Elsevier

Vibrational Spectroscopy

Volume 36, Issue 2, 6 December 2004, Pages 167-177
Vibrational Spectroscopy

2D IR analyses of rate processes in lipid–antibiotic monomolecular films

https://doi.org/10.1016/j.vibspec.2004.05.002Get rights and content

Abstract

Polarization modulation infrared reflection spectra of a 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA) monolayer on a subphase containing 5 mM tetracycline hydrochloride (TC) were collected under varying surface pressures at the air–water interface. Statistical correlation spectroscopy using 2D IR, βν and correlation analyses were performed on these spectra to gain a better understanding of the surface pressure-induced effects on the interaction between the phospholipid and antibiotic. Conventional 2D IR correlation maps revealed strong correlation behavior between the vibrational modes of the lipid and antibiotic. βν correlation plots provided information about the relative rates of occurrence of the coupled responses noted in the conventional 2D IR plots. These calculations indicated that molecular reorientation occurs at lower surface pressures for the modes in Ring A than for the modes in Ring C. A new model-dependent two-dimensional correlation method, exponential correlation analysis, confirmed the results from the previous correlation methods and confirmed that the lipid–antibiotic interactions occurred in a bimodal fashion, depending upon surface pressure. The conclusions of the correlation analysis of the surface-pressure induced changes in the DPPA–TC monolayer system lead to the following model for lipid–antibiotic interaction. Initial interaction between the tetracycline molecule and the DPPA molecule occurs at low surface pressures primarily between Ring A of the tetracycline molecule and the lipid headgroup region. However, with increasing surface pressure, the mode of interaction changes, and the strongest interaction at high surface pressures occurs between Ring C of tetracycline and the DPPA headgroup.

Introduction

Two dimensional infrared correlation spectroscopy (2D IR) has proven to be a valuable tool due to its ability to enhance spectral resolution and identify overlapped spectral features [1]. This has proven especially valuable in infrared spectroscopic studies of biomolecules, as this enables one, for example, to identify discrete and unique protein secondary structure conformations as well as interconversion of one form to another as a result of changes in external environment [2], [3], [4], [5], [6], [7]. Two-dimensional IR correlation analysis has also been used to analyze structure in monomolecular films. The phase behavior of phospholipid monolayers have been studied using 2D IR and it was shown how these methods could distinguish bands due to co-existing phases in a disorder-order phase transition in the monolayer [8], [9].

Standard 2D IR methods have been most successfully employed in simplifying complex spectra and facilitating band assignments through resolution enhancement [10]. In addition to these uses, 2D IR can also be used to determine the temporal order of events that occur in a set of dynamically varying spectra upon sample perturbation. The basis for this determination is the relative signs of the synchronous and asynchronous cross-peak at coordinate (ν1, ν2) in the 2D correlation plots [11].

While it is certainly possible to determine the relative sequence of molecular events based on standard 2D IR methods, this procedure tends to be difficult to implement for highly overlapped spectra and may lead to uncertainties. In order to more quantitatively describe the degree of coherence between spectral intensity changes and the sequence of molecular events in a set of dynamic spectra, we have recently developed a modified 2D IR correlation method called βν correlation analysis [12]. This method is a variation of asynchronous cross-correlation, in which dynamically varying spectra are correlated against a mathematical function with a varying phase angle. We recently applied βν correlation analysis to surface pressure-induced changes in the IRRAS spectra of phospholipid monolayers at the A/W interface, and showed how the relative rates of acyl chain and methyl group reorientation could be quantitatively determined [13] and have also applied this analysis in the study of conformational changes and relative reorientation rates of hydrophobic surfactant proteins SP-B and SP-C at the A/W interface [14].

Our current objective is to use polarization-modulation infrared reflection absorption spectroscopy (PM-IRRAS) to study the interactions of the antibiotic tetracycline with a phospholipid monolayer at the air–water interface. It has been shown that tetracycline induces significant changes on phospholipid monolayers and the strongest interactions are observed for the DPP A system due to specific dipole–dipole interactions [15], [16]. The aim of the current study is to unambiguously identifying the specific regions of interaction between the two molecules at the air–water interface.

To accomplish this aim, we use both conventional 2D IR and βν correlation analysis to analyze the PM-IRRAS spectra obtained from the lipid–antibiotic interactions. In addition, we introduce a new model-dependent 2D correlation method, correlation analysis. Our results are able to clearly identify the functional groups involved in this lipid–antibiotic interaction and the order in which their respective functional groups reorient upon increasing monolayer surface pressure.

Section snippets

Materials

Tetracycline hydrochloride (TC) was obtained from Sigma (St. Louis, MO, purity > 99%) while 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA) (>99%) was obtained from Avanti Polar Lipids (Alabaster, AL). The chemical structures of tetracycline hydrochloride and DPPA are shown in Fig. 1. HPLC grade chloroform (J.T. Baker, Phillipsburg, NJ) was used as the spreading solvent and typical DPPA concentrations of 1 mg/mL were used for making the spreading solutions. Ultrapure H2O obtained from a

Monolaver IR spectroscopv

Previous research using monomolecular films of DPPA on a tetracycline-containing subphase has indicated that specific inter-molecular interactions may occur between the polar head groups of the phospholipid in the condensed phase and the tetracycline molecules dissolved in the subphase [15], [16]. In order to test this hypothesis, we employed polarization-modulation infrared reflectance–absorption spectroscopy (PM-IRRAS) and obtained IRRAS spectra at the air–water (A/W) interface for monolayer

Conclusions

Polarization modulation infrared reflection spectra of a dipalmitoyl phosphatidic acid monolayer on a subphase containing 5 mM tetracycline hydrochloride were collected under varying surface pressures. Two-dimensional IR, βν and correlation analyses were performed on these PM-IRRAS spectra to gain a better understanding of the surface pressure-induced effects on the interaction between the phospholipid and antibiotic.

The synchronous 2D IR correlation map reveal strong correlation behavior

Acknowledgements

The work described here was supported by the US Public Health Service through National Institutes of Health grant EB001956 (R.A.D.).

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