Lipids

Biological Applications

7.2 Lipids

Infrared spectroscopy can provide valuable structural information about lipids, which are important molecular components of membranes. Many lipids contain phosphorus and are classified as phospholipids, with some examples being shown in Figure 7.1. Lipids are organized in bilayers of about 40–80 ˚A in thickness, where the polar head group points towards the aqueous phase and the hydrophobic tails point towards the tails of a second layer. The chains can be in an all-trans-conformation which is referred to as the gel phase, or a liquid crystalline phase is obtained when the chain also contains gauche C–C groups. The infrared spectra of phospholipids can be divided into the spectral regions that originate from the molecular vibrations of the hydrocarbon tail, the interface region and the head group [12, 13].

The major infrared modes due to phospholipids are summarized in Table 7.1.

The hydrocarbon tail gives rises to acyl chain modes. The most intense vibrations in the infrared spectra of lipid systems are the CH2stretching vibrations and these give rise to bands in the 3100 to 2800 cm⁻¹region. The CH2asymmetric and symmetric

H2C O C R1 O

CH O C R2

O

H2C O P O

O⁻ O X

where X = CH2 CH CO2H NH3

+

Phosphatidylserine X= CH2 CH2 N(CH⁺ 3)3 Phosphatidylcholine X= CH2 CH2 NH⁺ 3 Phosphatidylethanolamine and R1 and R2 are alkyl chains

Figure 7.1 Structures of phospholipids.

Biological Applications 139 Table 7.1 Major infrared bands of lipids. From Stuart, B.,

Biolog-ical Applications of Infrared Spectroscopy, ACOL Series, Wiley, Chichester, UK, 1997. University of Greenwich, and reproduced by permission of the University of Greenwich

Wavenumber (cm⁻¹) Assignment

3010 =C–H stretching

2956 CH3asymmetric stretching

2920 CH2asymmetric stretching

2870 CH3symmetric stretching

2850 CH2symmetric stretching

1730 C=O stretching

1485 (CH3)3N⁺asymmetric bending

1473, 1472, 1468, 1463 CH2scissoring

1460 CH3asymmetric bending

1405 (CH3)3N⁺symmetric bending

1378 CH3symmetric bending

1400 – 1200 CH2wagging band progression

1228 PO2−asymmetric stretching

1170 CO–O–C asymmetric stretching

1085 PO2−symmetric stretching

1070 CO–O–C symmetric stretching

1047 C–O–P stretching

972 (CH3)3N⁺asymmetric stretching

820 P–O asymmetric stretching

730, 720, 718 CH2rocking

stretching modes, at 2920 and 2851 cm⁻¹, respectively, are generally the strongest bands in the spectra. The wavenumbers of these bands are ‘conformation-sensitive’

and respond to changes of the trans/gauche ratio in the acyl chains. This is also the case for the infrared bands due to the terminal CH3groups at 2956 cm⁻¹ (asym-metric stretching) and 2873 cm⁻¹ (symmetric stretching). The=C–H stretching bands due to unsaturated acyl chains are found at 3012 cm⁻¹and the bands due to methylene and methyl groups occur in the 1500–1350 cm⁻¹region. At around 1470 cm⁻¹, there are bands due to CH2bending and the number and wavenumbers of these bands are dependent on acyl chain packing and conformation. While the asymmetric deformation modes of the CH3 group are obscured by the scissoring bands, the symmetric deformation mode appears at 1378 cm⁻¹.

In certain phospholipid membranes that contain unsaturated acyl chains, the typical lamellar liquid crystalline phase converts to a micellar non-lamellar phase upon heating [14]. Such a thermally induced transition involves a major struc-tural rearrangement. Temperature studies of the infrared spectra of phospholipids provide a sensitive means of studying such transitions in lipids. Figure 7.2 shows the temperature-dependence of the wavenumber of the symmetric CH2stretching

2850 2851 2852 2853 2854

Wavenumber (cm−1)

0 10 20 30 40 50 60 70

Temperature (ºC)

Figure 7.2 Temperature-dependence of the symmetric CH2 stretching band of phos-phatidylethanolamine. From Stuart, B., Biological Applications of Infrared Spectroscopy, ACOL Series, Wiley, Chichester, UK, 1997. University of Greenwich, and reproduced by permission of the University of Greenwich.

band in the spectra of lipid membranes obtained from phosphatidylethanolamine.

The increasing wavenumber with temperature indicates an increasing concentra-tion of gauche-bands in the acyl chains and this leads to the formaconcentra-tion of the non-bilayer phase at higher temperatures. Figure 7.2 shows a wavenumber shift of about 2 cm⁻¹at 18^◦C and this is associated with the gel-to-liquid crystal phase transition. An additional wavenumber shift of approximately 1 cm⁻¹ at 50^◦C is associated with a transition to the micellar phase. Both of these transitions have been observed to be reversible.

Spectral modes arising from the head group and interfacial region also provide valuable information [15]. Useful infrared bands for studying the interfacial region of lipid assemblies are the ester group vibrations, particularly the C=O stretching bands in the 1750–1700 cm⁻¹region. In diacyl lipids, this region consists of at least two bands originating from the two ester carbonyl groups. A band at 1742 cm⁻¹is assigned to the C=O mode of the first alkyl chain with a trans-conformation in the C–C bond adjacent to the ester grouping, while the 1728 cm⁻¹C=O wavenumber of the second alkyl chain suggests the presence of a gauche-band in that position.

The wavenumber difference observed reflects the structural inequivalence of the chains, with the first alkyl chain initially extending in a direction perpendicular to the second alkyl chain and then developing a gauche-bend in order to render the two chains parallel.

Biological Applications 141 SAQ 7.1

The infrared spectrum of phosphatidylserine in deuterium oxide (D₂O) was recorded and the deconvolved carbonyl region is shown below in Figure 7.3.

What does this band suggest about the conformation of phosphatidylserine?

1760

1780 1720 1680

Absorbance

Wavenumber (cm⁻¹)

Figure 7.3 The C=O stretching region of phosphatidylserine in D2O (cf. SAQ 7.1).

From Stuart, B., Biological Applications of Infrared Spectroscopy, ACOL Series, Wiley, Chichester, UK, 1997. University of Greenwich, and reproduced by permission of the University of Greenwich.

Quantitative infrared analysis can be carried out on blood serum to deter-mine the relative amounts of lipid present [16]. Triglycerides, phospholipids and cholesteryl esters are the classes of lipid that occur in blood serum and such com-pounds occur naturally in concentrations that make infrared analysis attractive.

These classes of compounds can be characterized by their carbonyl bands: the peak maxima appear at 1742 cm⁻¹ for the triglycerides, at 1737 cm⁻¹ for the phospholipids and at 1723 cm⁻¹for the cholesteryl esters. Although the carbonyl peaks are heavily overlapped, a least-squares method may be used to separate the components. The concentrations of these lipid components are usually in the range 0.03–0.3% in human blood, and standard solutions can be prepared in chloroform.