Bio-analytical applications of Fourier transform infrared spectroscopy

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Bio-analytical applications of Fourier transform infrared spectroscopy Michael Jackson and Henry H. Mantsch Winnipeg, Canada

Advances in instrumentation and data processing techniques in recent years have allowed infrared spectroscopy to be increasingly applied to complex biological systems. In this article we discuss the suitability of Fourier transform infrared spectroscopy for such studies, including the advantages it has over other biophysical techniques, and highlight some recent applications in the fields of biology and medicine.

Introduction

Every chemist is familiar with infrared spectroscopy as a powerful tool for the structural characterisation of complex organic materials. Indeed, IR spectroscopy represents one of the earliest and most widespread methods for structural characterisation. A perhaps less familiar (but no less powerful) area of application is to the complex materials comprising biological systems. The application of IR spectroscopy to biological and particularly biomedical systems has been limited until recently. Major difficulties included the almost blanket absorption of water in areas of interest to biologists and the overlap of important bands. The development of the Fourier transform infrared (FT-IR) spectrometer together with sophisticated data processing routines for spectral subtraction and band narrowing allowed these problems to be overcome and the 1980’s saw a dramatic increase in the application of IR spectroscopy to biological problems. In fact, in recent years a series of conferences has been organised devoted primarily to the applications of vibrational spectroscopy in biology and medicine [ 11.It is not our purpose to give a comprehensive survey in this article, but rather to highlight some of the major areas of application of FI’-IR spectroscopy in biology and medicine. More 0165-9936/92/$05.00

detailed information may be found in a number of previous review articles [2-51. FT-IR spectroscopy

of biological

molecules

Lipids

Lipids are amphipathic molecules (comprising a polar headgroup and apolar acyl chains) which spontaneously self-associate in water to form the bilayer structures which are the basis of all biological membranes. Such membranes are not static, rigid structures, but highly fluid and dynamic with most of the component lipids in a liquid crystalline state. Synthetic membranes of simple composition can be shown to exist in either this liquid crystalline state or in a more rigid, gel-like state, depending upon temperature. This thermal polymorphism is perhaps the most well studied aspect of lipid phase behaviour. Many absorptions in the IR spectrum of lipids have been shown to be diagnostic of phase and packing of acyl chains. The position and widths of the symmetric and antisymmetric CH:! stretching

4

3000

2950

2900

Wavenumber,

2850

2800

cm -’

Fig. 1. IR spectrum of dipalmitoyl phosphatidylcholine (DPPC) in the region of the C-H stretching bands in the gel phase at 20°C (solid line) and in the liquid crystalline phase at 45°C (broken line)

0 1992 Elsevier Science Publishers B.V. All rights reserved

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vibrations of the polymethylene groups of the lipid acyl chains in the infrared spectrum can provide information concerning the ratio of tram-gauche conformers and the degree of librational motion within the bilayer [2,5,6]. Thus, at the gel-liquid crystalline phase transition of the well investigated model membrane dipalmitoyl phosphatidylcholine (DPPC), increases in frequency of the symmetric and antisymmetric CH;! stretching band (from 2848 and 2917 cm-’ respectively, to 2850 and 2921 cm-‘) occur as the acyl chains convert from an all-tram configuration to a less well ordered mixture of tram and gauche conformers (see Fig. 1). This disordering of the bilayer allows more librational motion and is therefore accompanied by an increase in the width of both bands. In addition to changes in the CH2 stretching vibrations, significant changes are also apparent in vibrations arising from C=O stretching, CH2 scissoring, CH2 rocking and O-P-O stretching vibrations with variations in temperature. These changes can be related to alterations in hydrogen bonding and orientation of the headgroup and differences in chain packing [5]. For instance, below the phase transition of lipids, band narrowing methodologies such as Fourier derivation show the broad C=O band centered around 1730 cm-’ to consist of two overlapping absorptions at 1742 cm-’ and 1725 cm-‘. It was originally deduced that these two bands represented the sn-1 and sn-2 C=O acyl chain absorptions, respectively. However, replacing the sn-2 C=O group with a 13C-labelled carbonyl group which shifts the C=O stretching band by cu. 44 cm- \ ) clearly shows

I

1600

I

1750

I

I

1700

Wavenumber,

I

,

1650 cm

-'

Fig. 2. IR spectra in the carbonyl stretching region of aqueous liposomes of dimyristoyl phos hatidic acid with the ester carbonyl of the sn-2 chain I! C labelled in the gel phase (at 27°C) and in the liquid crystalline phase (at 45°C).

that both the sn-1 and sn-2 C=O groups give rise to two bands (see Fig. 2) [6]. In each case the low frequency band increases in intensity above the phase transition temperature. The lower frequency band from each C=O group is believed to reflect hydrogen bonding of the C=O group to water. The CH2 scissoring and bending vibrations can be used to assess packing of the acyl chains. Prior to the main gel-liquid crystalline phase transition of phosphatidylcholines a relatively minor gel-gel state transition, termed the pretransition, may be demonstrated. Below the pretransition of DPPC, IR spectra demonstrate a splitting of CH2 scissoring and bending bands which is indicative of orthorhombic packing of the acyl chains. However, increasing the temperature decreases the splitting of the bands until at the pretransition only one feature is seen, corresponding to acyl chains in a hexagonal subcell. Proteins

Proteins are long chain polymers formed from twenty commonly occurring amino acids. Folding of the protein polypeptide chain results in the formation of two major classes of regular secondary structures, a-helices and P-sheets. cc-Helical structures are characterised by a corkscrew-like arrangement of the polypeptide chain with hydrogen bonds formed between residues favourably aligned along the helix axis. On the other hand, P-sheets are extended, pleated structures, similar to those formed by nylon. The frequency of the amide I band (predominantly, i.e. cu. 80% a C=O stretching vibration) of proteins has been shown to be predictive of protein secondary structure: each regular secondary structure is associated with a characteristic hydrogen bonding pattern, which in turn may be expected to produce characteristic amide I frequencies for these structures. FT-IR spectra of proteins, known from X-ray diffraction studies to be predominantly a-helical, P-sheet or mixed a-helical/P-sheet in structure, are illustrated in Fig. 3. It can seen that after application of band narrowing techniques spectra of a-helical, P-sheet and mixed proteins show major features at 1648 cm-‘, 1635 cm-’ or both positions respectively. Typical frequency ranges for the major secondary structures in water soluble proteins, determined by comparison of amide I frequencies of proteins of known structure with X-ray diffraction data, are shown in Table I. While IR characterises water soluble proteins and the conformational rearrangements associated with ligand binding [ 11, in the analysis of membranes and membrane associated proteins and peptides IR spec-

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1648 4

1' 0

1675

16iO

16i5

Wavenumber,

cm-’

lt 0

Fig. 3. IR spectra of typical o-helical (myoglobin, a), mixed a-helical@-sheet (glutathione reductase, b) and P-sheet (concanavalin A, c) proteins in the region of the amide I bands.

troscopy has proved even more valuable [3,4]. Light scattering artifacts, problems with crystallisation and line broadening effects generally preclude the use of techniques such as circular dichroism (CD) spectroscopy, X-ray diffraction and NMR spectroscopy in the analysis of membrane proteins. No such problems are encountered with FT-IR spectroscopy and the technique has been applied to studies of intact cells, extracted biological membranes and the interactions of membrane proteins and peptides with synthetic membranes [2]. A brief survey of FT-IR spectroscopic studies of lipid-protein and lipid-peptide interactions [7] shows the predominant structural feature in membrane spanning proteins and peptides (as determined by FT-IR spectroscopy) to be almost always the a-helix, as indicated by amide I maxima in the range 1650-1657 cm-‘. The few exceptions known so far

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are alamethicin (a peptide antibiotic), bacteriorhodopsin (a proton translocating protein from the purple membranes of Halobacterium holobium), and pot-in (a membrane protein from Escherichia coli). Alamethicin is know to contain both 310- and a-helical structure. The IR is consistent with such a structure, the decreased strength of the hydrogen bond in the distorted 31~~helix increasing the frequency of the amide I maximum to 1662 cm-’ compared to the 1650-1657 cm-’ range for the standard a-helix. FT-IR spectra of bacteriorhodopsin also show an anomalously high amide I frequency, leading to the suggestion that the structures present in this protein are not classical a-helices, but some distorted helical structure. Many proteins and peptides are known which may exist either in aqueous solution or in a membrane associated form. FT-IR spectroscopy has the advantage of being able to probe the conformation of both forms. We shall illustrate this with an example from our laboratory. We have recently undertaken a study of the interaction of one such class of compounds, a series of positively charged antimicrobial peptides which are secreted by the skin of the African clawed frog Xerwpus laevis [8]. These peptides have been shown to form pores or channels in negatively charged membranes but not to cause haemolysis. We were able to demonstrate that while structureless in aqueous solution (amide I band at 1642 cm-‘), presumably as a result of repulsion between the positively charged amino acids, each peptide spontaneous1 adopted an a-helical structure (band at 1647 cm -7)

TABLE 1. Range of the frequency of the amide I band for secondary structural motifs in water-soluble proteins in *H20 Secondary structure

Frequency (cm-‘)

Blo-helix a-helix

1658-1668

Unordered p-sheet (intramolecular) P-sheet (intermolecular)

1648-1655 1640-1645 1630-1640 1675-1685 1610-1630 1680-1695

1700

1675

1650

Wavenumber,

1625

1600

cmel

Fig. 4. IR spectrum of the frog skin peptide PGL in aqueous solution (solid line) and in the presence of negatively charged liposomes (broken line). The strong band at 1673 cm-’ arises from the counter ion trifluoroacetate (TFA).

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with some P-sheet conformation (band at 1632 cm-‘) upon interaction with negatively charged lipids and inserted into the bilayer (Fig. 4). No such interaction was apparent with zwitterionic lipids. We have suggested that the lack of structural change in the presence of zwitterionic lipids forms the basis of the selectivity of these compounds for microbial cells. Higher animal cells have negligible amounts of negatively charged lipids in the outer layer of cell membranes, which prevents interaction with and incorporation into membranes. Bacterial membranes on the other hand have significant quantities of such lipids in the outer layers of their membranes, rendering them susceptible to these agents. Studies of this nature are not easily undertaken by NMR , CD spectroscopy or X-ray diffraction for the reasons discussed above and it is in this area that we expect to see FT-IR spectroscopy becoming increasingly applied.

Biomedical applications of FT-IR spectrosCOPY While the last decade saw a huge upsurge in the application of IR to biological problems, practical applications in the field of biomedical questions have been surprisingly few to date. Despite the limited work in this area, it has already been demonstrated that the technique may have useful applications in areas such as histopathology, bacterial identification and pharmaceutical screening. A number of studies [9,10] have shown that IR spectroscopy can reproducibly detect differences between malignant and non-malignant cells. Major differences were apparent in the symmetric and asymmetric PO2- stretching vibrations arising from cellular DNA. For example, the PO2- asymmetric stretching vibration consists of two overlapping absorptions, arising from non-hydrogen bonded (1240 cm-‘) and hydrogen bonded (1220 cm-‘) phosphodiester groups. Spectra of colon tissue known to contain malignant cells show an increase in the intensity of the 1220 cm-’ component, indicating increased hydrogen bonding of DNA phosphodiester groups in cancerous tissues. These differences are apparent in many malignant tissues and become even more apparent when the pressure dependence of these spectral parameters is examined. Other histopathological applications have been demonstrated based upon FT-IR microscopy, which allows spectroscopic and visual examination of tissue sections to be carried out. Subtraction of the spectrum of normal human kidney tissue recorded

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with an FT-IR microscope from that of a tissue section thought to have crystalline calcium oxalate deposits (kidney stones) produced a spectrum similar to that of calcium oxalate, confirming the diagnosis

[ill. This technique has also been applied to the study of the mineralisation of rat bone tissue [12]. FT-IR spectra showed marked differences in different areas of the same bone tissue, demonstrating a gradient in mineralisation with the lowest degree of mineralisation in the actively growing areas. Mineralisation in vitamin D-deficient rats was always less than that in healthy rats. This study has been extended to allow the degree of crystallinity of the hydroxyapatitecomponents to be qualitatively assessed in sections of bone tissue at 30 l_trnspatial resolution. As poorly crystalline hydroxyapatite is rapidly lost from bone tissue such studies may be useful in understanding not only the processes which are involved in development, but also bone healing following fracture and degenerative bone diseases such as osteoporosis. More recently, FT-IR microscopy has been used to study the composition of atherosclerotic plaques in rabbit arteries [ 131. The area probed in these experiments (20 x 20 mm, 5 mm thick) is smaller than the dimensions of arterial cells, which allows a profile of the arterial wall to be generated and a relationship between the composition of the cells and position within the tissue be determined. In addition, comparison of tissue at different stages of atherosclerosis may aid an understanding of the nature and progression of the disease. IT-IR has been shown to be useful not only for the identification of individual biomolecules, but also in the identification of cell types and species. Nauman et al. [ 141 utilised the high specificity of vibrational spectra and multivariate statistical analysis to develop a promising FT-IR procedure for bacterial differentiation and identification. This technique appears to provide reliable identification of pathogenic bacteria down to the species or even strain level [ 151.

Prospects for the future FT-IR spectroscopy is now a well established tool for characterisation of biological materials and in the field of medicine a number of potential histopathological applications have been demonstrated which allow rapid and less subjective diagnosis of disease states. We envisage a rapid expansion of IT-IR spectroscopy in this direction in the future.

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References 1 R.E. Hester and R.B. Grifftths (Editors), Spectroscopy of Biological Molecules, Proceedings of the 4th European Conference on the Spectroscopy of Biological Molecules, York, UK, September l-6, 1991, The Royal Society of Chemistry, Cambridge, 1991. Earlier conferences were held in Rimini, Italy (1989), Freiburg, Gemnany (1987) and Reims, France (1985). 2 R. Mendelsohn and H.H. Mantsch, in A. Watts and J.J.H.H.M. De Pont (Editors), Progress in ProteinLipid Interactions, Elsevier, Amsterdam, 1986, p. 103. 3 W.K. Surewicz and H.H. Mantsch, Biochim. Biophys. Acta, 952 (1988) 11.5. 4 M. Jackson, P.I. Haris and D. Chapman, J. Mol. Struct., 214 (1989) 329. 5 H.H. Mantsch and R.N. McElhaney, Chem. Phys. Lipids, 57 (1991) 213 6 W. Hubner, H.H. Mantsch and H.L. Casal, Appl. Spectrosc., 44 (1990) 732. 7 PI. Hat-is and D. Chapman, Biochem. Sot. Trans., 17 (1989) 161.

8 M. Jackson, H.H. Mantsch and J.H. Spencer, Biochemistry, (1992) in press. 9 P.T.T. Wong, E.D. Papavassilou and B. Rigas, Appl. Spectrosc., 45 (1991) 1563. 10 P.T.T. Wong, R.K. Wong, T.A. Caputo, T.A. Godwin and B. Rigas, Proc. Natl. Acad. Sci. U.S.A., 88 (1991) 10988. 11 D.A. Levison, P.R. Cracker and S.D. Allen, Eur. Spectrosc. News, 62 (1985) 18. 12 R. Mendelsohn, A. Hassankhani, E. Dicarlo, and A. Boskey, Calcified Tissue Znt., 44 (1989) 20. 13 D. R. Kodali, D.M. Small, J. Powell and K. Krishnan,Appl. Spectrosc., 45 (1991) 1310. 14 D. Nauman, V. Fijela, H. Labischinki and P. Giesbrecht, J. Mol. Struct., 174 (1988) 165 15 D. Nauman, D. Helm and H. Labischinki, Nature, 351 (1991) 81.

Dfs. Jackson and Mantsch are at the lnsfifute for Bodignostics, National Research Council of Canada, Winnipeg, Manitoba, R3B 1Y6, Canada.

Diode laser-induced fluorescence detection in chromatography A.J.G. Mank, H. Lingeman and C. Gooijer Amsterdam, The Netherlands

A new mode of laser-induced fluorescence detection in chromatography uses diode lasers. These lasers are compact, easy to handle, highly efficient, and relatively cheap, while their output can be stabilised extremely well. However, they lase at long wavelengths, where hardly any fluorophore can be excited. There is a trend towards shorter laser wavelengths: a 670 nm laser is now commercially available. The main challenge for analytical chemists is to develop appropriate covalent fluorescent probes that can be excited at such wavelengths and provide strong emission in the near infrared. Introduction Fluorescence

detection

Fluorescence spectroscopy is a versatile, sensitive, selective and easy-to-use analytical technique.

In the last decade many important advances have been made, primarily as a result of improvements in instrumentation such as the implementation of lasers. The first application of lasers in analytical molecular fluorescence was reported in 1971 by Jankow et al. [ 11. They used a commercial helium-cadmium (He-Cd) laser and a Cary Model 14 spectrophotometer, obtaining a detection limit of 2.10-13 M quinine bisulphate. Laser-induced fluorescence (LIF) detection in combination with chromatography was first reported by Diebold and Zare [2], also using a He-Cd laser. Focusing of the beam into a 4 ~1 droplet, suspended at the outlet of a chromatographic column, resulted in detection limits of 2.5 fmol for some aflatoxins. These spectacular detection limits made LIF detection the popular topic in analytical chemistry which it is today [3]. There are some obvious advantages of LIF compared to gas-discharge lamp excited fluorescence [4]. Lasers have a monochromatic output, which provides selective excitation and easy isolation of the fluorescence from Rayleigh- and Raman-scattering, but the most important advantage is the directionality of a laser beam. This characteristic can be used 0 1992 Elsevier Science Publishers

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