩ-cm, and surface tension of 71.9 mN/m. Ultrapure water is not having any hydrocarbon impurity as measured with VSFG spectroscopy. The pH of Phe solution of different concentrations (up to 12 mM) was in acidic range of 5.6 ± 0.1. In another set of experiments, Phe solution was neutralized to pH 7.3 using aqueous solution of disodium hydrogen phosphate (Sigma-Aldrich, purity ≥99%). Since Phe solution is prone to aggregation,1 freshly prepared solution was used for every single experiment.

4. RESULTS AND DISCUSSION We measured VSFG spectra to elucidate the structures and orientation of DPPC molecules, and to understand the effects of Phe on the same system. The CH stretching vibrations in the wavenumber range of 2800 to 3000 cm−1 were measured due to both CH2 and CH3 groups of hydrophobic alkyl chain of DPPC. Similarly, the VSFG spectra in the OH stretching region due to interfacial water molecules were measured in the wavenumber range of 3000 to 3600 cm−1. In this region, two broad peaks at ∼3200 and ∼3400 cm−1 are observed, which are assigned to the OH stretch of the hydrogen-bonded water molecules.2,17,27 These bands at around 3200 and 3400 cm−1 are commonly understood as the strongly hydrogen-bonded ice-like band in the region of IR absorption of ice, and the weakly hydrogen-bonded liquid-like band in the region of the IR absorption of liquid water, respectively.17 Measurements in the higher wavenumber range up to 3800 cm−1 were avoided, since the free OH oscillators (3710 to 3750 cm−1) are no C


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The Journal of Physical Chemistry C Phe subphase were measured at different concentrations (Phe =0 to 12 mM, DPPC = 1.3 × 1014 to 3.6 × 1014 molecules/ cm2) with mostly the ppp polarization scheme in the CH spectral region, and the ssp polarization in the OH region. These measurements were performed in both the acidic and neutral pH conditions. The pH of the Phe solution of different concentrations (up to 12 mM) in ultrapure Millipore water was in the acidic range of 5.6 ± 0.1. For the neutral pH condition, the Phe solution was neutralized to pH ∼ 7.3 using an aqueous solution of disodium hydrogen phosphate. In general, the concentration dependence of the VSFG intensity in the CH stretch region is better characterized by the νas(CH3) band intensity in the ppp polarization than the νs(CH3), and νFR(CH3) band intensities in the ssp polarization because of complications due to Fermi interaction,36 and interference from interfacial water OH stretch37 in the ssp spectra. Similarly, in our earlier work on the interaction of SDS with DPPC monolayer7 we found that the νas(CH3) band intensity in the ppp polarization represents the surface concentration more accurately than the νs(CH3), and the νFR(CH3) band intensities in the ssp polarization. Therefore, to understand the effect of the bulk Phe concentration, we have compared the VSFG spectra only in the ppp polarization. 4.2.1. CH Stretch Region. Acidic pH at 5.6. The Phe molecules are not sufficiently surface active38 to generate the VSFG spectra in the CH stretching region in the bulk concentrations employed. However, the vibrational spectra of Phe at different pH are reported in a bulk aqueous phase.16 But, the presence of Phe in the subphase affects the VSFG spectra of the DPPC monolayer. At a low DPPC surface density (1.8 × 1014 molecules/cm2), the CH stretching intensity in the ppp polarization is increased slightly at the Phe subphase (4 mM bulk solution) in comparison to the water subphase (Figure 1, graph A). The relative increase of the intensity at the Phe subphase is more obvious at its higher bulk concentration of 12 mM (Figure 1, graph B). Thus, the relative increase in the intensity at the Phe subphase is found to depend on the Phe bulk concentration. For the low DPPC surface density, the VSFG intensity in the CH region increases with the Phe bulk concentration up to 16 mM. But an increment at 16 mM is lower than that at 12 mM of Phe (not shown in the figure). The relative increase of the intensity depends on the DPPC surface density as well, in addition to the Phe bulk concentration. In comparison to the low DPPC surface density, the relative increase in the intensity is correspondingly less for both the Phe bulk concentrations of 4 mM (Figure 1, graph C) and 12 mM (Figure 1, graph D), at a high DPPC surface density (3.6 × 1014 molecules/cm2). For example, at the high DPPC surface density (Figure 1, graph D) the relative increase in the VSFG intensity in the presence of Phe (12 mM) is much less than at the low density (Figure 1, graph B). Please note that Figure 1 depicts four data sets (graphs A− D), and each set of experiments is performed under similar experimental conditions, and mostly on the same day for measuring a relative change in the VSFG intensity. Though efforts were made to maintain the same experimental conditions for all the sets in the graph, some variations could be observed in intensity between different sets performed on different days. The observed influence of the Phe molecules on the VSFG spectra of DPPC molecules suggests an interaction between these molecules, leading to some enhanced polar ordering of the alkyl tails of the DPPC molecules.

Figure 1. Dependence of the VSFG spectra of a DPPC monolayer in the CH stretch region at the water and Phe subphase at pH = 5.6 on the DPPC surface density and Phe bulk concentration. DPPC at 1.8 × 1014 molecules/cm2 with Phe at 4 mM (A) and 12 mM (B) and DPPC at 3.6 × 1014 molecules/cm2 with Phe at 4 mM (C) and 12 mM (D). Spectra in black, red, and blue are for Phe only, DPPC only, and DPPC + Phe, respectively. Points are experimental data in the ppp polarization, and solid curves are theoretical fits. Spectra are vertically displaced for clarity.

Neutral pH at 7.3. The VSFG spectra of a DPPC monolayer in the presence of the Phe subphase were measured at the physiological pH of ∼7.3, and at this pH also the Phe molecules exist mostly in the zwitterionic state. The VSFG intensity dependence on the Phe concentration was investigated at two different DPPC surface densities. Like the effects of Phe in the acidic condition, in the neutral condition as well, the effects of Phe on the VSFG intensity are qualitatively similar. Figure 2 depicts the effects of the Phe bulk concentrations (4 and 12 mM) on the VSFG spectra of a DPPC monolayer at its low (1.4 × 1014 molecules/cm2) and high (2.6 × 1014 molecules/ cm2) surface density. With increasing Phe concentration for a particular DPPC surface density, the relative increment in the intensity initially shows an increasing trend, then a decreasing trend, and finally the saturation at the high Phe bulk concentration, implying no effect of the higher concentrations of Phe on the DPPC monolayer. In some cases at a sufficiently higher concentration of Phe, the VSFG intensity rather decreases. At lower Phe concentration (4 mM) with the low surface density of DPPC, the relative increase in the intensity is significant (Figure 2, graph A). However, at 12 mM D


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DPPC surface density the VSFG intensity gets saturated at a relatively lower Phe concentration. It is observed that at high DPPC surface density (2.6 × 1014 molecules/cm2), the saturation occurs at a lower Phe concentration (≤8 mM) in the neutral condition than that in the acidic condition (>8 mM). Thus, we find that the effects of Phe, in both the acidic and neutral pH, are qualitatively similar on a DPPC monolayer. In both these cases, the Phe molecules from the subphase get adsorbed at the interface, and interact with the DPPC molecules, leading to some enhanced polar ordering of the alkyl tails of the DPPC molecules. However, we observed qualitatively different effects of Phe in acidic and neutral pH on the VSFG spectra in the OH stretch region. 4.2.2. OH Stretch Region. Acidic pH. The interaction between the Phe and DPPC molecules is also expected to influence the VSFG spectra of the interfacial water molecules. Unlike in the CH stretching region, wherein the VSFG intensity increases, in the OH stretching region it decreases with increasing Phe bulk concentration (shown in Figure 3, graphs A−D), and this decrease in the intensity is more pronounced at a greater Phe (DPPC) concentration for a particular DPPC (Phe) concentration. The figure depicts a larger decrease in the VSFG intensity in the ssp polarization at

Figure 2. Dependence of the VSFG spectra of a DPPC monolayer in the CH stretch region at the water and Phe subphase at pH = 7.3 on the DPPC surface density and Phe bulk concentration. DPPC at 1.4 × 1014 molecules/cm2 with Phe at 4 mM (A) and 12 mM (B) and DPPC at 2.6 × 1014 molecules/cm2 with Phe at 4 mM (C) and 12 mM (D). Spectra in black, red, and blue are for Phe only, DPPC only, and DPPC + Phe, respectively. Points are experimental data in the ppp polarization, and solid curves are theoretical fits. Spectra are vertically displaced for clarity.

concentration of Phe there is almost no increase in the intensity; and it gets saturated (Figure 2, graph B). The DPPC surface density has also a similar effect on the intensity as in the acidic pH. At a particular Phe concentration, the relative increment in the intensity is less for the high DPPC surface density. For example, at the low surface density of DPPC, the relative increase in the intensity is significant for 4 mM of the Phe concentration (Figure 2, graph A), as mentioned earlier, but at the high DPPC surface density, the increase is small (Figure 2, graph C). Similarly, at 12 mM concentration of Phe there is almost no increase in the intensity at the low DPPC surface density due to saturation (Figure 2, graph B), but a slight decrease is observed at the high DPPC density (Figure 2, graph D). Thus, in comparison to the low surface density, at the high density of DPPC the saturation of the VSFG intensity is found to occur at a relatively lower Phe concentration. At high DPPC surface density, the VSFG intensity gets saturated in both the neutral and acidic conditions with increasing Phe concentration. However, at low DPPC density the saturation of the VSFG intensity occurs at a relatively higher Phe concentration for both the neutral and acidic conditions. In general, for a neutral condition at a particular

Figure 3. Dependence of the VSFG spectra of DPPC monolayer in the OH stretch region in the ssp polarization due to the interfacial water molecules at the water and Phe subphase at pH = 5.6 on DPPC surface density and Phe bulk concentration. DPPC at 1.8 × 1014 molecules/cm2 with Phe at 4 mM (A) and 12 mM (B) and DPPC at 3.6 × 1014 molecules/cm2 with Phe at 4 mM (C) and 12 mM (D). Spectra in black, red, and blue are for Phe only, DPPC only, and DPPC + Phe, respectively. E


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fully aligned molecules due to a large number of the Phe molecules at the interface. Neutral pH. Unlike in the acidic pH, wherein the VSFG intensity in the OH stretching region decreases in the Phe subphase in comparison to the water subphase, at the neutral pH the VSFG intensity increases at a lower Phe bulk concentration (shown for 4 mM in Figure 4, graph A). But

the Phe bulk concentration of 12 mM (graph B) in comparison to 4 mM (graph A) at a low DPPC surface density (1.8 × 1014 molecules/cm2). Similar results of a larger decrease in the intensity for a greater Phe concentration (12 mM, graph D) in comparison to a low Phe concentration (4 mM, graph C) are observed even for a high DPPC surface density (3.6 × 1014 molecules/cm2). An increased DPPC surface density has similar effects as the increased Phe bulk concentration on the VSFG intensity. At a greater DPPC surface density (graph C for 4 mM and graph D for 12 mM of Phe concentration) the decrement in the intensity is more than lower DPPC density (graph A for 4 mM and graph B for 12 mM of Phe concentration) for a particular Phe concentration. The reduced VSFG intensity can be attributed, in general, to either decreased surface density/sampling depth or increased random orientation (decreased alignment of water molecules perpendicular to the interface) of interfacial water molecules.2 The interaction of the Phe molecules with the DPPC molecules should lead to an enhanced polar orientation, and not a random orientation of the interfacial water molecules. We observed, as described earlier, an enhanced polar orientation of the acyl chains of the DPPC molecules on interaction with the Phe molecules. Similarly, we do not expect any significant change in the surface density of the DPPC molecules in the presence of the Phe molecules. Thus, a decreased sampling depth of the interfacial water molecules should be responsible for the reduced VSFG intensity in the OH stretching frequency region. The results imply that the interfacial water molecules are replaced by the Phe molecules, which should be adsorbed from the subphase, and get incorporated in the DPPC layer. This process will lead to a decreased sampling depth of the interfacial water molecules and hence a decreased VSFG intensity in the OH stretch region. The exclusion of the interfacial water molecules by the Phe molecules is expected to be more effective for somewhat a packed DPPC monolayer that is at its greater surface concentration. Therefore, the Phe molecules are more effective in reducing the VSFG intensity in the OH stretching region for a larger surface coverage with the DPPC molecules. Similarly, a greater bulk concentration of Phe should be more effective in replacing the interfacial water molecules. Thus, we observed a decreasing VSFG intensity in the OH stretching region with the increasing Phe concentration. For the similar reason, the VSFG intensity in the CH stretching region is expected to increase with the increasing DPPC surface concentration. The interfacial Phe molecules intercalated in the DPPC layer will further compact the alkyl tails of the lipid, increasing the VSFG intensity of the CH3 band. The intercalation of the Phe molecules is in agreement with a reported increase in the area per lipid, and the surface pressure of the monolayer.8 But the intensity can increase up to a certain DPPC surface concentration, beyond which the phenyl ring of the Phe molecules cannot penetrate in the hydrophobic region of the DPPC monolayer, because of the steric factor, to influence the structure of the alkyl tails. Moreover, at a higher DPPC surface density the terminal CH3 group is already oriented to the large extent, and hence addition of the Phe molecules will have only a minor effect or no observable effect depending on its concentration. In fact, at a still higher concentration of Phe, the VSFG intensity due to the CH3 group is found to decrease, which can be due to aggregation of the CH3 group or some disruption of almost

Figure 4. Dependence of the VSFG spectra of DPPC monolayer in the OH stretch region in the ssp polarization due to the interfacial water molecules at the water and Phe subphase at pH = 7.3 on the surface density of DPPC and Phe bulk concentration. DPPC at 1.4 × 1014 molecules/cm2 with Phe at 4 mM (A) and 12 mM (B) and DPPC at 2.6 × 1014 molecules/cm2 with Phe at 4 mM (C) and 12 mM (D). Spectra in black, red, and blue are for Phe only, DPPC only, and DPPC + Phe, respectively.

with an increasing Phe concentration the relative value of the positive increment increases, and subsequently it decreases, and becomes nil (graph B), implying no effect of Phe concentration on the VSFG intensity. At still higher Phe concentration (up to 16 mM) the intensity remains unaffected at a lower DPPC surface density (1.4 × 1014 molecules/cm2), but shows a decrease in the intensity (graph D), at a higher DPPC surface density (2.6 × 1014 molecules/cm2). Thus, Figure 4 shows that at low density of DPPC (graphs A and B), for a change of the Phe concentration from 4 to 12 mM we can observe from a positive increment to almost no effect on the VSFG intensity. But at a high density of DPPC (graphs C and D) we can observe a change from the positive increment at the Phe concentration of 4 mM to a negative increment at that of 12 mM. An increase in the VSFG intensity in the OH region implies increased sampling depth of the interfacial water molecules at F


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concentration. Figure 5 depicts the dependence of a change in the surface pressure (Δπ) of DPPC, at the Phe subphase with

the neutral pH. However, at an acidic pH we observed a decreased depth of interfacial water molecules. These results indicate that the Phe molecules in different pH conditions interact differently with the DPPC monolayer. At the acidic pH, the decreased interfacial water molecules suggest that the Phe molecules intercalate deep in the DPPC monolayer excluding water molecules from the interface region. However, at the neutral pH the Phe molecules interact with the DPPC monolayer and condense it, but do not intercalate in the monolayer, and thus allow exposure of greater interfacial water molecules. This process will lead to an increased sampling depth of the interfacial water molecules, and hence increased VSFG intensity in the OH stretch region.2 These results suggest that the extent of intercalation of the Phe molecules from the bulk into a DPPC monolayer at the air−water interface depends on the pH of the Phe molecules. The Phe molecules at the neutral pH remain in the polar head region of the DPPC molecules, whereas that at acidic pH appear to intercalate relatively deeper, which is in agreement with a recent work.8 The penetration of the interface is found to be dependent on the state of the Phe molecules, and predicted still deeper for the neutral Phe molecules.1 The interaction between the Phe and DPPC monolayer is shown schematically in Scheme 2. This explanation on the effect of the Phe subphase on the VSFG spectra of DPPC at the air−water interface is supported by the surface pressure measurements as well.

Figure 5. Figure depicts the change in the surface pressure of DPPC monolayer on the Phe bulk concentration at pH = 5.6 for the DPPC surface density of 1.3 × 1014 and 2.5 × 1014 molecules/cm2. Points are experimental data, and solid lines are drawn to connect the points.

respect to the water subphase in acidic pH, on the bulk Phe concentration (0 to 12 mM) for the DPPC surface density of 1.3 × 1014 and 2.5 × 1014 molecules/cm2. The figure shows that for a particular DPPC concentration Δπ increases with the Phe bulk concentration, and it gets saturated at a higher Phe concentration. The values of Δπ depend on the DPPC surface density as well, and these are greater for higher DPPC density. Difference in Δπ from 4 to 12 mM of Phe is more marked in a lower than higher DPPC surface density; a similar effect is observed in the VSFG spectra in the CH frequency region (Figure 1). In contrast to the surface pressure dependence of DPPC on Phe in acidic condition, which has a positive deviation, the surface pressure in the neutral condition showed a negative deviation particularly for a higher DPPC surface coverage. Figure 6 depicts dependence of Δπ on the bulk Phe concentration for three different DPPC surface densities of 1.3 × 1014, 2.0 × 1014, and 2.7 × 1014 molecules/cm2. The figure shows that the surface pressure decreases with an increasing surface density of DPPC for a particular Phe concentration. The change in the value of Δπ is significant for the highest DPPC surface concentration (2.7 × 1014 molecules/ cm2) investigated. Thus, these surface pressure results support the VSFG results that the Phe molecules interact differently with the interfacial DPPC molecules in the acidic and neutral subphase conditions. Similar results have been reported on the interaction of the Phe molecules with the DPPC monolayer by the surface pressure−area (π−A) isotherm measurement.8 An increase in the surface pressure, implying a decrease in the surface tension, in an acidic condition suggests a replacement of the interfacial water molecules by the Phe molecules. Thus, the Phe molecules are adsorbed from the subphase, and get incorporated in the Langmuir DPPC layer. On the contrary in the neutral pH condition, the decrease in the surface pressure, implying an increase in the surface tension toward values of pure water, suggests condensation of lipids to some extent in the surface exposing water regions. Thus, it implies that the Phe molecules

Scheme 2. Schematic Representation of Interaction between Phe and DPPC Monolayer at an Air−Water Interface

4.3. Surface Pressure of DPPC Monolayer. The surface pressure of a Langmuir monolayer of DPPC at the water subphase, and Phe subphase was measured to get a better insight into the VSFG results. A known volume of DPPC in CHCl3 solution (corresponding to 3.7 × 1013 to 3.5 × 1014 molecules/cm2) was spread at the water subphase or an aqueous Phe solution subphase in a Teflon trough using a Hamilton syringe. We measured the surface pressure dependence of DPPC on its surface concentration at the air−water interface, which shows an expected increasing trend (shown in Supporting Information as Figure S2). This surface pressure− surface density plot of DPPC depicts qualitatively usual π−A isotherm in the reverse order of abscissa; that is, increasing values of the surface density imply decreasing surface area per molecule. We also measured the surface pressure of the DPPC monolayer in the presence of an aqueous Phe solution that is acidic in nature (pH = 5.6), and also in the neutral pH solution (pH ∼ 7.3). In acidic pH, we found that it increases with an increasing Phe concentration, and gets saturated at a higher G


Article


molecules induced by SDS molecules.3 Overall nature of interaction of Phe and SDS7 molecules with DPPC monolayer appears to be similar, and involves both hydrophobic, and electrostatic interactions. Still there is a difference between interactions of a DPPC monolayer with Phe than that with SDS.7 For Phe its polar head appears to play an important role, and determines whether the molecule will intercalate, or intercalate deeper in the monolayer or remain close to the polar head groups of the DPPC molecules.1 The hydrophobic interactions between hydrocarbon chains of DPPC and Phe molecules have a small effect because of a smaller alkyl chain length of the latter. However, instead the hydrophobic interactions between the chains of the DPPC molecules result, because the interactions of the Phe molecules bring these DPPC molecules closer. In contrast for the SDS molecules, the main driving force for their insertion into the DPPC monolayer is the hydrophobic interaction between their long hydrocarbon chains. The hydrophobic interactions are reflected in the VSFG spectra in the CH region. Since the hydrocarbon tail of SDS is much longer than that of Phe, relative increase in the CH3 intensity is much greater in the former. For the same reason, relative decrease in the vs(CH2) intensity is significant in the case of SDS. The effect of electrostatic interaction can be seen in the VSFG spectra in the OH stretch region. The OH intensity is invariably enhanced in the SDS subphase due to polar orientation of the interfacial water molecules, because of the charged headgroup of SDS. However, the intensity is either enhanced or decreased in the Phe subphase depending on the pH of the Phe molecules. In case of the acidic pH, the intensity is generally decreased, whereas in the neutral pH the intensity is increased at a low Phe concentration. In the SDS subphase the change in the OH intensity is mainly due to the polar orientation of the interfacial water molecules, whereas in the Phe subphase that is mainly due to a change in the sampling depth. Thus, the polar orientation of the interfacial water molecules is much more significant by SDS, and the VSFG intensity in the OH stretching region is mainly governed by SDS bulk concentration with a negligible/small role for the DPPC surface concentration. In contrast, the VSFG intensity of the DPPC monolayer at the air−water interface in the presence of the Phe subphase is influenced by concentration of both DPPC and Phe, although Phe molecules alone do not lead to much polar-ordering of water molecules. A small concentration of SDS (μM order) makes a significant change in the surface pressure of the DPPC monolayer.7 Similar changes in the surface pressure require much higher Phe concentration at the millimolar level. This implies that the interaction between SDS and DPPC is stronger than that between Phe and DPPC, because of the increased hydrophobic interactions between the long hydrocarbon chains of both SDS and DPPC molecules in comparison to a shorter chain length of Phe (as mentioned earlier). Similarly, electrostatic interaction of zwitterionic DPPC is much stronger with anionic surfactant SDS than with Phe (mostly zwitterionic).

Figure 6. Figure depicts the change in the surface pressure of DPPC monolayer on the Phe bulk concentration at pH = 7.3 for the DPPC surface density of 1.3 × 1014, 2.0 × 1014, and 2.7 × 1014 molecules/ cm2. Points are experimental data, and solid lines are drawn to connect the points.

interact with the DPPC monolayer in such a manner that the DPPC molecules come closer creating some vacant space for additional water molecules to be exposed. This situation is likely when the Phe molecules interact with mostly the head region of the DPPC molecules, but do not penetrate the monolayer. The surface pressure measurements suggesting the process of an exclusion and inclusion of the interfacial water molecules in the DPPC monolayer in the acidic and neutral conditions of the Phe subphase, respectively, corroborate the VSFG measurement in the OH stretching frequency region. As discussed earlier, the VSFG intensity of the interfacial water molecules for the DPPC Langmuir monolayer is reduced in the presence of the acidic Phe subphase, and increased in the neutral Phe subphase. The Phe molecules induced polar orientation does not seem to play any significant role in changing the VSFG intensity in the OH stretching frequency. As stated earlier, a decrease (in acidic pH) or increase (in neutral pH) in the sampling depth of the interfacial water molecules due to an interaction of Phe with the DPPC molecules is responsible for a decreased or increased VSFG intensity in the OH frequency region. An increase (in acidic pH) or decrease (in neutral pH) in the surface pressure supports the observed VSFG intensity. Thus, the VSFG intensity in the OH stretch region is enhanced mainly by altered sampling depth of the water molecules, with a negligible contribution from its polar orientation. In contrast, the VSFG intensity in the CH stretch region is enhanced mainly by the polar orientation of the hydrophobic chain of DPPC molecules. 4.5. Comparison between Phe-DPPC and SDS-DPPC Interactions. Both Phe and SDS are water-soluble with a longer hydrophobic chain length of the latter. Hence it is worth comparing interaction of Phe and SDS molecules with DPPC monolayer. The SDS molecules intercalate into the DPPC monolayer, and the VSFG intensity of the CH3 stretching frequency is increased (for low Phe concentration at μM level) and that of the CH2 is decreased in comparison to the water subphase.7 However, for high SDS concentration (at millimolar level) the VSFG intensity of the CH3 stretching frequency is decreased, probably due to the aggregation of the DPPC

5. CONCLUSIONS We investigated molecular-level interactions between a DPPC monolayer and Phe employing VSFG spectroscopy, and observed that both the polar headgroup and hydrophobic tails of the DPPC molecules are affected. Increased VSFG intensity of the vas(CH3) mode in the Phe subphase, at both the acidic and neutral pH, with respect to the water subphase H


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Phenylalanine with a Phospholipid Monolayer at the Water−Air Interface. J. Phys. Chem. B 2015, 119, 9038−9048. (2) Watry, M. R.; Tarbuck, T. L.; Richmond, G. L. Vibrational SumFrequency Studies of a Series of Phospholipid Monolayers and the Associated Water Structure at the Vapor/Water Interface. J. Phys. Chem. B 2003, 107, 512−518. (3) Harper, K. L.; Allen, H. C. Competition between DPPC and SDS at the Air-Aqueous Interface. Langmuir 2007, 23, 8925−8931. (4) Nakata, S.; Deguchi, A.; Seki, Y.; Fukuhara, K.; Goto, M.; Denda, M. Ability of Sodium Dodecyl Sulfate to Transiently Stabilize a Phospholipid Molecular Layer. Thin Solid Films 2016, 615, 215−220. (5) Meister, A.; Kerth, A.; Blume, A. Interaction of Sodium Dodecyl Sulfate with Dimyristoyl-sn-glycero-3-phosphocholine Monolayers Studied by Infrared Reflection Absorption Spectroscopy. A New Method for the Determination of Surface Partition Coefficients. J. Phys. Chem. B 2004, 108, 8371−8378. (6) Liu, B.; Hoopes, M. I.; Karttunen, M. Molecular Dynamics Simulations of DPPC/CTAB Monolayers at the Air/Water Interface. J. Phys. Chem. B 2014, 118, 11723−11737. (7) Saha, A.; SenGupta, S.; Kumar, A.; Naik, P. D. Interaction of Sodium Dodecyl Sulfate with Lipid Monolayer Studied by SumFrequency Generation Spectroscopy at Air−Water Interface. J. Phys. Chem. C 2017, 121, 13175−13182. (8) Cutró, A. C.; Hollmann, A.; Cejas, J.; Maturana, P.; Disalvo, E. A.; Frías, M. A. Phenylalanine Interaction with Lipid Monolayers at Different pHs. Colloids Surf., B 2015, 135, 504−509. (9) Kotecka, K.; Krysinski, P. Effect of Tetracycline Antibiotic on the Monolayers of Phosphatidylcholines at the Air−Water Interface. Colloids Surf., A 2015, 482, 678−686. (10) Goto, T. E.; Caseli, L. The Interaction of Mefloquine Hydrochloride with Cell Membrane Models at the Air−Water Interface is Modulated by the Monolayer Lipid Composition. J. Colloid Interface Sci. 2014, 431, 24−30. (11) Guzman, E.; Santini, E.; Zabiegaj, D.; Ferrari, M.; Liggieri, L.; Ravera, F. Interaction of Carbon Black Particles and Dipalmitoylphosphatidylcholine at the Water/Air Interface: Thermodynamics and Rheology. J. Phys. Chem. C 2015, 119, 26937−26947. (12) Chen, X.; Allen, H. C. Interactions of Dimethylsulfoxide with a Dipalmitoylphosphatidylcholine Monolayer Studied by Vibrational Sum Frequency Generation. J. Phys. Chem. A 2009, 113, 12655− 12662. (13) Adler-Abramovich, L.; Vaks, L.; Carny, O.; Trudler, D.; Magno, A.; Caflisch, A.; Frenkel, D.; Gazit, E. Phenylalanine Assembly into Toxic Fibrils Suggests Amyloidetiology in Phenylketonuria. Nat. Chem. Biol. 2012, 8, 701−706. (14) Singh, V.; Rai, R. K.; Arora, A.; Sinha, N.; Thakur, A. K. Therapeutic Implication of L-Phenylalanine Aggregation Mechanism and Its Modulation by D-Phenylalanine in Phenylketonuria. Sci. Rep. 2015, 4 (1), 3875. (15) Griffith, E. C.; Vaida, V. Ionization state of L-Phenylalanine at the Air−Water Interface. J. Am. Chem. Soc. 2013, 135, 710−716. (16) Olsztynska, S.; Komorowska, M.; Vrielynck, L.; Dupuy, N. Vibrational Spectroscopic Study of L-Phenylalanine: Effect of pH. Appl. Spectrosc. 2001, 55, 901−907. (17) Tian, C. S.; Shen, Y. R. Recent Progress on Sum-Frequency Spectroscopy. Surf. Sci. Rep. 2014, 69, 105−131. (18) Sung, W.; Kim, D.; Shen, Y. R. Sum-Frequency Vibrational Spectroscopic Studies of Langmuir Monolayers. Curr. Appl. Phys. 2013, 13, 619−632. (19) Zhuang, X.; Miranda, P. B.; Kim, D.; Shen, Y. R. Mapping Molecular Orientation and Conformation at Interfaces by Surface Nonlinear Optics. Phys. Rev. B: Condens. Matter Mater. Phys. 1999, 59, 12632−12640. (20) Wang, H.-F.; Gan, W.; Lu, R.; Rao, Y.; Wu, B.-H. Quantitative Spectral and Orientational Analysis in Surface Sum Frequency Generation Vibrational Spectroscopy (SFG-VS). Int. Rev. Phys. Chem. 2005, 24, 191−256.

suggests a microscopic ordering of the hydrocarbon chains of DPPC due to an interaction with the Phe molecules. The measured VSFG spectra in the CH stretch region did not show a significant difference between interactions of Phe at acidic pH of 5.6 and at neutral pH of 7.3 with the DPPC monolayer. However, the VSFG spectra in the OH stretch region, and the surface pressure of the DPPC monolayer are significantly different at these two pHs. The VSFG intensity in the OH region of the DPPC monolayer is reduced at the acidic pH and enhanced at the neutral pH. The contrasting effect is mainly due to a change in the sampling depth, which is decreased at the acidic pH, and increased at the neutral pH in the Phe subphase. This explanation is supported by a change in the surface pressure of the DPPC monolayer in the presence of Phe. The surface pressure is increased at the acidic pH and decreased at the neutral pH in the Phe subphase. The increased surface pressure can result from the intercalation of the Phe molecules in the DPPC monolayer leading to the replacement of some interfacial water molecules with the Phe molecules, and thus reducing the sampling depth for the VSFG spectra. However, the decreased surface pressure induced by the neutral Phe could be explained by a direct interaction of Phe with the lipid head groups, condensing some lipids at the interface, which would promote the formation of a lipid free space for the additional water molecules to be exposed. This condition leads to an increase in the sampling depth, and thus enhanced VSFG spectra in the OH region in the presence of the neutral Phe molecules. A comparison between DPPC−Phe and DPPC− SDS interactions suggests that hydrophobic interaction is relatively much stronger, and plays a significant role in the latter due to the long alkyl chain lengths. The hydrophobic interactions lead to an increase in the conformational order of the alkyl chain of DPPC, such as a decrease in the gauche, and increase in trans conformers. The orientation of the interfacial water molecules of the DPPC monolayer in the SDS subphase at the air−water interface is controlled mostly by the SDS bulk concentration.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.7b11049. VSFG spectra and the surface pressure of the DPPC monolayer at different surface density of DPPC and tables with spectral fitted parameters (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(A.K.) E-mail: [email protected]. Telephone: 91-2225590302. Fax: 91-22-25505151, 25505331. ORCID

Awadhesh Kumar: 0000-0003-4377-6610 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge assistance from Dr Sipra Choudhury in surface pressure measurements. REFERENCES

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