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Difference infrared spectroscopy of aqueous model and biological membranes using an infrared data station
Journal o f Biochemical and Biophysical Methods, 2 (1980) 315--323
315
© Elsevier/North-Holland Biomedical Press
DIFFERENCE INFRARED SPECTROSCOPY OF AQUEOUS MODEL AND BIOLOGICAL MEMBRANES USING AN INFRARED DATA STATION
D. CHAPMAN l, J.C. GOMEZ-FERN~,NDEZ 1,*, F.M. GONI 1,** and M. BARNARD 2 1 Royal Free Hospilal, School o f Medicine, University o f London, 8 Hunter Street, London W C I N IBP, and 2 Perkin Elmer Corporation, Beaconsfield, Bucks., U.K.
(Received 14 September 1979; accepted 19 December 1979)
A Perkin-Elmer infrared Data Station associated with a simple IR spectrometer (model 298) is shown to give excellent results with aqueous model and biomembrane systems. Examples are presented of difference spectra obtained with lipid--water systems, reconstituted lipid--protein systems and a natural biomembrane. The spectra of the lipid after water subtraction and of the intrinsic protein after lipid subtraction from a model reconstituted Ca2+-ATPase membrane system are shown. The potential for studying intrinsic protein conformations is emphasised. Key words: in frared spectroscopy ; model membranes; biomembranes, Ca2+-ATPase.
INTRODUCTION I n f r a r e d s p e c t r o s c o p y can p r o v i d e a w e a l t h o f i n f o r m a t i o n a b o u t struct u r a l f e a t u r e s o f biological s y s t e m s and it has b e e n e x t e n s i v e l y used f o r t h e q u a l i t a t i v e a n d q u a n t i t a t i v e s t u d y o f lipids and m e m b r a n e s [ 1 - - 3 ] , p r o t e i n s and nucleic acids [4] and c a r b o h y d r a t e s [5]. H o w e v e r , t h e p r o b l e m o f t h e intense w a t e r a b s o r p t i o n s has l i m i t e d t h e use o f c o n v e n t i a l I R s p e c t r o s c o p y w i t h a q u e o u s biological materials. R e c e n t l y s o m e w o r k has b e e n r e p o r t e d [ 6 - - 8 ] using a F o u r i e r t r a n s f o r m I R s p e c t r o m e t e r . T h e d a t a handling facility o f t h e F T - I R s p e c t r o m e t e r e n a b l e d a s p e c t r u m o f w a t e r to be s u b t r a c t e d f r o m t h e s p e c t r a o f lipids a n d m e m b r a n e s in a q u e o u s m e d i a . T h e resulting d i f f e r e n c e s p e c t r a s h o w e d t h e f i n g e r p r i n t region o f t h e lipids a n d m e m branes. I n d e p e n d e n t l y o f t h e s e a u t h o r s similar investigations w e r e u n d e r t a k e n using a c o n v e n t i o n a l o p t i c a l null I R s p e c t r o m e t e r , a P e r k i n - E l m e r M o d e l * Permanent address: Departamento de Bioqufmica, Facultad de Medicina, Murcia, Spain. ** Permanent address: Departamento de Bioqufmica, Facultad de Ciencias, Bilbao, Spain. Abbreviations: IR, infrared spectroscopy; FT-IR, Fourier-transform infrared spectroscopy; DMPC, dimiristoylphosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine.
316 298, coupled to an IR Data Station. The Data Station was used to store data and facilitate the subtraction of the water spectrum from the spectra of aqueous samples. It was also possible to subtract the spectrum of the lipid in water from a r econs t i t ut e d lipid--protein--water system to obtain the absorption bands due to the intrinsic protein structure existing within the lipid bilayer matrix. MATERIALS AND EQUIPMENT Phospholipids (DMPC and DPPC) were obtained from Fluka (Buchs, Switzerland). All the chemicals used were Analytical Grade. DMPC and DPPC samples were dispersed in excess water (80 mol of water per mole of lipid). Rabbit sarcoplasmic reticulum (SR), sarcoplasmic reticulum--ATPase and DMPC--ATPase recombinants were prepared as previously described [9]. SR membranes were f ur t he r purified by centrifugation for 6 h at 2 5 0 0 0 0 X g in a sucrose gradient (20--60%, w/v). SR and the DMPC-ATPase r e c o m b i n a n t were washed three times in 1.2 M KC1 by centrifugation at 105 000 X g, 30 min, to remove the buffer used in their preparation. The spectra o f all samples were recorded on a Perkin-Elmer 298 Infrared S p e c t r o p h o t o m e t e r which is microprocessor controlled making it ideally suited to couple to a m o d e r n data handling system. The interface accessory enables it to be c o n n e c t e d directly to any c o m p u t e r with a standard V24 or RS232C co mmu ni cat i ons interface. Spectral i nform at i on is processed by the interface and o u t p u t as a digital ordinate value. For these experiments a Perkin-Elmer Data Station was used. The Data Station consists of a processing module with an integral dual micro floppy disc drive unit, a k e y b o a r d module which combines a conventional alphanumeric l a y o u t with funct i on keys and a numeric key pad, and a visual display unit. The software for the system is stored on micro f l oppy discs. Most routines are called up by labelled f u n ctio n keys so t hat a routine, e.g. scan, can be activated by the push of a b u t t o n . All keyboar d entries and spectra are m o n i t o r e d on the screen. METHODOLOGY All the samples under investigation were h y d r a t e d so t hat it was necessary to use barium fluoride cells. A 7 pm barium fluoride cell was used to record the standard water spectrum and the spectra of the lipids and m e m b r a n e were recorded as capillary films between barium fluoride windows at a t e m p e r a t u r e of 37 or 46 ° C. The spectra were recorded and stored on a micro f l o p p y disc. It was possible to use the Data Station to subtract the water spectrum f r o m the spectra of the h y d r a t e d lipids and membrane. Transmittance spectra were converted into absorbance and the difference between them was calculated. A scaling factor was i n t r o d u c e d by specifying the water band to be cancelled. The resulting difference spectrum was in
317
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Lm
W A V E ~t U~IB E R ( c~CI) |.~l,
b
tJ~ ¢_9
c:>
W A V E N U [i B E R ( cM -I)
Fig. 1. Infrared spectra of water and a sample of DMPC dispersed in excess water at 37°C. (a) Spectrum of water (broken line) and phospholipid--water dispersion (continuous line). (b) The difference spectrum obtained after subtraction of the water absorption bands.
318 L~
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/
&m
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. . . .
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Fig. 2. Infrared spectra of a reconstituted DMPC---Ca2+-ATPase system (57 : 1 molar ratio) obtained at 37°C. (a) P h o s p h o l i p i d - - p r o t e i n - - w a t e r system. (b) Difference spect r u m after subtraction of the water absorption bands from a. (c) Difference s p e c t r u m after subtraction of the phospholipid absorption bands from b.
absorbance and was converted back to transmittance. This transmittance spectrum was p l o t t e d on the Model 298 Infrared S p e c t r o p h o t o m e t e r chart f o r comparison with the original spectrum. It was n o t necessary to do any signal averaging to obtain these spectra as an adequate signal-to-noise ratio was obtained in single scans. Lipids dispersed in excess water and natural and reconst i t ut ed membranes (as pellets after centrifugation) were transferred to a BaF2 plate with a spatula or a microsyringe and spread giving a thin film between b o t h plates. A b o u t 1 mg o f lipid was used in all cases. Care was taken to avoid an excess of water as this could make the subtraction of the water spectrum difficult. It was checked th at the t e m p e r a t u r e was constant within I ° C during the acquisition of the spectrum. T he water spectrum used t o subtract from t hat of the sample was obtained under similar conditions of t e m p e r a t u r e and pathlength to those of the sample.
Phospholipid--water systems Phospholipid--water systems are a useful model b i o m e m b r a n e system as
320
t h e y can spontaneously form lipid bilayer structures similar to the lipid matrix of natural biomembranes. We see (Fig. 1) the effect of water subtraction on a pure phospholipid DMpC sample dispersed in excess water and scanned at 37°C (i.e. above its transition temperature, To). The 1740 cm -j band, assigned to C=O stretching, appears now detached from the water signal; a substantial improvement is also found in the C--H stretching vibration bands, in the region near 2900 cm -~. It is possible to compare and contrast the band positions of the lipid in a solvent or in the solid state with those of the lipid in water, thereby providing information about hydrogenbonding interactions with phosphate groups and water.
Reconstitu ted pro tein--lipid systems A more detailed model biomembrane system involves reconstitution processes. In this case an intrinsic protein can be extracted from a natural biomembrane and included into a pure synthetic lipid--water system. We have examined the IR spectra of reconstituted sarcoplasmic reticulum membranes using the Ca2÷-ATPase, and incorporating this into dimyristoyllecithin. The spectra in Fig. 2 shows t h a t not only is it possible to examine the spectra after subtracting the water bands but that also we have been able to subtract the spectrum of the lipid, thereby showing clearly the absorption bands due to the intrinsic protein structure. This is illustrated in Fig. 2c where spectra from a DMPC--ATPase recombinant (57 : 1 molar ratio) are plotted before and after subtraction of water and DMPC. Bands in the 1800--1500 cm -1 region are prominent. Two major peaks are found, one at 1660 cm -1 due to the amide I vibration (C=O stretching), and one at 1545 cm -1 due to the amide II vibration (in plane NH bend). The shoulder centred at 1640 cm -1 was suspected to be due to an imbalance of the strong H20 absorption in this region, or traces of buffer. (Buffer solutions themselves give complicated absorption in this region.) To check this the reconstituted ATPase was washed with 2H20. The spectrum 2H20 is shown in Fig. 3. The difference spectrum after subtraction of absorption due to the 2H20 and to the DMPC lipid is also shown. In this spectrum there is no evidence for the additional peak at 1640 cm -~. Strong bands occur at 1650 and 1545 cm -1. (It is important to appreciate that shifts in the water absorption bands can occur because of the presence of the buffer so that care is necessary when the difference spectra are obtained. A study of the spectrum of the material in 2H20 is an added precaution.)
Natural biomem branes The same principle can be applied to natural biomembranes. As an example we show in Fig. 4 the spectrum from sarcoplasmic reticulum biomembrane after water and lipid substraction. The spectrum of the protein
321
t.o
[.Bf
ao
LL
I
'~
/
I} A V E II U rl B [ R (c~C1)
Fig. 3. Infrared spectrum on a reconstituted D M P C - - C a 2 + - A T P a s e
system (57 : I) molar
ratio obtained at 37°C after washing three times in 1.2 M KC] in 2H20. The spectrum of 2H20 is given by the dotted line. The difference spectrum corresponding to the protein after subtraction of bands due to 2H20 and due to the DMPC is given by the full line.
obtained in this way for SR--ATPase is similar to the corresponding difference spectrum from the DML--ATPase recombinant. Amide I and amide II bands are the most prominent at 1650 cm-' and at 1545 cm -1. A band at 1067 cm -1, that corresponds to C--O and C--C stretching, is also prominent. Receritly a study of the IR spectra of the Halobiurn halobacterium purple membrane showed the amide I band to be unusually high i.e. at 1665 cm -1. It was suggested that this is outside the normal range of frequencies (1650-1655 cm -1) reported for alpha-helical proteins and polypeptides. A possibility that this may indicate unusual alpha helices in the bacteriorhodopsin protein has been put forward [10]. Our studies of the sarcoplasmic reticulum biomembrane and its reconstituted systems show that whilst the sarcoplasmic reticulum biomembrane shows the amide I band in the normal range, the reconstituted ATPase systems do not, and may have similar unusual helical structures. Further studies will be made to check this.
322 L~
I.W
c::
ILm
W A V E ~i U ri B E P, ( cM- I )
Fig. 4. The infrared difference spectrum of sarcoplasmic reticulum biomembrane after subtraction of absorptions due to the water and phospholipid absorption bands.
DISCUSSION Difference IR spectroscopy has considerable potential for a number of research fields using relatively simple and not costly equipment. In this respect the advantage of IR over other techniques such as X-ray diffraction, DSC or NMR is the large a m o u n t of information which can be obtained in the short period of time needed for scanning a spectrum, the small a m o u n t of sample and the inexpensive equipment required. With model lipid--water systems the technique allows effects of ions and drugs to be studied. This may provide information concerning changes of fluidity, water structure, hydrogen bonding and drug conformations. Studies of the reconstituted systems and natural biomembranes have the potential for revealing information concerning conformational changes of intrinsic proteins as a consequence of temperature or triggering processes. Many other materials apart from biomembranes can be explored using this technique. Polysaccharides, cell walls, nucleic acids and proteins in aqueous solutions can also be studied.
323 SIMPLIFIED DESCRIPTION OF THE METHOD AND ITS APPLICATIONS The infrared Data Station associated with a simple IR spectrophotometer provides a relatively inexpensive means of studying biological samples in the presence of water and can be applied to the study of biomembranes, protein structures, polysacharides and other biological materials. Only small amounts of samples (less than 1 mg) are required. The possibility of subtracting absorption bands, due to different components, of a complex system allows the selective study of the individual components within the biomembrane structure, such as information about the protein conformation. ACKNOWLEDGEMENTS
We wish to acknowledge support from the Wellcome Trust and the Medical Research Council. J.C.G.-F. thanks the Wellcome Trust for a Fellowship. REFERENCES 1 Chapman, D. (1965) J. Am. Oil Chem. Soc. 42,353--371 2 Chapman, D. (1965) The Structure of Lipids, 1st edn., pp. 53--132. Methuen, London 3 Wallach, D.F.H. and Winzler, R.J. (1974) in Evolving Strategies and Tactics in Membrane Research, pp. 140--189. Springer-Verlag, Berlin 4 Thomas, G.K. and Kyogoku, Y. (1977) in Infrared and Raman Spectroscopy (Brame, E.G. and Grasselli, J.G., eds.), Vol. 1, Part C, pp. 717--872. Marcel Dekker, New York 5 Spedding, H. (1963) in Advances in Carbohydrate Chemistry (Wolfram, M.L. and Tipson, R.S., eds.), Vol. 19, pp. 23--49. Academic Press, New York 6 Cameron, D.G. and Mantsch, H.H. (1978) Biochem. Biophys. Res. Commun. 83, 886--892 7 Cameron, D.G., Casal, H.L. and Mantsch, H.H. {1979) J. Biochem. Biophys. Methods 1, 21--36 8 Casal, H.L., Smith, I.C.P., Cameron, D.G. and H.H. Mantsch (1979) Biochim. Biophys. Acta 5 5 0 , 1 4 5 - - 1 4 9 9 Gbmez-Fern~ndez, J.C., Goni, F.M., Bach, D., Restall, C.J. and Chapman, D. (1979) FEBS Lett. 9 8 , 2 2 4 - - 2 2 8 10 Rothschild, K.J. and Clark, N.A. (1979) Science 2 0 4 , 3 1 1 - - 3 1 2
315
© Elsevier/North-Holland Biomedical Press
DIFFERENCE INFRARED SPECTROSCOPY OF AQUEOUS MODEL AND BIOLOGICAL MEMBRANES USING AN INFRARED DATA STATION
D. CHAPMAN l, J.C. GOMEZ-FERN~,NDEZ 1,*, F.M. GONI 1,** and M. BARNARD 2 1 Royal Free Hospilal, School o f Medicine, University o f London, 8 Hunter Street, London W C I N IBP, and 2 Perkin Elmer Corporation, Beaconsfield, Bucks., U.K.
(Received 14 September 1979; accepted 19 December 1979)
A Perkin-Elmer infrared Data Station associated with a simple IR spectrometer (model 298) is shown to give excellent results with aqueous model and biomembrane systems. Examples are presented of difference spectra obtained with lipid--water systems, reconstituted lipid--protein systems and a natural biomembrane. The spectra of the lipid after water subtraction and of the intrinsic protein after lipid subtraction from a model reconstituted Ca2+-ATPase membrane system are shown. The potential for studying intrinsic protein conformations is emphasised. Key words: in frared spectroscopy ; model membranes; biomembranes, Ca2+-ATPase.
INTRODUCTION I n f r a r e d s p e c t r o s c o p y can p r o v i d e a w e a l t h o f i n f o r m a t i o n a b o u t struct u r a l f e a t u r e s o f biological s y s t e m s and it has b e e n e x t e n s i v e l y used f o r t h e q u a l i t a t i v e a n d q u a n t i t a t i v e s t u d y o f lipids and m e m b r a n e s [ 1 - - 3 ] , p r o t e i n s and nucleic acids [4] and c a r b o h y d r a t e s [5]. H o w e v e r , t h e p r o b l e m o f t h e intense w a t e r a b s o r p t i o n s has l i m i t e d t h e use o f c o n v e n t i a l I R s p e c t r o s c o p y w i t h a q u e o u s biological materials. R e c e n t l y s o m e w o r k has b e e n r e p o r t e d [ 6 - - 8 ] using a F o u r i e r t r a n s f o r m I R s p e c t r o m e t e r . T h e d a t a handling facility o f t h e F T - I R s p e c t r o m e t e r e n a b l e d a s p e c t r u m o f w a t e r to be s u b t r a c t e d f r o m t h e s p e c t r a o f lipids a n d m e m b r a n e s in a q u e o u s m e d i a . T h e resulting d i f f e r e n c e s p e c t r a s h o w e d t h e f i n g e r p r i n t region o f t h e lipids a n d m e m branes. I n d e p e n d e n t l y o f t h e s e a u t h o r s similar investigations w e r e u n d e r t a k e n using a c o n v e n t i o n a l o p t i c a l null I R s p e c t r o m e t e r , a P e r k i n - E l m e r M o d e l * Permanent address: Departamento de Bioqufmica, Facultad de Medicina, Murcia, Spain. ** Permanent address: Departamento de Bioqufmica, Facultad de Ciencias, Bilbao, Spain. Abbreviations: IR, infrared spectroscopy; FT-IR, Fourier-transform infrared spectroscopy; DMPC, dimiristoylphosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine.
316 298, coupled to an IR Data Station. The Data Station was used to store data and facilitate the subtraction of the water spectrum from the spectra of aqueous samples. It was also possible to subtract the spectrum of the lipid in water from a r econs t i t ut e d lipid--protein--water system to obtain the absorption bands due to the intrinsic protein structure existing within the lipid bilayer matrix. MATERIALS AND EQUIPMENT Phospholipids (DMPC and DPPC) were obtained from Fluka (Buchs, Switzerland). All the chemicals used were Analytical Grade. DMPC and DPPC samples were dispersed in excess water (80 mol of water per mole of lipid). Rabbit sarcoplasmic reticulum (SR), sarcoplasmic reticulum--ATPase and DMPC--ATPase recombinants were prepared as previously described [9]. SR membranes were f ur t he r purified by centrifugation for 6 h at 2 5 0 0 0 0 X g in a sucrose gradient (20--60%, w/v). SR and the DMPC-ATPase r e c o m b i n a n t were washed three times in 1.2 M KC1 by centrifugation at 105 000 X g, 30 min, to remove the buffer used in their preparation. The spectra o f all samples were recorded on a Perkin-Elmer 298 Infrared S p e c t r o p h o t o m e t e r which is microprocessor controlled making it ideally suited to couple to a m o d e r n data handling system. The interface accessory enables it to be c o n n e c t e d directly to any c o m p u t e r with a standard V24 or RS232C co mmu ni cat i ons interface. Spectral i nform at i on is processed by the interface and o u t p u t as a digital ordinate value. For these experiments a Perkin-Elmer Data Station was used. The Data Station consists of a processing module with an integral dual micro floppy disc drive unit, a k e y b o a r d module which combines a conventional alphanumeric l a y o u t with funct i on keys and a numeric key pad, and a visual display unit. The software for the system is stored on micro f l oppy discs. Most routines are called up by labelled f u n ctio n keys so t hat a routine, e.g. scan, can be activated by the push of a b u t t o n . All keyboar d entries and spectra are m o n i t o r e d on the screen. METHODOLOGY All the samples under investigation were h y d r a t e d so t hat it was necessary to use barium fluoride cells. A 7 pm barium fluoride cell was used to record the standard water spectrum and the spectra of the lipids and m e m b r a n e were recorded as capillary films between barium fluoride windows at a t e m p e r a t u r e of 37 or 46 ° C. The spectra were recorded and stored on a micro f l o p p y disc. It was possible to use the Data Station to subtract the water spectrum f r o m the spectra of the h y d r a t e d lipids and membrane. Transmittance spectra were converted into absorbance and the difference between them was calculated. A scaling factor was i n t r o d u c e d by specifying the water band to be cancelled. The resulting difference spectrum was in
317
o
i
l.m
J r
I
Lm
W A V E ~t U~IB E R ( c~CI) |.~l,
b
tJ~ ¢_9
c:>
W A V E N U [i B E R ( cM -I)
Fig. 1. Infrared spectra of water and a sample of DMPC dispersed in excess water at 37°C. (a) Spectrum of water (broken line) and phospholipid--water dispersion (continuous line). (b) The difference spectrum obtained after subtraction of the water absorption bands.
318 L~
t.m z
/
&m
. . . .
I
. . . .
I
. . . . . . . . .
I
. . . . . . . . .
,---t
W A V E rl U l.l B E R ( c~ - I )
b
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. . . .
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! " " "
319
C
I.W t.~
cD
o
N A V E~I U I"iB E R ( cM-1)
Fig. 2. Infrared spectra of a reconstituted DMPC---Ca2+-ATPase system (57 : 1 molar ratio) obtained at 37°C. (a) P h o s p h o l i p i d - - p r o t e i n - - w a t e r system. (b) Difference spect r u m after subtraction of the water absorption bands from a. (c) Difference s p e c t r u m after subtraction of the phospholipid absorption bands from b.
absorbance and was converted back to transmittance. This transmittance spectrum was p l o t t e d on the Model 298 Infrared S p e c t r o p h o t o m e t e r chart f o r comparison with the original spectrum. It was n o t necessary to do any signal averaging to obtain these spectra as an adequate signal-to-noise ratio was obtained in single scans. Lipids dispersed in excess water and natural and reconst i t ut ed membranes (as pellets after centrifugation) were transferred to a BaF2 plate with a spatula or a microsyringe and spread giving a thin film between b o t h plates. A b o u t 1 mg o f lipid was used in all cases. Care was taken to avoid an excess of water as this could make the subtraction of the water spectrum difficult. It was checked th at the t e m p e r a t u r e was constant within I ° C during the acquisition of the spectrum. T he water spectrum used t o subtract from t hat of the sample was obtained under similar conditions of t e m p e r a t u r e and pathlength to those of the sample.
Phospholipid--water systems Phospholipid--water systems are a useful model b i o m e m b r a n e system as
320
t h e y can spontaneously form lipid bilayer structures similar to the lipid matrix of natural biomembranes. We see (Fig. 1) the effect of water subtraction on a pure phospholipid DMpC sample dispersed in excess water and scanned at 37°C (i.e. above its transition temperature, To). The 1740 cm -j band, assigned to C=O stretching, appears now detached from the water signal; a substantial improvement is also found in the C--H stretching vibration bands, in the region near 2900 cm -~. It is possible to compare and contrast the band positions of the lipid in a solvent or in the solid state with those of the lipid in water, thereby providing information about hydrogenbonding interactions with phosphate groups and water.
Reconstitu ted pro tein--lipid systems A more detailed model biomembrane system involves reconstitution processes. In this case an intrinsic protein can be extracted from a natural biomembrane and included into a pure synthetic lipid--water system. We have examined the IR spectra of reconstituted sarcoplasmic reticulum membranes using the Ca2÷-ATPase, and incorporating this into dimyristoyllecithin. The spectra in Fig. 2 shows t h a t not only is it possible to examine the spectra after subtracting the water bands but that also we have been able to subtract the spectrum of the lipid, thereby showing clearly the absorption bands due to the intrinsic protein structure. This is illustrated in Fig. 2c where spectra from a DMPC--ATPase recombinant (57 : 1 molar ratio) are plotted before and after subtraction of water and DMPC. Bands in the 1800--1500 cm -1 region are prominent. Two major peaks are found, one at 1660 cm -1 due to the amide I vibration (C=O stretching), and one at 1545 cm -1 due to the amide II vibration (in plane NH bend). The shoulder centred at 1640 cm -1 was suspected to be due to an imbalance of the strong H20 absorption in this region, or traces of buffer. (Buffer solutions themselves give complicated absorption in this region.) To check this the reconstituted ATPase was washed with 2H20. The spectrum 2H20 is shown in Fig. 3. The difference spectrum after subtraction of absorption due to the 2H20 and to the DMPC lipid is also shown. In this spectrum there is no evidence for the additional peak at 1640 cm -~. Strong bands occur at 1650 and 1545 cm -1. (It is important to appreciate that shifts in the water absorption bands can occur because of the presence of the buffer so that care is necessary when the difference spectra are obtained. A study of the spectrum of the material in 2H20 is an added precaution.)
Natural biomem branes The same principle can be applied to natural biomembranes. As an example we show in Fig. 4 the spectrum from sarcoplasmic reticulum biomembrane after water and lipid substraction. The spectrum of the protein
321
t.o
[.Bf
ao
LL
I
'~
/
I} A V E II U rl B [ R (c~C1)
Fig. 3. Infrared spectrum on a reconstituted D M P C - - C a 2 + - A T P a s e
system (57 : I) molar
ratio obtained at 37°C after washing three times in 1.2 M KC] in 2H20. The spectrum of 2H20 is given by the dotted line. The difference spectrum corresponding to the protein after subtraction of bands due to 2H20 and due to the DMPC is given by the full line.
obtained in this way for SR--ATPase is similar to the corresponding difference spectrum from the DML--ATPase recombinant. Amide I and amide II bands are the most prominent at 1650 cm-' and at 1545 cm -1. A band at 1067 cm -1, that corresponds to C--O and C--C stretching, is also prominent. Receritly a study of the IR spectra of the Halobiurn halobacterium purple membrane showed the amide I band to be unusually high i.e. at 1665 cm -1. It was suggested that this is outside the normal range of frequencies (1650-1655 cm -1) reported for alpha-helical proteins and polypeptides. A possibility that this may indicate unusual alpha helices in the bacteriorhodopsin protein has been put forward [10]. Our studies of the sarcoplasmic reticulum biomembrane and its reconstituted systems show that whilst the sarcoplasmic reticulum biomembrane shows the amide I band in the normal range, the reconstituted ATPase systems do not, and may have similar unusual helical structures. Further studies will be made to check this.
322 L~
I.W
c::
ILm
W A V E ~i U ri B E P, ( cM- I )
Fig. 4. The infrared difference spectrum of sarcoplasmic reticulum biomembrane after subtraction of absorptions due to the water and phospholipid absorption bands.
DISCUSSION Difference IR spectroscopy has considerable potential for a number of research fields using relatively simple and not costly equipment. In this respect the advantage of IR over other techniques such as X-ray diffraction, DSC or NMR is the large a m o u n t of information which can be obtained in the short period of time needed for scanning a spectrum, the small a m o u n t of sample and the inexpensive equipment required. With model lipid--water systems the technique allows effects of ions and drugs to be studied. This may provide information concerning changes of fluidity, water structure, hydrogen bonding and drug conformations. Studies of the reconstituted systems and natural biomembranes have the potential for revealing information concerning conformational changes of intrinsic proteins as a consequence of temperature or triggering processes. Many other materials apart from biomembranes can be explored using this technique. Polysaccharides, cell walls, nucleic acids and proteins in aqueous solutions can also be studied.
323 SIMPLIFIED DESCRIPTION OF THE METHOD AND ITS APPLICATIONS The infrared Data Station associated with a simple IR spectrophotometer provides a relatively inexpensive means of studying biological samples in the presence of water and can be applied to the study of biomembranes, protein structures, polysacharides and other biological materials. Only small amounts of samples (less than 1 mg) are required. The possibility of subtracting absorption bands, due to different components, of a complex system allows the selective study of the individual components within the biomembrane structure, such as information about the protein conformation. ACKNOWLEDGEMENTS
We wish to acknowledge support from the Wellcome Trust and the Medical Research Council. J.C.G.-F. thanks the Wellcome Trust for a Fellowship. REFERENCES 1 Chapman, D. (1965) J. Am. Oil Chem. Soc. 42,353--371 2 Chapman, D. (1965) The Structure of Lipids, 1st edn., pp. 53--132. Methuen, London 3 Wallach, D.F.H. and Winzler, R.J. (1974) in Evolving Strategies and Tactics in Membrane Research, pp. 140--189. Springer-Verlag, Berlin 4 Thomas, G.K. and Kyogoku, Y. (1977) in Infrared and Raman Spectroscopy (Brame, E.G. and Grasselli, J.G., eds.), Vol. 1, Part C, pp. 717--872. Marcel Dekker, New York 5 Spedding, H. (1963) in Advances in Carbohydrate Chemistry (Wolfram, M.L. and Tipson, R.S., eds.), Vol. 19, pp. 23--49. Academic Press, New York 6 Cameron, D.G. and Mantsch, H.H. (1978) Biochem. Biophys. Res. Commun. 83, 886--892 7 Cameron, D.G., Casal, H.L. and Mantsch, H.H. {1979) J. Biochem. Biophys. Methods 1, 21--36 8 Casal, H.L., Smith, I.C.P., Cameron, D.G. and H.H. Mantsch (1979) Biochim. Biophys. Acta 5 5 0 , 1 4 5 - - 1 4 9 9 Gbmez-Fern~ndez, J.C., Goni, F.M., Bach, D., Restall, C.J. and Chapman, D. (1979) FEBS Lett. 9 8 , 2 2 4 - - 2 2 8 10 Rothschild, K.J. and Clark, N.A. (1979) Science 2 0 4 , 3 1 1 - - 3 1 2