[12] Resonance Raman and infrared difference spectroscopy of retinal proteins

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[12] R e s o n a n c e R a m a n a n d I n f r a r e d D i f f e r e n c e S p e c t r o s c o p y of R e t i n a l P r o t e i n s B y FRIEDRICH SIEBERT

Introduction Vibrational spectroscopy such as resonance Raman (RR) and infrared difference spectroscopy (IRD) has mostly been applied to retinal proteins having the retinal covalently bound to the protein via a protonated Schiff base. In all the cases studied, the side chain of a lysine constitutes the amino group. The vertebrate and invertebrate visual pigments and four different proteins located in the plasma membrane of the bacterium Halobacterium halobium belong to this class of proteins. The absorption of light by the chromophore causes its isomerization which, in turn, induces the protein structural changes responsible for the various functions of these systems. The proton pump bacteriorhodopsin, the chlorine pump halorhodopsin, the sensory pigments sensory rhodopsin and P-480 constitute the retinal proteins of Halobacterium halobium. Several reviews on these systems have been published. TM As far as the visual pigments are concerned, the special volume (Vol. 13) ofPhotobiochemistry andPhotobiophysics (1986) and the articles by Balogh-Nair and Nakanishi, 5 Ottolenghi,2 and Sandorfy and VoceUe6 provide a survey. Vibrational spectroscopy can help to determine what are the structures of the chromophores in these pigments and in the intermediates of their photoreactions, what kind of interaction exists between the chromophore and the protein, and what are the structural changes evoked in the protein by the photoreaction. These topics are dealt with in two recent reviews on the application of RR spectroscopy7,8 and in a review on the 1 D. Oesterhelt and J. Tittor, Trends Biochem. Sci. 14, 57 (1989). 2 M. Ottolenghi, in "Advances in Photochemistry" (J. N. Pitts, G. S. Hammond, K. Gollnik, and D. Grosjean, eds.), p. 97. Wiley (Interscience), New York, 1980. 3 W. Stoeckenius and R. A. Bogomolni, Annu. Rev. Biochem. 52, 587 (1982). 4 j. K. Lanyi, Annu. Rev. Biophys. Biophys. Chem. 15, 11 (1986). 5 V. Balogh-Nair and K. Nakanishi, in " N e w Comprehensive Biochemistry, Volume 3: Stereochemistry" (C. Tamm, ed.), p. 283. Elsevier Biomedical, Amsterdam, 1982. 6 C. Sandorfy and D. Vocelle, Can. J. Chem. 64, 2251 (1989). 7 M. Stockburger, T. Alshuth, D. Oesterhelt, and W. G~rtner, in "Spectroscopy of Biological Systems" (J. H. Clark and R. E. Hester, eds.), p. 483. Wiley, New York, 1986. 8 R. A. Mathies, S. O. Smith, and I. Palings, in "Biological Application of Raman Spectrometry, Volume 2: Resonance Raman Spectra of Polyenes and Aromatics" (T. G. Spiro, ed.), p. 59. Wiley, New York, 1987.

METHODS IN ENZYMOLOGY, VOL. 189

Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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application of IRD spectroscopy.9 Such problems may also apply to other retinal-containing proteins. The basic principles of the two methods are described here and some important applications discussed.

Resonance Raman Spectroscopy Normal Raman scattering is a weak effect, and recording a complete spectrum usually requires many hours. However, if the molecule has an absorption band near the wavelength of the probing beam (usually a continuous wave laser), the scattered photon is emitted from the electronic excited state and the scattering cross section for vibrations of this molecule is increased by several orders of magnitude. Resonance Raman spectroscopy has, therefore, the unique advantage of selectivity in that, in the spectrum of such complex systems as retinal proteins, only vibrations of the chromophore will be reflected. In the retinal proteins, however, a photoreaction is evoked by the absorption of light. Resonance Raman scattering is, therefore, always connected with the generation of intermediates of the photoreaction, and special techniques have been developed to obtain spectra of these intermediates as well as of the initial state. The basic methods are described briefly. Depending on the system being investigated, there are two basic techniques for overcoming these difficulties: namely, time-resolved technique and pump-probe technique. The first technique uses either a capillary flow system 1°-12or a rotating cell. 13,14The main purpose of both methods is to continuously bring fresh sample into the laser beam. The laser power has to be low enough to accumulate negligible amounts of photoproducts during the short time the sample resides within the cross-section of the laser beam (usually a few microseconds). The pump-probe technique was first applied to rhodopsin by Oseroff and Callender. 15In this application a photoequilibrium between three species of the photoreaction of rhodopsin can be established at 80 K (rhodopsin, bathorhodopsin, and isorhodopsin). By altering the wavelength of the "pump" laser beam, which usually

9 M. S. Braiman and K. J. Rothschild, Annu. Rev. Biophys. Biophys. Chem. 17, 541 (1988). 10 R. H. Callender, A. Doukas, R. K. Crouch, and K. Nakanishi, Biochemistry 15, 1621 (1976). 11 R. A. Mathies, A. R. Oseroff, and L. Stryer, Proc. Natl. Acad. Sci. U.S.A. 73, 1 (1976). ~2R. A. Mathies, T. B. Freedman, and L. Stryer, J. Mol. Biol. 109, 367 (1977). ~3 W. Kiefer and H. J. Bernstein, Appl. Spectrosc. 25, 500 (1971). 14 M. Stockburger, W. Klusmann, H. Gattermann, G. Massig, and R. Peters, Biochemistry 18, 4886 (1979). 15 A. R. Oseroff and R. H. Callender, Biochemistry 13, 4243 (1974).

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has the higher intensity, the composition of the photoequilibrium is changed. By tuning the wavelength of the "probe" laser beam to a position where one of the three species contributes most to the absorption spectrum, the RR scattering of this compound will selectively be enhanced. The probe beam should have a lower intensity in order not to alter the composition of the photoequilibrium. For systems which undergo a cyclic photoreaction, such as bacteriorhodopsin and halorhodopsin, the pump-probe technique can be applied at room temperature. The photostationary state will be dominated by the initial state and the intermediate with the slowest decay time. The time-resolved and pump-probe techniques can, of course, be combined. The composition of intermediates can be altered by altering the power and wavelength of the pump laser. Again, by adjusting the probe beam, the RR scattering from the species of interest will be enhanced. In general, however, irrespective of the method employed, the RR spectrum will contain contributions from several species. Employing time-resolved techniques, the RR spectrum of the initial state can usually be obtained by illuminating the sample with a very weak probe beam only. This spectrum can then be used to deduce from the composite spectra the spectra of the intermediates of the photoreaction. By moving the focus of the probe beam from the focus of the pump beam, it is possible to collect spectra at times after the excitation of the sample by the pump beam. The time is given by the distance between pump and probe beam divided by the velocity of the flow. This is an additional method for altering the relative amounts of intermediates in the cross section of the probe beam. Intermediates arising several microseconds up to several milliseconds after excitation can be measured in this way. More technical details about the methods can be found in the review papers mentioned above. One can obtain detailed molecular information by comparing the RR spectra of retinal proteins with those of model compounds. The basic investigation by the Mathies group has contributed greatly to our understanding of the vibrational spectra of isomers of retinal 16'~7and of protonated and unprotonated retinylidene Schiff bases.lS.~9 An essential part of this study was the collaboration with the Lugtenburg group, providing the 16 B. Curry, A. Broek, J. Lugtenburg, and R. A. Mathies, J. Am. Chem. Soc. 104, 5274 (1982). 17 B. Curry, I. Palings, A. Broek, J. A. Pardoen, P. P. J. Mulder, J. Lugtenburg, and R. A. Mathies, J. Phys. Chem. 88, 688 (1984). i8 S. O. Smith, A. B. Myers, R. A. Mathies, J. A. Pardoen, C. Winkel, E. M. M. Van den Berg, and J. Lugtenburg, Biophys. J. 47, 653 (1985). 19 B° CLlrry, I. Palings, J. A. Pardoen, J. Lugtenburg, and R. A. Mathies, Adv. Infrared Raman Spectrosc. 12, ll5 (1985).

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13C- and 2H-labeled retinals. 2°,21 By developing an empirical molecular force field, almost all bands in the RR spectra could be assigned to specific vibrations of the molecules and characteristic bands for the various retinal isomers could be deduced. The effects of forming the Schiff base and its protonation have also been studied. Since retinals, as well as their protonated and unprotonated Schiff bases, are photolabile, the capillary flow technique proved to be essential. In Fig. l, the RR spectra of bacteriorhodopsin under various illumination conditions using the rotating cell technique are shown. 14Spectrum (a) in Fig. 1 was collected with a weak probe beam of 514 nm only, reflecting the light-adapted species of bacteriorhodopsin only, BR568, which has an absorption maximum at 568 nm. By applying a broad pump beam of 514 nm and a coaxial probe beam of 450 nm, an almost pure spectrum of the long-lived intermediate M412 could be obtained [spectrum (b) in Fig. 1], having an absorption maximum at 412 nm. Only the shoulder at 1530 cm -1 indicates a small percentage of the initial state BR568. If the intensity of the probe beam is increased, spectrum (c) in Fig. 1 is obtained. This spectrum contains, in addition to BR568, the M412 intermediate, as can be seen from the band at 1567 cm -1. I f a pump beam of 450 is now applied, driving the M412 intermediate back to BR568, spectrum (d) (Fig. 1) is obtained. These experiments demonstrate the principles of the pump-probe technique in combination with time-resolved techniques. The spectra (Fig. 1) are all dominated by a strong band between 1500 and 1600 cm -1, caused by the ethylenic vibration of the retinal. Its position is correlated with the absorption maximum of the retinylidene Schiff base, owing to the dependency of the ethylenic force constants on ~r-electron delocalization. 22 The band at 1642 cm -l (Fig. la) was shown to shift to 1624 cm -1 in 2H20, demonstrating that it is caused by the C = N stretching vibration of the Schiff base and that "the Schiff base is protonated. The vibrations of this group have been especially investigated, both theoretically and experimentally. 23-28It appears unclear whether protonation increases or de2o j. Lugtenburg, Pure Appl. Chem. 57, 753 (1985). 21 j. Pardoen, C. Winkel, P. Mulder, and J. Lugtenburg, Recl. Tray. Chim. Pays-Bas 103, 135 (1984). 22 M. E. Heyde, D. Gill, R. G. Kilponen, and L. Rimai, J. Am. Chem. Soc. 93, 6776 (1971). 23 H. Deng and R. H. Callender, Biochemistry 26, 7418 (1987). 24 H. S. R. Gilson, B. H. Honig, A. Croteau, G. Zarrilli, and K. Nakanishi, Biophys. J. 53, 261 (1988). 25 T. Baasov, N. Friedman, and M. Sheves, Biochemistry 26, 3210 (1987). 26 j. j. L6pez-Garriga, G. T. Babcock, and J. F. Harrison, J. Am. Chem. Soc. 108, 7131 (1986).

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Av (cm-') FIG. 1. Pump-probe resonance Raman spectra of bactefiorhodopsin at room temperature using a rotating cell system. (a) Spectrum of the initial state BR568 taken with a weak probe beam of 514 nm only; (b) spectrum of the long-lived intermediate M412, taken with a 514-nm pump beam a 457-nm probe beam; (c) spectrum obtained with strong probe beam of 514 nm, showing the presence of BR568 and M412; (d) as (c), but with an additional pump beam of 457 nm, driving the M412 intermediate back to BR568. [Reproduced from M. Stockburger, W. Klusmann, H. Gattermann, G. Massig, and R. Peters, Biochemistry 18, 4886 (1979).]

128

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creases the C~---N force constant. Also, the contribution of the NH bending vibration to the C ~ N frequency is not completely understood. The understanding of this coupling is especially important, since it would provide a means to investigate the interaction of the protonated Schiff base group with its environment. Figure 2 demonstrates the pump-probe technique at -160 °, where the primary photoreaction of rhodopsin has been investigated) 9 Spectrum (A) in Fig. 2 was obtained with a probe beam of 585 nm. The sample was also illuminated with an all-lines argon laser pump beam. If the spectrum was recorded without the pump beam [spectrum (B), Fig. 2] only isorhodopsin was present. A pure rhodopsin spectrum was obtained by the capillary flow technique [spectrum (C), Fig. 2]. If appropriate amounts of spectra (B) and (C) are subtracted from spectrum (A), a pure spectrum of bathorhodopsin, the first intermediate which can be stabilized at low temperature, could be obtained. The downshift of the ethylenic mode (1545 versus 1536 cm -~) is in accordance with the red-shifted absorption maximum of bathorhodopsin. The strong bands below I000 cm -1 are of special interest. They could be assigned to hydrogen-out-of-plane (HOOP) vibrations of the retinal (i.e., the hydrogens move perpendicular to the polyene plane), and the high intensities were attributed to twists around single bonds of the polyene.3°,31 This means that the chromophore, already isomerized from 11-cis to all-trans, could not yet adopt a relaxed planar configuration owing to steric interaction with the protein, which is rigid at low temperature. A similar effect was observed for the 80 K photoproduct of bacteriorhodopsin, the K intermediate, 32and interpreted in a similar way. A peculiarity in the bathorhodopsin spectrum is the anomalous decoupling of the 1I- and 12-HOOP vibrations, causing the band at 920 cm-~; it was attributed to a specific interaction with a negatively charged residue or dipole of the protein. In recent illustrative applications of RR spectroscopy, demonstrating the selectivity of the method, the visual pigments in the retina of toad, anglefish, gecko, and bullfrog have been investigated) 3 In this case, the 27 j. j. L6pez-Garriga, G. T. Babcock, and J. F. Harrison, J. Am. Chem. Soc. 108, 7241 (1986). 28 j. j. L6pez-Garriga, S. Hanton, G. T. Babcock, and J. F. Harrison, J. Am. Chem. Soc. 108, 7251 (1986). G. Eyring and R. A. Mathies, Proc. Natl, Acad. Sci. U.S.A. 76, 33 (1979). 3o G. Eyring, B. Curry, A. Broek, J. Lugtenburg, and R. Mathies, Biochemistry 21, 384 (1982). 3~ I. Palings, E. M. M. Van den Berg, J. Lugtenburg, and R. A. Mathies, Biochemistry 28, 1498 (1989). 32 M. Braiman and R. A. Mathies, Proc. Natl. Acad. Sci. U.S.A. 79, 403 (1982). 33 B. A. Barry and R. A. Mathies, Biochemistry 26, 59 (1987).

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Frequency, cm -1 FIG. 2. Resonance Raman spectra of photostationary states of rhodopsin at -160 °, demonstrating the pump-probe technique. (A) Spectrum with 585-nm probe beam in the presence of an all-lines argon laser pump beam; (B) as (A), but without the pump beam, reflecting a pure isorhodopsin spectrum; (C) rapid-flow RR spectrum of rhodopsin taken with 600-nm probe beam (from Ref. 11); (D) RR spectrum of pure bathorhodopsin obtained by subtracting the appropriate amounts of isorhodopsin (B) and rhodopsin (C) from spectrum (A). [Reproduced from G. Eyring and R. A. Mathies, Proc. Natl. Acad. Sci. U.S.A. 76, 33 (1979).]

130

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pump-probe technique at low temperature has been combined with the Raman microscope technique, to select the outer segment of the photoreceptor of interest. As in normal bathorhodopsin, in the low-temperature photoproducts of all pigments the unusual decoupling of the I 1- and 12HOOP vibrations is observed. This shows that the factor regulating the absorption maximum (ranging from 430 to 502 nm in this investigation) is not responsible for this special interaction with the protein. Investigations on the toad blue photoreceptor pigment (actually the isopigment, containing 9-cis-retinal) showed that the chromophore behaves like the 9-cis model compound in solution. 34 This was interpreted by the lack of two internal carboxyl groups, which in the red rod pigments provide a special interaction regulating the absorption maximum and altering the RR spectrum. Resonance Raman spectroscopy has greatly contributed to our understanding of the photoreaction of bacteriorhodopsin, and the reader is referred to the reviews mentioned above. Two applications are of more general interest. The RR spectrum of vacuum-dried bacteriorhodopsin was measured, and it was concluded that water interacts with the Schiff base. 35 Support for this interaction was obtained by the observation that the C = N stretching vibration of the Schiff base, being located near the bending vibration of water, is broader in H20 than in 2H20. Since protonation of the Schiff base requires a polar environment, the water molecules may serve this purpose. In a combination of protein chemistry methods and RR spectroscopy, it was possible to identify the retinal-binding lysine. 36 It is possible to recombine bacteriorhodopsin from its two chymotryptic fragments. One fragment was obtained from bacteriorhodopsin isolated from bacteria grown in a synthetic medium containing [e-15N]lysine, the other was from unmodified lysine. Only in the RR spectra of the recombinant in which lysine-216 was labeled was a downshift of the C = N stretching vibration observed. This showed unequivocally that lysine-216 is the binding site. By recording the RR spectra of the M412 intermediate, a change of the binding site during the photoreaction could be excluded. Infrared Difference Spectroscopy At first sight, infrared spectroscopy would seem to be less suitable for the investigation of complex biological systems. Because the method is G. R. Loppnow, B. A. Barry, and R. A. Mathies, Proc. Natl. Acad. Sci. U.S.A. 86, 1515 (1989). 35 p. Hildebrandt and M. Stockburger, Biochemistry 23, 5539 (1984). 36 K. J. Rothschild, P. V. Argade, T. N. Earnest, K.-S. Huang, E. London, M.-J. Liao, H. Bayley, H. G. Khorana, and J. Herzfeld, J. Biol. Chem. 257, 8592 (1982).

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not selective, all groups of a protein contribute to the infrared absorbance, and it would be impossible to analyze the spectrum, which is constituted of many overlapping bands, in terms of single prosthetic groups or single amino acid side chains and their molecular changes. Only the vibrations of the amide group (amide I and amide II band) have been used to deduce information on the secondary structure. Infrared spectroscopy can, however, be rendered selective, that is, reflecting only functional groups, by forming the difference spectra between two different functional states. Only those groups which undergo molecular changes between these two states appear in the difference spectra, the remainder canceling each other by the subtraction. Infrared difference spectroscopy was first applied to retinal proteins (rhodopsin and bacteriorhodopsin) using timeresolved methods. 37-4° Somewhat later, static Fourier-transform infrared (FTIR) difference spectroscopy was applied to these systems.41-46 With the development of faster scanning FTIR spectrophotometers, time-resolved FTIR spectroscopy became accessible. 47,48The basic techniques of IRD are described here, with special emphasis on sample preparation and methods to assign bands to specific groups of the systems under investigation. Infrared difference spectroscopy requires that the two spectra being subtracted differ only in the altered molecular states. In principle, it would be possible to record, for instance, spectra of a retinal-binding protein with and without retinal. The corresponding difference spectrum should reflect, in addition to the infrared bands of the retinal, the protein groups involved in binding. Since only a small percentage of the total system will contribute, the absorbance changes in the difference spectra will range from 10 -4 t o 10 -2 at the most. Making the measurement of such small differences reliable requires, besides the different molecular states, 37 F. Siebert and W. M~intele, Biophys. Struct. Mech. 6, 147 (1980). 38 F. Siehert, W. Mantele, and W. Kreutz, Biophys. Struct. Mech. 6, 139 (1980). 39 F. Siebert, W. M~intele, and W. Kreutz, Can. J. Spectrosc. 26, l l 9 (1981). 4o W. M~intele, F. Siebert, and W. Kreutz, this series Vol. 88, p. 729. 41 K. J. Rothschild, M. Zagaeski, and W. A. Cantore, Biochem. Biophys. Res. Commun. 103, 483 0981). 42 K. J. Rothschild and H. Marrero, Proc. Natl. Acad. Sci. U.S.A. 79, 4045 (1982). 43 K. Bagley, G. Dollinger, L. Eisenstein, A. K. Singh, and L. Zimanyi, Proc. Natl. Acad. Sci. U.S.A. 79, 4972 (1982). F. Siebert and W. M~intele, Eur. J. Biochem. 1311, 565 (1983). 45 F. Siebert, W. M~intele, and K. Gerwert, Eur. J. Biochem. 136, 119 (1983). 46 K. J. Rothschild, W. A. Cantore, and H. Marrero, Science 219, 1333 (1983)° 47 M. S. Braiman, P. L. Ahl, and K. J. Rothschild, Proc. Natl. Acad. Sci. U.S.A. 84, 5221 (1987). 48 K. Gerwert, Ber. Bunsen-Ges. Phys. Chem. 92, 978 (1988).

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samples almost identical in other respects. This is possible for soluble proteins, and investigations on enzyme-substrate interactions using IRD have been published49; however, this will be very difficult to realize for suspensions of membrane proteins. One has to realize that because water strongly absorbs infrared radiation in almost the total spectral range of interest, the concentrations have to be very high (of the order of 100 mg/ml) and the cuvette has to be very thin (4 /~m). It is possible to make reproducible samples for soluble proteins under such conditions, but this is not feasible for suspensions owing to heterogeneity. Another method of sample preparation appears to be advantageous for membrane proteins: 100 to 200 p.g of the membranes are dried onto one window of the infrared cuvette, rehydrated with the required amount of water, and sealed with a second window. Most IRD spectroscopy investigations on retinal proteins have been performed on such types of samples. Infrared attenuated total reflection (ATR) spectroscopy has recently been applied to bacteriorhodopsin. 5°,51 This method offers the advantage that, as the penetration depth of the infrared radiation is of the order of its wavelength only, samples which are deposited onto the ATR crystal can be bathed in aqueous solutions and the solutions can be exchanged in one experiment. Special precautions have to be taken to avoid detachment of the film from the crystal surface. The difficulties in preparing highly reproducible samples of membrane proteins has limited investigations to photobiological systems. Here, the different states of the system can easily be evoked by irradiation with visible light. Intermediates of the photoreaction can be trapped by lowering the temperature or by adjusting the pH. The small absorbance changes in the difference spectra make it mandatory to employ FTIR spectroscopy, which exhibits a much higher sensitivity owing to the multiplex advantage and the larger energy through-put. Static IRD spectra are obtained by first recording a single-beam spectrum of the nonilluminated sample, then producing the desired intermediate, and subsequently recording the single-beam spectrum of the illuminated sample. By forming the logarithm of the ratio of the two single-beam spectra, the absorbance difference spectrum is obtained. Usually, to obtain a satisfactory signal49 C. W. Wharton, R. S. Chittok, J. Austin, and R. E. Hester, in "Spectroscopy of Biological Molecules--New Advances" (E. D. Schmid, F. W. Schneider, and F. Siebert, eds.), p. 95. Wiley, New York, 1988. 50 H. Marrero and K. J. Rothschild, FEBS Lett. 223, 289 (1987). 51 H. Marrero and K. J. Rothschild, Biophys. J. 52, 629 (1987).

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to-noise ratio, 100 to 1000 scans have to be coadded for one single-beam spectrum. Figure 3 shows the static FTIR difference spectra of the photoreaction of rhodopsin. 52 The intermediates have been stabilized at low temperatures: the batho-, lumi-, metarhodopsin I, and metarhodopsin II intermediates at 80, 173,240, and 273 K, respectively. The usual convention is that the bands of the initial state point downward and the bands of the photoproduct point upward. It is noteworthy that the strong bands below 1000 cm -l of bathorhodopsin, which have been observed in the RR spectra and attributed to HOOP vibrations, appear also as strong bands in the infrared. In a recent investigation, this striking behavior was shown to be caused by a coupling of C - C modes to the HOOP vibrations if the retinal is twisted. 53 In the infrared, strong HOOP bands can also be taken as evidence of a twisted chromophore. Figure 3 shows that in the spectra of the later intermediates more and more protein molecular changes occur. This can be deduced from the increasing bands between 1600 and 1800 era-1.

One difficulty of IRD spectroscopy is discriminating between bands caused by the chromophore and those arising from the protein. Isotopic labeling of the chromophore is essential. A convenient method to clearly deduce the labeling effect is subtraction of the unlabeled from the labeled difference spectrum. Protein bands cancel in the subtraction, and bands of the chromophore which are affected by the specific labeling show up and can be ~ssigned. 54,55 The overlap of bands of the initial state and of the photoproduct is a common problem in difference spectroscopy. It can only be overcome if the absolute spectrum of one species is known. If the absorbance strength of bands of one species is much lower, an approximate absolute spectrum can be obtained. 54 Also, a comparison of the IRD spectra with corresponding RR spectra may be useful to deduce the true band positions in the difference spectra. These methods have been applied to obtain information on the geometry of the retinal in the intermediates of bacteriorhodopsin and rhodopsin and have been used to assign their C~---N stretching frequencies .54,55 The main advantage of IRD is its capability to detect protein molecular 52 U. M. Ganter, E. D. Schmid, D. Perez-Sala, R. R. Rando, and F. Siebert, Biochemistry 28, 5954 (1989). 5~ K. Fahmy, M. F. Grossjean, F. Siebert, and P. Tavan, J. Mol. Struct. 214, 257 (1989). 54 K. Gerwert and F. Siebert, EMBO J. 5, 805 (1986). 55 U. M. Ganter, W. G~irtner, and F. Siebert, Biochemistry 27, 7480 (1988).

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changes. In most cases, to assign bands unequivocally to specific groups ern methods of genetic engineering may provide additional tools by expressing nonbacterial systems in bacteria or by introducing point mutations. Such applications are now briefly discussed. of the protein, isotopic labeling is a prerequisite. Whereas for bacterial systems this is feasible, other systems pose greater difficulties. The modProtonation changes of carboxyl groups during the photocycle of bacteriorhodopsin have been detected4~,56and were later assigned by isotopic labeling to aspartic acids) 7,58 In the same way, molecular changes of tyrosines could be detected) 9-62 By the use of point mutations, some of the spectral changes could be attributed to a specific tyrosine in the amino acid chain. With the same technique, some of the aspartic acids could be assigned to specific positions in the peptide chain. 63 By incorporating deuterated lysine into bacteriorhodopsin, vibrational modes at which lysine participates could be detected. 64 It is reasonable to assume that these modes are caused by the retinal-binding lysine. The analysis may provide information on the reaction of lysine upon the isomerization of the retinal. In another application the role of water in rhodopsin was investigated. By exchanging H2180for H2160, water molecules could be detected which undergo a slight change during the reaction of rhodopsin to bathorhodopsin. It was assumed that these water molecules are in the immediate neighborhood of the Schiff base and may help to stabilize its protonation state. A prior RR investigation may provide a link between the retinal proteins discussed in this chapter and the retinal-binding proteins. In this investigation the pigments of lobster shell were examined. From the correlation of the C ~ C stretching frequency with the absorption maximum it 56 F. Siebert, W. M~intele, and W. Kreutz, FEBS Lett. 141, 82 (1982). 57 M. Engelhard, K. Gerwert, B. Hess, W. Kreutz, and F. Siebert, Biochemistry 24, 400 (1985). 58 L. Eisenstein, S.-L. Lin, G. Dollinger, K. Odashima, J. Termini, K. Konno, W.-D. Ding, and K. Nakanishi, J. Am. Chem. Soc. 109, 6860 (1987). 59 S.-L. Lin, P. Ormos, L. Eisenstein, R. Govindjee, K. Konno, and K. Nakanishi, Biochemistry 26, 8327 (1987). 6o K. J. Rothschild, P. Roepe, P. L. Ahl, T. N. Earnest, R. A. Bogomolni, S. K. Das Gupta, C. M. Mulliken, and J. Herzfeld, Proc. Natl. Acad. Sci. U.S.A. 83, 347 (1986). 6, p. Roepe, P. L. Ahl, S. K. Das Gupta, J. Herzfeld, and K. J. Rothschild, Biochemistry 26, 6696 (1987). 62 p. Roepe, P. Scherrer, P. L. Ahl, S. K. Das Gupta, R. A. Bogomolni, J. Herzfeld, and K. J. Rothschild, Biochemistry 2,6, 6708 (1987). 63 M. S. Braiman, T. Mogi, T. Marti, L. J. Stern, H. G. Khorana, and K. J. Rothschild, Biochemistry 27, 8516 (1988). 64 E. McMaster and A. Lewis, Biochem. Biophys. Res. Commun. 156, 86 (1988).

136

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was concluded that the color is dominated by excitonic coupling of carotenoids. 65-67 This example may provide some hints of how RR spectroscopy could also be applied to the retinal-binding proteins. Infrared difference spectroscopy may be more difficult to implement; however, owing to its capability to provide direct information on chromophore-protein interactions, it appears worthwhile. 6~ V. R. Salares, N. M. Young, P. R. Carey, and H. J. Bernstein, J. Raman Spectrosc. 6, 282 (1977). V. R. Salares, N. M. Young, H. J. Bernstein, and P. R. Carey, Biochemistry 16, 4751 (1977). 67 V. R. Salares, R. Mendelsohn, P. R. Carey, and H. J. Bernstein, J. Phys. Chem. 80, 1137 (1976).

[13] A n a l y s i s of W a t e r - S o l u b l e C o m p o u n d s : G l u c u r o n i d e s

By ARUN B. BARUA Introduction

Retinyl/3-glucuronide and retinoyl fl-glucuronide, the glucuronic acid conjugates of retinol and retinoic acid, respectively, occur naturally in bile, 1-3 several other tissues, 3-5 and human blood. 6,7 Unlike retinol 0 - 2 . 5 /.tM concentration), retinoic acid, 6-~° retinyl glucuronide, and retinoyl glucuronide occur in much smaller concentrations (1-3 nM) in human blood. 6'7 The concentration of these conjugates in other tissues is not known but appears to be low. Until recently, retinyl fl-glucuronide (1) and retinoyl fl-glucuronide (II) were not available in sufficient quantities to study their metabolism and possible role in the functions of vitamin A. This chapter describes (1) chemical synthesis of retinyl/3-glucuronide and retinoyl fl-glucuronide, and (2) extraction of the two compounds from I R. D. Zachman, P. E. Dunagin, and J. A. Olson, J. Lipid Res. 7, 3 (1966). 2 p. E. Dunagin, E. H. Meadows, and J. A. Olson, Science 148, 86 (1965). 3 M. H. Zile, R. C. Inhorn, and H. F. DeLuca, J. Biol. Chem. 267, 3537 (1982). 4 K. Lippel and J. A. Olson, J. Lipid Res. 9, 168 (1968). 5 M. E. Cullum and M. H. Zile, J. Biol. Chem, 260~ 10590 (1985). 6 A. B. Barua and J. A. Olson, Am. J. Clin. Nutr. 43, 481 (1986). 7 A. B. Barua, R. O. Batres, and J. A. Olson, Am. J. Clin. Nutr. 50, 370 (1989). 8 M. G. DeRuyter, W.,E. Lambert, and A. P. DeLeenheer, Anal. Biochem. 98, 402 (1979). 9 A. P. DeLeenheer, W. E. Lambert, and I. Claeys, J. Lipid Res. 23, 1362 (1982). l0 j. L. Napoli, B. C. Pramanik, J. B. Williams, M. 1. Dawson, and P. D. Hobbs, J. Lipid Res. 26, 387 (1985).

METHODS IN ENZYMOLOGY, VOL. 189

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