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Colloids and Surfaces B: Biointerfaces 55 (2007) 241–250
Infrared spectroscopy studies of cation effects onlipopolysaccharides in aqueous solution
Sanjai J. Parikh, Jon Chorover ∗Department of Soil, Water and Environmental Science, The University of Arizona, Tucson, AZ 85721, United States
Received 6 December 2006; received in revised form 20 December 2006; accepted 20 December 2006Available online 30 December 2006
bstract
The conformation of amphiphilic lipopolysaccharides (LPS) influences the behavior of free and cell-bound LPS in aqueous environments,ncluding their adhesion to surfaces. Conformational changes in Pseudomonas aeruginosa serotype 10 LPS aggregates resulting from changes inolution pH (3, 6, and 9), ionic strength [I] 1, 10, and 100 mmol L−1, and electrolyte composition (NaCl and CaCl2) were investigated via attenuatedotal reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy. ATR-FTIR data indicate that LPS forms more stable aggregates in NaClelative to CaCl2 solutions. Time- and cation-dependent changes in ATR-FTIR data suggest that LPS aggregates are perturbed by Ca2+ complexation
t lipid A phosphoryl groups, which leads to reorientation of the lipid A at the surface of a ZnSe ATR internal reflection element (IRE). PolarizedTR-FTIR investigations reveal orientation of LPS dipoles approximately perpendicular to the IRE plane for both Na- and Ca-LPS. The resultsndicate that changes in solution chemistry strongly impact the conformation, intermolecular and interfacial behavior of LPS in aqueous systems. 2007 Elsevier B.V. All rights reserved.
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eywords: LPS; ATR-FTIR spectroscopy; Lipid A; Polarized FTIR; Endotoxin
. Introduction
Lipopolysaccharides (LPS) are amphiphilic moleculesFig. 1a) anchored to the surface of Gram-negative bacteria [1].he hydrophobic lipid A portion of the molecule (Fig. 1b) is
mbedded in the outer membrane, with very little structural vari-tion between different bacteria [1]. The O-antigen (hydrophilicolysaccharide region) extends from the bacterial surface anday facilitate adhesion to environmental surfaces. For exam-
le, Jucker et al. [2] have shown that H-bonding interactions of-antigen with oxide surfaces contribute to bacterial adhesion.ost LPS research has been motivated by the immunoreac-
ive properties of these biomacromolecules [3–8] that are thuslso referred to as endotoxins. Given the importance of surfi-ial macromolecules to bacterial deposition in environmentaledia (e.g., mineral and organic particles in soils, water filtra-
ion systems), there is also a need to elucidate the role of LPSn environmental processes such as conditioning film formationnd bacterial adhesion.
∗ Corresponding author. Tel.: +1 520 626 5635; fax: +1 520 621 1647.E-mail address: [email protected] (J. Chorover).
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Bacterial LPS occur in either “smooth” or “rough” form [1].PS in the rough form lack the O-antigen. The O-antigen (20–70
epeating units of three to five sugar molecules) of the smoothorm can protrude up to 30 or more nanometers from the cellurface. The O-antigens of Pseudomonas aeruginosa LPS areelieved to extend up to 40 nm from the cell, depending on solu-ion chemistry [5]. The core region of LPS (present in both theough and smooth form) consists of 5–10 negatively chargedugar units [9]. Due to longer protrusion from the cell, LPS withhe O-antigen (i.e., smooth LPS) are likely responsible for inter-ctions of Gram-negative bacteria with environmental surfaces9,10]. Surface affinity of LPS is variable, depending on bacterialtrain and substrate composition. Specifically, LPS with longer-antigens are adsorbed more extensively and less reversibly
9]. Although negatively charged bacteria exhibit electrostaticepulsion with many like-charged environmental surfaces, it haseen suggested that polymer bridging by LPS can transcend thenergy barrier by forming hydrogen bonds with mineral surfaces9].
As a result of cell turnover and lysis, LPS occurs in both “cell-ound” and “free” forms in natural aquatic systems [11]. Theipid A of cell-bound LPS is contained within the outer mem-rane of the cell and, therefore, it does not participate directly
242 S.J. Parikh, J. Chorover / Colloids and Surfac
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ig. 1. Schematic diagram of (a) smooth lipopolysaccharide and (b) lipid A [1].
n cell adhesion to other surfaces. Conversely, adsorption of freePS to surfaces may be mediated by functional groups asso-iated with either hydrophilic or hydrophobic portions of theolecule. Bacterial extracellular polymeric substances, which
nclude free LPS [12], contribute to the formation of “con-itioning films” on environmental surfaces [13–15], which inurn modify subsequent cell adhesion processes [16,17]. Evenn free LPS, however, exposure of the lipid A is limited by LPSmphiphilic properties that promote intermolecular associationsnd the formation of supramolecular structures above a criticalggregation concentration (CAC) [18–21]. Indeed, such aggre-ates of free LPS have been used above the CAC to representell-bound forms under the assumption that, in both cases, onlyhe O-antigen is exposed for interaction with environmental sur-aces. For example, Jucker et al. [2] measured the adsorption ofPS micelles to TiO2, Al2O3, SiO2, and glass beads. However,
he nature of restructuring of such aggregates that may occurpon association with surfaces is unknown.
LPS contains weakly acidic (hydroxyl, phosphoryl, amide)unctional groups associated with the lipid A, core and O-antigenFig. 1a). Changes in aqueous phase pH, ionic strength andonic composition are expected to affect LPS ionization andackground ion complexation reactions in a manner that isomparable to that for model polyelectrolytes [22–24]. Con-ormation of individual LPS molecules (i.e., monomers) isikely to be affected by electrostatic repulsion between anionicunctional groups on the O-antigen. Increased background elec-rolyte concentration, decreased pH and increased prevalence ofivalent (relative to monovalent) background counterions are allxpected to diminish such repulsion and to promote coiling.
Human-health-related studies have shown the importance ofalcium in the conformation and aggregation of LPS, howeverone of these studies have attempted to model the behavior of
PS under conditions representing natural waters. Crystallinealcium silicate hydrate is effective for removing LPS from solu-ion [25,26], and the strong affinity of Ca2+ for phosphate groupsn the lipid A region has been implicated as a key mediator ofTa1a
es B: Biointerfaces 55 (2007) 241–250
his process [25]. The presence of Ca2+ has also been shown toncrease LPS aggregation (compared to Na+), even acting as abridge” between LPS subunits to form supramolecular vesicletructures [27–29]. Other researchers have also demonstrated atrong affinity between Ca2+ and the phosphate groups in theipid A region of rough LPS [18,30,31].
Studies on rough LPS show that at low pH insoluble com-lexes result from diminished hydration and charge repulsionf the lipid A region. At high pH, diminished hydrogen bond-ng and increased hydration of the lipid A results in micellarggregates [30,32]. It is not clear how changes in pH affect theolubility of smooth LPS, as the solubility of these molecules isictated largely by the hydrophilic O-antigen chain. Coughlint al. [30] also suggest that pH and salt content can influenceggregate structure of rough LPS. These solution chemistryffects are likely to also affect smooth LPS aggregation andurface interaction but there are few reports in the literature.astowsky et al. [33] performed a molecular modeling study on
he conformation of smooth LPS that indicated a high degreef flexibility for the O-antigen chain, with the maximum lengthf four repeating units (sugar rings) measuring 9.6 nm. How-ver, in that study the effect of solution chemistry was notnvestigated.
Other studies of smooth LPS conformation have been lim-ted to aggregation without considering the effects of pH, I, oron composition [19,20,34]. There have been LPS conforma-ional studies focused on rough LPS [3,6], some of which didnvestigate the effect of solution chemistry [18,30–32]. Whileomparisons to these studies are appropriate, due to the greaterexibility and greater ionic nature of the O-antigen it is expected
hat smooth LPS will vary their conformation to a much greaterxtent with changes in solution chemistry. Few studies havenvestigated these effects for smooth LPS under conditions rep-esentative of natural aqueous systems [2,9,35–37]. Changes inPS monomer conformation and aggregation are expected toave a significant effect on adhesion of both free and cell-boundPS to environmental surfaces. Therefore, the objective of thistudy was to probe the effects of solution chemistry on the con-ormational behavior of smooth LPS in aqueous systems usingn in situ spectroscopic method.
. Research approach: attenuated totaleflectance-Fourier transform infrared (ATR-FTIR)pectroscopy
ATR-FTIR spectroscopy can provide nondestructive,olecular-scale information on biofilms [38–41], bacterial cells
38,42–44], and biomacromolecules, such as LPS [6] immersedn aqueous solutions of variable chemistry. We previously usedTR-FTIR to determine the effects of solution chemistry on
unctional group ionization and associated conformationalhange in extracellular bacterial polymers [45] and to assessechanisms of their adsorption at the �-FeOOH surface [46].
he spectrum derives from infrared-absorbing moieties thatre probed by an evanescent wave. The wave propagates ca.02–103 nm (depending on crystal type, incident beam angle,nd wavelength) beyond the interface of the crystalline internal
S.J. Parikh, J. Chorover / Colloids and Surf
Table 1Pertinent IR assignments for LPS
Wavenumber (cm−1) IR band assignment Reference
2915–2923 νas(CH2)§ [40,73]2848–2854 νs(CH2)† [40]1460–1470 δ(CH2)‡ [53]1250–1265 Metal-complexed/dehydrated
νas(PO2)[53,72,74,75]
1230–1245 Metal-complexed/hydratedνas(PO2
−)[53,72,74,75]
1200–1225 Hydrated νas(PO2−) [53,72,74,75]
1106 νs(PO2−) [53,72]
1020, 1050–1085 ν(C–O, C–O–C) [40,76]960–983 ν(PO2
−) [77]
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§ νas = asymmetric stretching vibration.† νs = symmetric stretching vibration.‡ δ = bending vibrations.
eflection element (IRE) and into an aqueous suspension ofiomacromolecules (Table 1). Beam penetration depth (dp)aries according to Eq. (1):
p = λ
2π[(sin2 θ) − (n1/n2)2]1/2 (1)
here λ is wavelength (nm) of incident radiation, n1 and n2re the refractive index (RI) values for the IRE and sample,espectively, and θ is the effective angle of incidence [47]. TheI value for the ZnSe IRE used in the present study (nZnSe)
s 2.4. The RI range for LPS (nLPS) likely falls in the rangeeported for lipid A (1.50 for pure lipid A; 1.33 with 90%ater) [48], bacterial cells (1.38) [49,50], proteins (1.5) [51],
nd polymer bilaminate films (1.52) [52]. As a result of theimited wave penetration depth, ATR spectra pertain to theegion in close proximity to the sample-IRE interface. Forxample, the dp for bacteria samples on a 45◦ ZnSe IRE is52 nm at 2920 cm−1, 1299 nm at 1240 cm−1, and 1520 nm at060 cm−1.
The use of polarized infrared light for ATR-FTIR spec-roscopy can provide additional information regarding therientation of dipoles at the IRE–liquid interface [53–58]. Polar-zed FTIR requires spectra to be collected independently usingarallel (a||) and perpendicular (a⊥) incident light. Qualitativenalysis of molecular orientation can be carried out throughpectral subtraction of the ⊥ (corrected for electric field compo-ents) from the || spectra [54]. Isotropic arrangement of dipolest the crystal interface will result in a horizontal line. Parallelrientation results in positive peaks in the spectral subtraction,hereas negative peaks indicate perpendicular orientation. The
atio of infrared absorbance a|| to a⊥ is known as the dichroicatio (R):
a||a⊥
= R (2)
ll of the orientation information of dipoles is contained withinhis ratio [54,55]. From R and the electric field components (Ex,y, Ez) obtained via Fresnel’s equations [55] the mean segmen-
al order parameter used in IR spectroscopy (Sk) can be obtained
dNio
aces B: Biointerfaces 55 (2007) 241–250 243
53,55]. When Sk = 0 isotropic arrangement of molecules isresent, Sk = −0.5 results for perfect || alignment, and Sk = 1hen alignment is perfect ⊥ to the surface [53]. The theoryf polarized FTIR, and its applications to studies of lipids andPS, is discussed in Brandenburg and Seydel [55] and Reiter etl. [53].
. Experimental methods
.1. LPS preparation
A single batch of freeze-dried P. aeruginosa serotype 10PS (batch 123K4144; Sigma Inc.) was used for all experi-ents. Analysis of LPS by size exclusion high performance
iquid chromatography revealed high purity and very low pro-ein contamination. Triplicate measurements of the LPS criticalggregation concentration (CAC) were determined via both elec-rical conductivity (EC) [59] and ultraviolet visible spectroscopyUV–vis) [60] techniques for LPS dispersed in Barnstead nanop-re (BNP) water. LPS solutions were prepared above the CACy dissolving 4.0 mg of freeze-dried LPS in 1.0 g of NaClr CaCl2 electrolyte solution at the target ionic strength (I)ith pH adjusted to specific values using HCl or NaOH at
he same I. Samples were vortexed, sonicated for 10 min, andtored overnight at 4 ◦C prior to re-equilibration the follow-ng day to room temperature. All measurements were carriedut at 23 ± 2 ◦C, three different values of pH (3, 6 and 9),nd three different values of I (1, 10 and 100 mmol L−1). Thisxperimental matrix produced nine replicated samples that werexamined by ATR-FTIR for each of the two background elec-rolyte compositions (NaCl and CaCl2), resulting in a total of8 different aqueous chemistry conditions. To resolve thresholdpectral behavior resulting from changes in cation composition,n additional sample set was prepared for infrared studies at0 mmol L−1 ionic strength and pH 6 with incremental variationn the aqueous phase charge fraction of Na+ versus Ca2+.
.2. ATR-FTIR spectroscopy and analysis
FTIR spectra were collected using a Nicolet 560 Magna IRpectrometer (Madison, WI). For each solution chemistry condi-ion, a 1 mL aliquot of LPS solution (4 mg mL−1) was depositedn a 45◦ ZnSe IRE (Spectra-Tech ARK ATR cell). Spectra wereollected beginning at 0, 15, 30, 60, and 120 min after introduc-ion of LPS solution into the ATR cell. Polarized spectra wereollected in triplicate using a ZnSe polarizer at 0◦ and 90◦ inde-endently for Na-LPS and Ca-LPS (10 mM, pH 6). The dichroicatio (R) was calculated for prominent peaks using peak heightnd a mean value from triplicate spectra was used for furthernalysis. All FTIR spectra were collected with 400 scans at acm−1 resolution (collection time: 495 s) using the correspond-
ng LPS-free electrolyte solution as background. Peak locationsere verified via second derivative analysis and peak areas were
etermined via curve fitting using Grams/AI software (Salem,H). Spectral areas of Gaussian/Lorentzian fitted peaks or peakntensities were used to quantify the effect of solution chemistryn LPS conformation and aggregation.
2 urfaces B: Biointerfaces 55 (2007) 241–250
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44 S.J. Parikh, J. Chorover / Colloids and S
. Results
.1. Critical aggregate concentration measurements
Excellent agreement between EC and UV–vis methods wasbserved for P. aeruginosa ser 10 LPS CAC measurements. ECeasurements in BNP water yielded a CAC of 12.7 mg L−1 withstandard deviation of 0.47. Corresponding UV–vis determina-
ion resulted in a CAC value of 13.2 mg L−1 with a standardeviation of 0.88. The CAC measurements are in agreementith values found in the literature for LPS aggregation [19–21].hese values ensure that all experiments were performed above
he CAC.
.2. ATR-FTIR spectroscopy
ATR-FTIR spectra show absorbances corresponding to vibra-ional modes of distinct ser 10 LPS moieties (Table 1).he lipid A is represented by stretching vibrations of–H (2820–2940 cm−1; 1460–1470 cm−1) and phosphate
1200–1265 cm−1, 1106 cm−1, and 960–983 cm−1). The–O–C stretching of polysaccharides on the O-antigen occurs at050–1085 cm−1. Spectral data between 2700 and 1500 cm−1
ave been removed from all figures; this region is devoid of use-ul information and its deletion permits an expanded view ofore important data.The type of background cation (Na+ versus Ca2+) strongly
ffects ATR-FTIR results (Figs. 2 and 3). Relative to Na-PS, spectra for Ca-LPS exhibit several sharper and more
ntense absorbances in the region 1500–900 cm−1, and theylso show significant changes in relative intensity of vari-us bands. When LPS are immersed in a NaCl background,TR-FTIR spectra show strong pH dependence, and a smallerffect of I (Fig. 2). The distinct peak at 1060 cm−1 (Fig. 2a)ndicates a greater relative contribution of polysaccharidesO-antigen; C–O, C–O–C) at pH 3 relative to pH 6 and 9.a-LPS spectra also show a relative increase in fatty acid
CH2) and phosphate (PO2−) group absorbances of the lipid
with increased I. Other notable effects include resolutionf a triplet band (1263, 1228, 1207 cm−1) in the phosphatePO2
−) region of the spectrum at pH 9 in 100 mmol L−1 NaClFig. 2c).
The solubility of LPS in CaCl2 solution decreased at high Ind low pH. White precipitates were formed in 100 mmol L−1 IaCl2 at pH 3 and, to a lesser degree, at pH 6. Strong PO2
−1243, 1213, 1106, 964 cm−1) and CH2 vibrations (∼2920,2852, 1471 cm−1) arising from lipid A are present in alla-LPS spectra (Fig. 3). Contributions of the O-antigen are rela-
ively diminished, with only small peaks observed at 1085, 1060,018, and 983 cm−1. Exceptions are 100 mmol L−1 I CaCl2 (pHand 6) samples where broad bands with peaks at 1085 and
060 cm−1 that emerged (Fig. 3a and b) coincident with therecipitates, are indicative of O-antigen polysaccharides. In con-
rast, spectra for 1 mmol L−1 CaCl2 (pH 6 and 9) show a largeontribution from νas(CH2) (2920 and 2852 cm−1) with muchower absorbances from νas(PO2−) and ν(C–O, C–O–C) (Fig. 3bnd c).
h6ww
ig. 2. ATR-FTIR spectra of P. aeruginosa ser 10 LPS in 1, 10, 100 mmol L−1
aCl at: (a) pH 3, (b) pH 6, and (c) pH 9 (spectra collected at 120 min).
Maximum absorbance values for Ca-LPS were significantly
igher than for Na-LPS. For example, at I = 10 mmol L−1, pHand equivalent LPS concentrations, absorbance at 2920 cm−1as 0.010 for Na and 0.082 for Ca, at 1240 cm−1 absorbanceas 0.007 for Na and 0.213 for Ca, and at 1060 cm−1 absorbance
S.J. Parikh, J. Chorover / Colloids and Surfaces B: Biointerfaces 55 (2007) 241–250 245
FC
wtAti
Fig. 4. Kinetic data: ATR-FTIR spectra for P. aeruginosa ser 10 LPS in1f
mc(iadvlNacia
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ig. 3. ATR-FTIR spectra of P. aeruginosa ser 10 LPS in 1, 10, 100 mmol L−1
aCl2 at: (a) pH 3, (b) pH 6, and (c) pH 9 (spectra collected at 120 min).
as 0.008 for Na and 0.019 for Ca. In addition, Ca-LPS spec-
ra showed greater time-dependency than did Na-LPS spectra.bsorbance values for Na-LPS spectra are constant from 0o 120 min (Fig. 4a) whereas the Ca-LPS absorbance valuesncreased with time (Fig. 4b).
ap1t
0 mmol L−1, pH 6 solution: (a) NaCl and (b) CaCl2. Spectra are collectedor 8 min, 15 s; and times noted indicate beginning of data collection.
Variation in the charge fraction of Ca2+ (ECa, defined as theoles of Ca charge normalized to the total moles of cation
harge) in CaCl2/NaCl solutions at pH 6 and at constant I10 mmol L−1) shows increasing IR absorbance values withncreasing charge fraction of Ca when spectra are plotted oncommon scale (Fig. 5a). Spectral changes are apparent whenata are plotted on non-common scale to maximize absorbancealues for all spectra (Fig. 5b). The latter clearly shows the evo-ution from a broad phosphate peak (1248–1204 cm−1) whena+ is the predominant cation (ECa ≤ 0.07) to a doublet (1243
nd 1213 cm−1) when Ca2+ is present (ECa ≥ 0.08). Increasingharge fraction of Ca2+ leads to greater peak separation andntensity, and distinct peaks are observed at ECa values as lows 0.08.
Polarized ATR-FTIR spectra for Na-LPS and Ca-LPS10 mM, pH 6) are given in Fig. 6. The ⊥ and || spectra
re similar for both Na-LPS and Ca-LPS, however greatereak intensities are observed for phosphate moieties (1243,213, 1106 cm−1) in the Ca-LPS ⊥ spectrum. Prior to sub-raction, the ⊥ spectra was multiplied by the isotropic dichroic
246 S.J. Parikh, J. Chorover / Colloids and Surfaces B: Biointerfaces 55 (2007) 241–250
Fig. 5. ATR-FTIR spectra (collected at 120 min) for P. aeruginosa ser 10 LPSat pH 6, 10 mmol L−1 ionic strength and variable charge fraction (ECa) of Carelative to Na: (a) y-axis (absorbance) constant for all spectra (common scale),a
rtSL(cipvtf(
pgp
F −1
pv
5
iatastHaihtOC
nd (b) y-axis manipulated to show major peaks in spectra (non-common scale).
atio (Riso) of 1.15 to correct for the difference in IR lightransmitted through the polarizer in the ⊥ and || positions.pectral subtractions produce weak downward peaks for Na-PS (Fig. 6a) and strong sharp downward peaks for Ca-LPS
Fig. 6b), with the strongest peaks corresponding to PO2−, indi-
ating orientation of both Na-LPS and Ca-LPS at the crystalnterface. Quantitative analysis of molecular orientation waserformed via calculation of R and Sk (Table 3). Na-LPS Ralues range from 0.74 to 1.03 for prominent IR peak loca-ions, and from 0.65 to 0.90 for Ca-LPS. Sk values rangerom 0.04 to 0.43 for Na-LPS and −0.12 to 0.52 for Ca-LPSTable 3).
Representative peak-fitting results are shown in Fig. 7, witheak locations selected on the basis of known vibrations. Inte-rated peak areas are used for calculation of IR absorbance ratiosresented in the Section 5.
sLab
ig. 6. Polarized FTIR spectra of for P. aeruginosa ser 10 LPS in 10 mmol L ,H 6 solution: (a) NaCl and (b) CaCl2. A*: dichroic spectra (⊥–||) were obtainedia spectral subtraction after multiplication of the ⊥ spectra by Riso (1.15).
. Discussion
The results suggest that solution-chemistry-induced changesn conformational properties of smooth LPS are intermedi-te between those reported for rough LPS [18,30–32] andhose expected for acidic polyelectrolytes [22–24,61,62]. Wettribute this to the prevalence in smooth LPS of O-antigenaccharides. The aggregation of LPS monomers is driven byhe hydrophobic effect whereby the favorable energetics of-bonded water promotes the exclusion non-polar moieties
nd the coalescence of the amphiphilic molecules [63]. Dur-ng aggregation, LPS molecules are expected to internalize theydrophobic lipid A because contact between the non-polar lipidail and water molecules is minimized while hydration of the-antigen is promoted [2,18–20,34]. Counter ion (e.g., Na+,a2+)-dipole and H-bonding at O-antigen saccharide groups
hould also contribute to aggregate stability. H-bonding betweenPS monomers of ionizable groups of the KDO (ethanolaminend carboxyl groups) or phosphate groups of the lipid A areelieved to stabilize rough LPS aggregates in NaCl [30], whereas
S.J. Parikh, J. Chorover / Colloids and Surfaces B: Biointerfaces 55 (2007) 241–250 247
Fig. 7. ATR-FTIR spectra for P. aeruginosa ser 10 LPS in 10 mmol L−1, pH6ip
tptoi
5
b(lrte[mwa
FL
n(Asm
5
sarcttwcwapν
btierally increased with increasing Ca concentration (Table 2).The only exception to this trend was the 100 mmol L−1 CaCl2sample at pH 3, which is also the sample that showed limitedsolubility. The relative intensities of corresponding IR peaks
Table 2FTIR absorbance ratio of Ca2+–νas(PO2
−):H2O–νas(PO2−) for P. aeruginosa
ser 10 LPS in CaCl2 (data collection at 2 h)
pH 3 pH 6 pH 9
solution for (a) NaCl and (b) CaCl2 (collected at 120 min). The gray linesndicate Gaussian fits to spectra and dotted lines represent data fits based oneak deconvolution.
he formation of cation-phosphate bonds (e.g., in CaCl2) couldotentially disrupt aggregate structure [25]. Steric and elec-rostatic repulsion between the saccharide groups must bevercome to promote aggregation. Thus, increasing I or decreas-ng pH are expected to facilitate this process.
.1. Effect of solution chemistry on Na-LPS conformation
With increasing pH and I, Na-LPS spectra show increasedand intensities for ν(CH2, CH3) relative to ν(C–O, C–O–C)Fig. 2), indicating a progressively larger contribution from theipid A moiety. One possible explanation is that increasing Iesults in an increase in accumulation of LPS monomers and,herefore, in lipid moieties bonded at the IRE interface. How-ver, the CAC of lipids generally decreases with increasing I
63–65], which should result in a lower concentration of freeonomers in solution. Thus, the negative effect of I on CACould be expected to result in an increase in the ν(CH2, CH3)t lower I, whereas the opposite is observed here. In addition,
111
ig. 8. ATR-FTIR spectral kinetics: absorbance ratios for P. aeruginosa ser 10PS in 10 mmol L−1, pH 6 solution (NaCl and CaCl2).
o time-dependent changes in Na-LPS spectra were observedas might be expected for macromolecular bonding to the IRE).lternatively, increasing pH and I both contribute to proton dis-
ociation of weakly acidic hydroxyls on saccharides and thisay diminish IR absorbance in the polysaccharide region [45].
.2. Effect of solution chemistry on Ca-LPS conformation
Na-LPS ATR-FTIR data were unchanged with time (Fig. 4a),uggesting that LPS structures pre-equilibrated in NaCl solutionre not affected by introduction to the IRE. Conversely, Ca-LPSesults showed time-dependent changes that were indicative ofonformational restructuring in the ATR cell (Fig. 4b). Indeed,he integrated ratios of peaks assigned to νas(CH2) or νas(PO4
−)o �(C–O, C–O–C) increased with time for Ca-LPS samples,hile remaining constant for Na-LPS (Fig. 8). Time-dependent
hanges in conformation of the lipid A itself are also evidenthen Ca2+ is present. Examination of the phosphate to fatty
cid absorbance ratio for 10 mmol L−1 CaCl2 (Fig. 9) shows arogressive increase in the contribution of νas(PO4
−), relative to(CH2, CH3) up to ca. 3000 s and then, although spectral areas ofoth functional groups are still increasing with time, their respec-ive rates of increase are equivalent so that their absorbance ratios unchanged. The ratio of Ca2+-bound to hydrated PO2
− gen-2+
mM CaCl2 0.90 0.90 0.990 mM CaCl2 1.00 0.98 1.0600 mM CaCl2 0.91a 1.10a 1.09
a Low solubility samples, may not be representative.
248 S.J. Parikh, J. Chorover / Colloids and Surfac
F(
f(io(pb
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dtb
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5
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R
ig. 9. IR peak area ratios for phsophate:fatty acid of P. aeruginosa ser 10 LPS10 mmol L−1 CaCl2) as a function of time at pH 3, 6, and 9.
or Ca-LPS are much greater than those observed for Na-LPSFig. 4). This is true even for IR peaks that do not increase inntensity with time (e.g., 1060 cm−1) in the Ca-LPS system. Thebserved increase in IR absorbance indicates surface reactione.g., sorption) between Ca-LPS and the ZnSE IRE. ATR-FTIReak amplification has previously been observed for compoundsinding at an IRE interface [45,46,66,67].
Since the beam penetration depth exceeds the size of LPSonomers, the relative intensity increase for CH2 and PO2
−roups of the lipid A (Fig. 8) can not be attributed only to theirreferential accumulation in proximity to the IRE; a concurrentncrease in absorbance for O-antigen functionalities should alsoesult. However, the results do suggest that LPS micelles/tubesre destabilized in the presence of Ca2+ (relative to Na-LPS),hich is in agreement with previously published studies investi-ating aggregation of rough LPS [66]. In a molecular modelingtudy, Obst et al. [31] reported that Ca2+ ions bind to bothhosphate and carboxyl groups of rough LPS to form a stableidentate complex. This fixes the distance between the ionizedroups, stabilizes and rigidifies the head group (inner core andlucosamine backbone), and changes the conformation of thelucosamine backbone of the lipid A [18,30,31].
Our interpretation of the Ca-LPS ATR results is that aftereposition on the IRE, LPS aggregates disassemble such thathe lipid A re-orients on the IRE surface. This may be facilitatedy lipid A adsorption to the ZnSe IRE either by hydropho-
aas
able 3ichroic ratio (R) and mean segmetal order paramter (Sk) for prominant peaks for P.
arameter νas(CH2) 2915 νs(CH2) 2848 δ(CH2) 1471 νas(PO2) 124
Na-LPS 0.86 ± 0.17 0.74 ± 0.11 – –Ca-LPS 0.80 ± 0.09 0.88 ± 0.15 0.76 ± 0.05 0.90 ± 0.18
k
Na-LPS 0.04 0.17 – –Ca-LPS 0.08 0.01 0.14 −0.12
iso (isotropic arrangement) for LPS is 1.15.
es B: Biointerfaces 55 (2007) 241–250
ic interaction or, more likely, by Ca2+ bridging between theegatively charged ZnSe IRE surface and PO2
−/COO− groups.ince the isoelectric point for ZnSe is pH < 4 [69], the IRE isegatively charged at pH 6 and 9, which would favor cationridging interactions. Wang [25] suggested that LPS aggre-ates are re-organized in the presence of a Ca silicate surfaceo form two-dimensional micelles anchored at the interface bya2+ binding to phosphate groups and our results support thisodel. In this scenario, Ca-LPS molecules exhibit conforma-
ional change giving rise to their adsorption to the IRE surfacen the time scales of minutes to hours (Figs. 8 and 9).
.3. Polarized ATR-FTIR spectra
The dichroic spectra (Fig. 6, spectral subtraction) suggest ⊥rientation of Na-LPS and Ca-LPS at the ZnSe interface [54].elative differences in peak intensities between Na-LPS anda-LPS are due to the greater IR absorbance of Ca-LPS. In thevent of no molecular orientation a flat line would be producedpon subtraction of the ⊥ (multiplied by Riso) from the || spec-ra. Quantitative analysis via calculation of R and Sk parametersre in general agreement regarding orientation of LPS. Devi-tion from Riso (1.15) in either direction indicates || (positiveeviation) or ⊥ (negative deviation) orientation to the surface54]. The analysis for Na-LPS and Ca-LPS reveal R < Riso, againndicating ⊥ orientation.
Analysis of Sk does not give a clear indication of LPS arrange-ent. Complete orientation of LPS at the crystal interface would
esult in Sk values of −0.5 (||) or 1 (⊥) and isotropic arrangementesults in a Sk vale of 0 [53], the data presented in Table 3 showsk values typically between 0 and 0.5. Therefore || orientationf dipoles can be ruled out. From Sk analysis, both Na-LPSnd Ca-LPS molecules appear partially ⊥ oriented. Polarizedata analysis is complicated by the physical/chemical proper-ies of LPS molecules and aggregates. Large molecules withunctional group heterogeneity present uncertainties in polarizedTIR spectral interpretation [53]. For LPS samples, the variousunctional groups may exhibit different orientations, which canimit the power of the analysis. However, from the polarized ATRate presented here it can be surmised that ⊥ orientation of LPSolecules is preferred for LPS interactions with a ZnSe surface.
Results of the non-polarized studies suggest that Ca-LPSggregates are disrupted leading to orientation of LPS moleculest the crystal surfaces, whereas Na-LPS aggregates are moretable and aggregate structures become oriented on the surface.
aeruginosa ser 10 LPS polarized spectra
3 νas(PO2) 1213 νs(PO2−) 1106 ν(C–O) 1018 ν(PO2
−) 981
0.76 ± 0.34 0.74 ± 0.29 0.79 ± 0.53 1.03 ± 0.810.83 ± 0.14 0.70 ± 0.15 0.65 ± 0.09 0.85 ± 0.24
0.23 0.19 0.43 0.17−0.01 0.52 0.32 0.17
Surf
Tprrs
5
(gsMa(fs
PtsfghasfpCdisirbai
F(a
miLoEtdFpo
wibrFis(sbPiemi[a(C
6
S.J. Parikh, J. Chorover / Colloids and
he polarized FTIR data do not validate or refute this inter-retation conclusively. Validation of Ca-LPS orientation mayequire kinetic observations of Ca-LPS aggregate disruption andeorientation at small time intervals via polarized ATR-FTIRpectroscopy.
.4. Phosphate group hydration
Our results suggest that phosphate groups of the lipid AFig. 1b) play a central role in LPS conformation and aggre-ation in CaCl2 solution. Calcium (along with Mg2+) has beenhown to have a strong affinity for lipid A phosphates [27,70].
olecular dynamics simulations have revealed that Ca2+ ionsre very strongly bound to the negatively charged head groupsPO2
− and COO−) of rough LPS [31]. We suggest that key dif-erences in behavior of Na- and Ca-LPS can be attributed to thetronger binding of Ca2+ with PO2
−.Binding of metal cations can lead to diminished hydration of
O2− [72] and this is reflected in FTIR spectra. PO2
− vibra-ions are observed in the region from 1200 to 1265 cm−1, withpecific frequencies correlating inversely with the degree ofunctional group hydration [71]. Highly hydrated PO2
− groupsive rise to peaks at 1200–1225 cm−1, whereas moderatelyydrated and non-hydrated groups produce peaks at 1230–1245nd 1250–1265 cm−1, respectively [72]. Brandenburg et al. [72]howed PO2
− frequency shifts to higher wavenumber resultedrom Mg2+ binding to PO2
−, which was thought to result in dis-lacement of solvation waters. In the present work, binding ofa2+ to PO2
− in the lipid A results in the formation of a strongoublet peak (Fig. 3). Relative to Na-LPS (Fig. 2), Ca-LPS showncreased absorbance in the PO2
− region and reduction in theignal of moderately hydrated phosphate. This implies that bind-ng of Ca2+ to phosphate groups displaces hydration waters and
esults in the strong band at 1243 cm−1. PO2− groups that are notonded to Ca2+ remain hydrated and give rise to the absorbancet 1213 cm−1. This strong binding of Ca2+ to the lipid A regions expected to have a substantial effect on conformation of LPS
ig. 10. IR absorbance ratio of phosphate to fatty acid vs. charge fraction of CaECa) in pH 6, I of 10 mmol L−1 (varying contributions of NaCl and CaCl2) P.eruginosa ser 10 LPS samples.
iscICaoscfaoIbtof
A
mS
aces B: Biointerfaces 55 (2007) 241–250 249
onomers and aggregates. Spectra of Na/Ca-LPS with vary-ng Ca2+ charge fraction in solution reveal its strong influencePS aggregation/conformation (Fig. 5). Fig. 10 shows the ratiof LPS PO2
− to saccharide groups as a function of ECa. WithCa values greater than 0.18 there is a large increase in spec-
ral contributions from PO2−. Although the ratio is sensitively
ependent on ECa even at low values, the lack of a plateau inig. 10 indicates that competition from Na+ for LPS binding sitesersists to ECa values up to at least 0.40 (equimolar contributionsf Ca2+ and Na+).
Our data are complementary to those of Coughlin et al. [68],ho determined that equimolar Ca2+ converted Na-LPS tubes
nto bilayers. Variation in the contribution of Na+ and Ca2+ toackground electrolyte at constant I (10 mmol L−1) and pH 6eveals that the emergent impacts of Ca–PO2 bonding on ATR-TIR spectra occurs even at low charge fraction of the bivalent
on (Fig. 5). Increased charge fraction of Ca2+ leads to progres-ive reorientation of LPS (CH2 and PO2
− absorbances increase)Fig. 5a) and separation of the broad phosphate peak into twoharp absorbances assigned to hydrated (1213 cm−1) and Ca-ound (1243 cm−1) PO2
− (Fig. 10). The persistence of hydratedO2
− (1213 cm−1), even at high ECa (Fig. 10), results fromncreased residence time of water molecules (due to the pres-nce of Ca2+) in the inner core and glucosamine backbone of theolecule [31]. This indicates incomplete shielding of the Ca2+
ons, required for intermolecular binding via bivalent cations31]. However, as Ca2+ concentrations are increased there isn increase in the ratio of Ca2+-bound to hydrated phosphateTable 2 and Fig. 10), indicating removal of H2O and increaseda2+ binding.
. Conclusions
Changes in LPS aggregation as a function of solution chem-stry were observed using ATR-FTIR spectroscopy. ATR-FTIRpectra reveal strong effects of counterion composition on theonformational properties of LPS in proximity to a ZnSe IRE.ncreased spectral contributions from the lipid A are observed fora- relative to Na-LPS, and these effects become increasinglypparent with time (up to 2 h) that Ca-LPS is in the presencef the ZnSe surface. We propose that LPS aggregates are lesstable in the presence of Ca2+, as the bivalent ion forms strongomplexes with phosphate groups of the lipid A. This complexormation apparently results in a reorientation of LPS at the neg-tively charged ZnSe surface and this also enhances absorbancef lipid A functionalities within the region probed by the incidentR beam. Polarized FTIR indicate perpendicular orientation ofoth Na-LPS and Ca-LPs at a ZnSe interface. The results indicatehat bivalent ions strongly affect the conformational propertiesf LPS, even when they are present at relatively low chargeraction in aqueous solution.
cknowledgements
We are grateful to David A. White II for performing the CACeasurements. This research was supported by the Nationalcience Foundation CRAEMS program (Grant CHE-0089156).
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50 S.J. Parikh, J. Chorover / Colloids and S
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