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Fourth-derivative spectrophotometry of proteins
508
T I B S - December 1984
References l Das, M. and Fox, C. F. (1979) Annu. Rev. Biophys. Bioeng. 8, 165-193 2 Christiansen, G. and Griffith, J. (1977) Nucleic Acids Res. 4, 1837-1851 3 McGhee, J. D. and von Hippel, P. H. (1975) Biochemistry 14, 1297-1303 4 McGhee, J. D. and yon Hippel, P. H. (1975) Biochemistry 14, 1281-1296 5 Jackson, V. (1978) Cell 15,945-954 6 Chatteraj, D. K. and Inman, R. B. (1974) J. Mol. Biol. 87, 11-22 7 Peters, K. and Richards, F. M. (1977) Annu. Rev. Biochem. 41,523-551 8 Lawley, P. D. and Brookes, P. (1%7) Z Mol. Biol. 25, 143-160 9 Thomas, J. O., Sternberg, N. and Weisberg, R. (1978) Z Mol. Biol. 123, 14%161 10 Smith, K. C. (1%2) Biochem. Biophys. Res. Commun. 8, 157-163 11 Varghese, A. J. (1976) inAging, Carcinogenesis and Radiation Biology (Smith, K. C., ed.), pp.
207-223, Plenum Press 12 Markovitz, A. (1972) Biochim. Biophys. Acta 281,522-534 13 Kunkel, G. R. and Martinson, H. G. (1978) Nucleic Acids Res. 5, 4263-4272 14 Cao, T. M. and Sung, M. T. (1982) Biochern/stry 21, 3419--3427 15 Mandel, R., Kolomijtseva, G. and Brahms, J. G. (1979) Eur. J. Biochem. %, 257-265 16 Lica, L. and Ray. D. S. (1977)J. Mol. Biol. 115, 45-59 17 Hillel, Z. and Wu, C.-W. (1978) Biochemistr)' 17, 2954-2%1 18 Park, C. S., HilleL Z. and Wu, C.-W. (1980) Nucleic Acids Res. 8, 5895-5912 19 Gilmour, D. S. and Lis, J. T. (1984) Proc. Natl Acad. Sci. USA 81, 4275-4279 20 Harrison, C. A., Turner, D. H. and Hinkle, D. C. (1982) Nucleic Acids Res. 10, 2399-2414 21 Lin, S.-Y. and Riggs, A. D. (1974) Proc. Natl Acad. Sci. USA 71,947-951 22 Widom, J. and Baldwin, R. L. (1983) J. Mol. Biol. 171,419-437
Fourth-derivative spectrophotometry of proteins Esteve Padr6s, Mireia Dufiach, Antoni Morros, Manuel Sab6s and Joan Mafiosa Fourth-derivative spectrophotometry offers several advantages over classical absorption or difference spectrophotometry in examining the characteristics of aromatic amino acids in proteins. The basic principles o f the technique and its applications are outlined.
In elucidating protein structure, it is useful to study the individual characteristics of aromatic amino acids, such as the formation of hydrogen bonds by the O H group of tyrosine or responses to changes in the environment of the protein. Ideally, the electronic absorption spectrum of an aromatic residue in the near-ultraviolet range (240-320 nm) would be a narrower peak corresponding to a single transitionL Unfortunately, in reality, solvent-solute interactions and rotational and vibrational transitions cause the peak to broaden, and so peaks of individual c o m p o n e n t s overlap. Fourth-derivative spectrophotometry was developed in an attempt to resolve these individual components 2,3.
Basic principles The first-order derivative curve, and any higher order curve obtained by repeating the process the requisite number of times, is p r e p a r e d by computing The authors are at the Departarnent de Biofisica, Facultat de Medicina, Universitat AutOnoma de Barcelona, BELLA TERRA (Barcelona), Spain.
the difference b e t w e e n the absorption of a sample at a certain wavelength A (h) and that of the same sample at a wavelength shifted a finite interval (Ah, a differencing interval), A (k + Ah). The difference value obtained is assigned to the mid-point b e t w e e n A (h) and A (h + AX), that is at (X + Ah/2). For Gaussian curves, the intensity of the nth derivative is inversely proportional to the nth p o w e r of the bandwidth. Thus the derivative analysis favours the narrow bands to the detriment of the broader ones. Theoretically, the higher the order of the derivative, the greater the resolution, but a difficulty arises in the form of a decrease in the signal-to-noise ratio 2. T h e intensity of bands in the difference curve is directly proportional to the size of the differencing intervals used, but the noise is independent, being 2 n times the original noise level. The choice o f derivative must therefore be a compromise between these two opposing factors. W e have chosen the fourth derivative as it provides better resolution than the firsP or second s derivatives and has the advantage of all
© 1984,ElsevierSciencePublishersB.V..Amsterdam 0376- 5(~67/84/$(E.00
23 Levinson, J. W., Liebes, L. F. and McCormic, J. J. (1976) Biochim. Biophys. Acta 447, 260273 24 Levina, E. S., Bavykin, S. G., Shick, V. V. and Mirzabekov, A. D. (1981) Anal. Biochem. 110, 93--101 25 Mirzabekov, A. D., Bavykin, S. G., Karpov, V.L., Preobrazhenskaya, O. V., Ebralidze, K.K., Tuneev, V. M., Melinkova, A.F., Goguadze, E.G., Chenchick, A.A. and Beabealashvili, R. S. (1982) Co/d Spring Harbor Symp. Quant. Biol. 47, 503-510 26 Chenchick, A. A., Beabealashvili, R. S. and Mirzabekov, A. D. (1981) FEB Lett. 128, 46-50 27 Hearst, J. E. (1981) Ann. Rev. Biophys. Bioeng. 10, 69-86 28 Schwartz. D. C., Saffran, W., Welsh, J., Haas, R., Goldenberg, M. and Cantor, C. R. (1983) Cold Spring Harbor Syrup. Quant. Biol. 47, 189-195 29 Becker, M. M. and Wang, J. C. (1984) Nature 309. 682~87
even-number derivatives - the maxima of the derivatives are at the same wavelengths as the original absorption spectrum. Some commercial spectrophotometers are now capable of obtaining the fourth derivative directly. As an example of the resolving ability of the fourth-derivative technique, Fig. 1 shows the absorption and the fourthderivative spectra of bacteriorhodopsin (8 T r p , l l Tyr,13 Phe) in two very different situations: as an aqueous suspension at 20°C, and as a dry film at - 1 9 6 ° C . In the former case, the broadening mechanisms yield an absorption spectrum exhibiting very little fine structure, whereas in the dry film at - 1 9 6 ° C the absence of solvent-solute interactions as well as the decrease in the rotational and vibrational energies allows the appearance of some fine structure. Despite the great differences between the corresponding absorption spectra, the fourth-derivative spectrum of the aqueous sample shows all the major features of that of the dry film, except for the shift of the whole spectrum, which is due to the different temperature. Note that the maxima of the fourth-derivative spectrum correspond closely to the maxima or shoulders of the absorption spectrum.
Fourth-derivative spectra of the aromatic models The application of this technique to study aromatic amino acid models demonstrates that the different vibrational transitions can be well resolved 6.7. Figure 2 shows the absorption and the fourth-derivative spectra of the three aromatic models in water. For the tyrosine model, it can be seen that the
509
T 1 B S - D e c e m b e r 1984
at 286.7 nm in dimethylformamide. The parameter R changes from 0.95 in chloroform to 1.60 in methanoP.
0.8
- 0.3 M NaCI,
o• 0,6 ¢-
..Q
o 0.4 .Q
< 0.2
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== ~,
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:~ -2
,.;
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240
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I
/
I
I
i
i
260 280 300 320 Wavelength (nm)
Fig. l. Absorption (a) and Jburth-derivative spectra (b) of bacteriorhodopsin. ( ) Spectra obtained from an aqueous sample at 2(PC. ( . . . . ) Spectra obtained J ? o m a dry .film at -t96~'C. The spectra were taken on a PerkinEhner model 320 spectrophotometer, using the following instrumental parameters: slit width, 1 nm; time ~onstant, ] s; scan rate, 30 nm/min; and derivative interval, 4 nm. Low-temperature spectra were obtained with an OxJord D N 1704 cryostat,
Applications to proteins In proteins containing no tryptophan, the contributions of phenylalanine and tyrosine are well separated. For tyrosine, the wavelength of the peaks and the parameter R depend on the strength of the hydrogen bonds formed by the OH group of tyrosine, and R also depends on the heterogeneity of the tyrosine environments. For phenylalanine, the wavelength of the peaks mainly depends on the polarity of the environmenP. When tryptophan and tyrosine are both present, the method is unable to completely separate its contributions; however, if the ratio of tryptophan to tyrosine is at least 1:4, the fourth-derivative peaks of tl3'ptophan are dominant, chiefly in the minimum of the longest wavelength. The simultaneous consideration of R and the wavelengths of the peaks allows an estimation to be made of the tryptophan environment7. Taking the fourth-derivative spectrum of bacteriorhodopsin as an example (Fig. 1), the above observations imply that the peaks in the 280-300 nm range are indicative of the trypi
.
.
.
.
.
.
,
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(a)
0.2
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most intense peak corresponds to the shoulder of the absorption spectrum, and it originates from the O - O vibrational band of the phenolic rings. The secondary main peak corresponds to the maximum of the absorption spectrum, whereas the small peak originates from a minor vibrational band. Similar reasoning holds for the other two models, except that the tryptophan spectrum in the near-ultraviolet originates from two electronic transitions, ~L, and ~Lb, a fact that complicates its interpretation7. Changes in the environment of the aromatic residues produce changes in the fourth-derivative spectra, reflecting the alterations suffered by the electronic energy levels Lg. In order to quantify these~changes, we have used the wavelengths of the maxima and minima and a geometrical parameter R, which is defined as the ratio between the amplitude of the two main peaks 6,7. For example, the main peak of the fourthderivative spectrum of the tyrosine model appears at 282.3 nm in water, and
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.
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Wavelength (nm) Fig. 2. Absorption ( . . . . ) and fourth-derivative spectra ( ) o f the aromatic models in water, p H 7.0 at 20°C. (a) 1 mM AcPheOEt," (b) O. 7 mM AcTyrOEt; (c) 0.18 mm AcTrpOEt.
pH 3 -0.3MNaCI, pH 7
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Wavelength (nm) Fig. 3. Fourth-derivative spectra ~d" an equimolecular mixture o f non-interacting histones H2A and H 2 B ( . . . . ), and of the dimer (H2A) ( H2B) ( ), at the same concentration.
tophan environment. The value of 294.3 nm for the longest-wavelength minimum of the aqueous sample is the same as that obtained for the model AcTrpOEt in dioxane v. It can be inferred, therefore, that on average the tryptophan residues in bacteriorhodopsin are buried within the molecule. This is in accordance with the known location of tryptophans in bacteriorhodopsinHL To date we have applied this technique to the study of conformational changes in proteins, such as thermal denaturation of histone H1 and cytochrome c 6,7, thermal denaturation of RNase A ~ and SDS denaturation of bacteriorhodopsin 7.11 as well as the study of tryptophan environments in the L550 and M412 intermediates of bacteriorhodopsin ~2. The study of proteinprotein interactions can also be undertaken using the fourth-derivative technique, as indicated by the spectral changes observed on formation of the melittin (bee-venom toxin) tetramer', or the (H2A)(H2B) histone dimer. Figure 3 shows the fourth-derivative spectra of an equimolecular mixture of histones H2A and H2B, under interacting and non-interacting conditions. Notable differences exist between the two spectra, basically in the tyrosine range (270300 nm), although differences are also found in the phenylalanine region (250-265 nm). These changes are interpreted as the consequence of some aromatic residues being buried upon formation of the histone dimer.
510 The applications of the fourth-derivative method can be extended to cases in which broad peaks overlap with those of interest. For example, ionized tyrosine has a broad absorption maximum at 295 nm, which obscures the bands arising from the un-ionized residues. The fourth-derivative operation cancels out the broad band, thus allowing the study of un-ionized tyrosine residues in the alkaline region 6. On the other hand, when there is appreciable non-selective light-scattering, this technique still gives a horizontal baseline, thus permitting the study of moderately turbid samples.
Condusions The examples outlined in this article illustrate that fourth-derivative spectrophotometry can be used to monitor the environment of the aromatic residues in a variety of protein systems. The tech-
TIBS - December 1984
nique offers several advantages over classical absorption or difference spectrophotometry, such as the possibility of studying phenylalanine separately from tyrosine and tryptophan and, under certain conditions, tryptophan separately from tyrosine. Likewise, the method is capable of clearly revealing small peaks not readily apparent in the absorption or difference spectra and it is particularly useful when broad, perturbing bands are present.
Acknowledgement The authors acknowledge the financial support from the Comisi6n Asesora de Investigaci6n Cientifica y T6cnica (grant 403/81).
References 1 Laskowski, M. (1970) in Spectroscopic Approaches to Biomolecular Conformation (Urry, D. W., ed.). pp. 1-21, American Med.
Open Question Cytochrome P-450 and glutathione: what is the significance of their interrelationship in lipid peroxidation?
Ass., USA 2 Butler, W. L. (1979) Methods Enzymol. 56, 501-515 3 Talsky, G., Mayring, U and Kreuzer, H. (1978) Angew. Chem. Int. Ed. Engl. 17,785--799 4 Brandts, J. F. and Kaplan, L. J. (1973) Biochemistry 12, 2011-2024 5 Ichikawa, T. and Terada, H. (1979) Biochim. Biophys. Acta 580, 120-128 6 Padr6s, E., Morros, A., Mafiosa, J. and Dufiach, M. (1982) Eur. J. Biochem. 127, 117122 7 Duhach, M., Sab6s, M. and Padr6s, E. (1983) Eur. J. Biochem. 134, 123-128 8 Horwitz, J., Strikland, E. H. and Billups, C. (1970) J. Am. Chem. Soc. 92, 2119-2129 9 Donovan, J. W. (1969) in Physical Principles and Techniques of Protein Chemistry (Leach, S. J., ed.), part A, pp. 101-170, Academic Press 10 Sherman, W. V. (1981) Photochem. Photobiol. 33, 367-371 ll Padr6s, E., Dufiach, M. and Sab6s, M. (1984) Biochim. Biophys. Acta 769, 1-7 12 Sab6s, M., Dufiach, M., Mafiosa, J., Morros, A. and Padr6s, E. (1984) Photobiochem. Photobiophys. 8, 97-101
atoms or radicals. Malondialdehyde is formed during the propagation phase of both LOOH-independent and LOOHdependent lipid peroxidation. The respective initiation processes differ and are not well understood.
Cytochrome P-450
Enzymically induced lipid peroxidation is frequently studied using NADPH and the microsomal membrane. The enzymes involved are components of the cytochrome P-450 system: NADPH cytochrome P-450 reductase and cytoAalt Bast and Guido R. M. M. Haenen chrome P-450. Stimulation of lipid peroxidation by the reductase can occur via transfer of electrons, not to cytochrome P-450, but to iron or chelated iron (forBoth cytochrorne P-450 and glutathione participate in the metabolism of xenobiotics. Their interrelationship is described here, as well as current findings indicating their mation of reduced (chelated) iron) or to xenobiotics (with subsequent redox cycmutual involvement in lipid peroxidation. ling)2. The role of cytochrome P-450 in lipid peroxidation is more controversial Lipid peroxidation is the process by malondialdehyde (MDA). Propagation and is discussed here. which oxidative degradation of poly- was defined as breakdown of LOOH to High concentrations of the cytounsaturated fatty acids (LH) occurs. give reactive intermediates and products chrome P-450 system are found in the Because of the biomedical implications of lipid peroxidation (e.g. malon- microsomal membrane. The enzyme of lipid peroxidation (its toxicity), the dialdehyde) with reformation of system has attracted researchers in the process has been the subject of stren- LOOH. These definitions have been fields of biochemistry, pharmacology uous research. The effects of both adopted frequently by others, with sub- and toxicology. It plays a pivotal role in enzymic and non-enzymic systems in sequent confusion. For, in general the metabolism of endogenous comchemical terms, initiation means radical pounds (steroids, prostaglandins) and in lipid peroxidation have been studied. Lipid peroxidation was first consid- production and propagation means radi- the biotransformation of xenobiotics. In ered as two distinct sequential phases by cal transfer, m more accurate represen- our western society there is an everSvingen et al.~: initiation and propaga- tation of the reaction is shown in Fig. 1, increasing demand on cytochrome P-450 tion. Initiation was defined as the forma- with classification of LOOH-indepen- to cope with xenobiotics. However, the tion of lipid hydroperoxides (LOOH) dent lipid peroxidation and LOOH- involvement of cytochrome P-450 does accompanied by a minimal formation of dependent lipid peroxidation. Both not guarantee detoxification. It has been types of peroxidation have an initiation hypothesized that cytochrome P-450 A. Bast and G. R. M. M. Haeen are at the Faculty of Pharmacy, Dept. of Pharmacology and Phar- phase and a propagation phase. During played an early role in the detoxification macotherapy, State University of Utrecht, Catharijne- the latter, reactants are converted to of oxygen, during chemical evolution. It products with no net consumption of was probably present in living organisms singel 60, 3511 GH Utrecht, The Netherlands.
(~) 1984,ElsevierSciencePublishelsB.V., Amsterdam 0376- 5(~7/84/$02.00
T I B S - December 1984
References l Das, M. and Fox, C. F. (1979) Annu. Rev. Biophys. Bioeng. 8, 165-193 2 Christiansen, G. and Griffith, J. (1977) Nucleic Acids Res. 4, 1837-1851 3 McGhee, J. D. and von Hippel, P. H. (1975) Biochemistry 14, 1297-1303 4 McGhee, J. D. and yon Hippel, P. H. (1975) Biochemistry 14, 1281-1296 5 Jackson, V. (1978) Cell 15,945-954 6 Chatteraj, D. K. and Inman, R. B. (1974) J. Mol. Biol. 87, 11-22 7 Peters, K. and Richards, F. M. (1977) Annu. Rev. Biochem. 41,523-551 8 Lawley, P. D. and Brookes, P. (1%7) Z Mol. Biol. 25, 143-160 9 Thomas, J. O., Sternberg, N. and Weisberg, R. (1978) Z Mol. Biol. 123, 14%161 10 Smith, K. C. (1%2) Biochem. Biophys. Res. Commun. 8, 157-163 11 Varghese, A. J. (1976) inAging, Carcinogenesis and Radiation Biology (Smith, K. C., ed.), pp.
207-223, Plenum Press 12 Markovitz, A. (1972) Biochim. Biophys. Acta 281,522-534 13 Kunkel, G. R. and Martinson, H. G. (1978) Nucleic Acids Res. 5, 4263-4272 14 Cao, T. M. and Sung, M. T. (1982) Biochern/stry 21, 3419--3427 15 Mandel, R., Kolomijtseva, G. and Brahms, J. G. (1979) Eur. J. Biochem. %, 257-265 16 Lica, L. and Ray. D. S. (1977)J. Mol. Biol. 115, 45-59 17 Hillel, Z. and Wu, C.-W. (1978) Biochemistr)' 17, 2954-2%1 18 Park, C. S., HilleL Z. and Wu, C.-W. (1980) Nucleic Acids Res. 8, 5895-5912 19 Gilmour, D. S. and Lis, J. T. (1984) Proc. Natl Acad. Sci. USA 81, 4275-4279 20 Harrison, C. A., Turner, D. H. and Hinkle, D. C. (1982) Nucleic Acids Res. 10, 2399-2414 21 Lin, S.-Y. and Riggs, A. D. (1974) Proc. Natl Acad. Sci. USA 71,947-951 22 Widom, J. and Baldwin, R. L. (1983) J. Mol. Biol. 171,419-437
Fourth-derivative spectrophotometry of proteins Esteve Padr6s, Mireia Dufiach, Antoni Morros, Manuel Sab6s and Joan Mafiosa Fourth-derivative spectrophotometry offers several advantages over classical absorption or difference spectrophotometry in examining the characteristics of aromatic amino acids in proteins. The basic principles o f the technique and its applications are outlined.
In elucidating protein structure, it is useful to study the individual characteristics of aromatic amino acids, such as the formation of hydrogen bonds by the O H group of tyrosine or responses to changes in the environment of the protein. Ideally, the electronic absorption spectrum of an aromatic residue in the near-ultraviolet range (240-320 nm) would be a narrower peak corresponding to a single transitionL Unfortunately, in reality, solvent-solute interactions and rotational and vibrational transitions cause the peak to broaden, and so peaks of individual c o m p o n e n t s overlap. Fourth-derivative spectrophotometry was developed in an attempt to resolve these individual components 2,3.
Basic principles The first-order derivative curve, and any higher order curve obtained by repeating the process the requisite number of times, is p r e p a r e d by computing The authors are at the Departarnent de Biofisica, Facultat de Medicina, Universitat AutOnoma de Barcelona, BELLA TERRA (Barcelona), Spain.
the difference b e t w e e n the absorption of a sample at a certain wavelength A (h) and that of the same sample at a wavelength shifted a finite interval (Ah, a differencing interval), A (k + Ah). The difference value obtained is assigned to the mid-point b e t w e e n A (h) and A (h + AX), that is at (X + Ah/2). For Gaussian curves, the intensity of the nth derivative is inversely proportional to the nth p o w e r of the bandwidth. Thus the derivative analysis favours the narrow bands to the detriment of the broader ones. Theoretically, the higher the order of the derivative, the greater the resolution, but a difficulty arises in the form of a decrease in the signal-to-noise ratio 2. T h e intensity of bands in the difference curve is directly proportional to the size of the differencing intervals used, but the noise is independent, being 2 n times the original noise level. The choice o f derivative must therefore be a compromise between these two opposing factors. W e have chosen the fourth derivative as it provides better resolution than the firsP or second s derivatives and has the advantage of all
© 1984,ElsevierSciencePublishersB.V..Amsterdam 0376- 5(~67/84/$(E.00
23 Levinson, J. W., Liebes, L. F. and McCormic, J. J. (1976) Biochim. Biophys. Acta 447, 260273 24 Levina, E. S., Bavykin, S. G., Shick, V. V. and Mirzabekov, A. D. (1981) Anal. Biochem. 110, 93--101 25 Mirzabekov, A. D., Bavykin, S. G., Karpov, V.L., Preobrazhenskaya, O. V., Ebralidze, K.K., Tuneev, V. M., Melinkova, A.F., Goguadze, E.G., Chenchick, A.A. and Beabealashvili, R. S. (1982) Co/d Spring Harbor Symp. Quant. Biol. 47, 503-510 26 Chenchick, A. A., Beabealashvili, R. S. and Mirzabekov, A. D. (1981) FEB Lett. 128, 46-50 27 Hearst, J. E. (1981) Ann. Rev. Biophys. Bioeng. 10, 69-86 28 Schwartz. D. C., Saffran, W., Welsh, J., Haas, R., Goldenberg, M. and Cantor, C. R. (1983) Cold Spring Harbor Syrup. Quant. Biol. 47, 189-195 29 Becker, M. M. and Wang, J. C. (1984) Nature 309. 682~87
even-number derivatives - the maxima of the derivatives are at the same wavelengths as the original absorption spectrum. Some commercial spectrophotometers are now capable of obtaining the fourth derivative directly. As an example of the resolving ability of the fourth-derivative technique, Fig. 1 shows the absorption and the fourthderivative spectra of bacteriorhodopsin (8 T r p , l l Tyr,13 Phe) in two very different situations: as an aqueous suspension at 20°C, and as a dry film at - 1 9 6 ° C . In the former case, the broadening mechanisms yield an absorption spectrum exhibiting very little fine structure, whereas in the dry film at - 1 9 6 ° C the absence of solvent-solute interactions as well as the decrease in the rotational and vibrational energies allows the appearance of some fine structure. Despite the great differences between the corresponding absorption spectra, the fourth-derivative spectrum of the aqueous sample shows all the major features of that of the dry film, except for the shift of the whole spectrum, which is due to the different temperature. Note that the maxima of the fourth-derivative spectrum correspond closely to the maxima or shoulders of the absorption spectrum.
Fourth-derivative spectra of the aromatic models The application of this technique to study aromatic amino acid models demonstrates that the different vibrational transitions can be well resolved 6.7. Figure 2 shows the absorption and the fourth-derivative spectra of the three aromatic models in water. For the tyrosine model, it can be seen that the
509
T 1 B S - D e c e m b e r 1984
at 286.7 nm in dimethylformamide. The parameter R changes from 0.95 in chloroform to 1.60 in methanoP.
0.8
- 0.3 M NaCI,
o• 0,6 ¢-
..Q
o 0.4 .Q
< 0.2
I
i
I
I
I
i
I
i
(b) 4
== ~,
2 0
:~ -2
,.;
-4 I
240
I
I
/
I
I
i
i
260 280 300 320 Wavelength (nm)
Fig. l. Absorption (a) and Jburth-derivative spectra (b) of bacteriorhodopsin. ( ) Spectra obtained from an aqueous sample at 2(PC. ( . . . . ) Spectra obtained J ? o m a dry .film at -t96~'C. The spectra were taken on a PerkinEhner model 320 spectrophotometer, using the following instrumental parameters: slit width, 1 nm; time ~onstant, ] s; scan rate, 30 nm/min; and derivative interval, 4 nm. Low-temperature spectra were obtained with an OxJord D N 1704 cryostat,
Applications to proteins In proteins containing no tryptophan, the contributions of phenylalanine and tyrosine are well separated. For tyrosine, the wavelength of the peaks and the parameter R depend on the strength of the hydrogen bonds formed by the OH group of tyrosine, and R also depends on the heterogeneity of the tyrosine environments. For phenylalanine, the wavelength of the peaks mainly depends on the polarity of the environmenP. When tryptophan and tyrosine are both present, the method is unable to completely separate its contributions; however, if the ratio of tryptophan to tyrosine is at least 1:4, the fourth-derivative peaks of tl3'ptophan are dominant, chiefly in the minimum of the longest wavelength. The simultaneous consideration of R and the wavelengths of the peaks allows an estimation to be made of the tryptophan environment7. Taking the fourth-derivative spectrum of bacteriorhodopsin as an example (Fig. 1), the above observations imply that the peaks in the 280-300 nm range are indicative of the trypi
.
.
.
.
.
.
,
t
(a)
0.2
0.24
-0.2
most intense peak corresponds to the shoulder of the absorption spectrum, and it originates from the O - O vibrational band of the phenolic rings. The secondary main peak corresponds to the maximum of the absorption spectrum, whereas the small peak originates from a minor vibrational band. Similar reasoning holds for the other two models, except that the tryptophan spectrum in the near-ultraviolet originates from two electronic transitions, ~L, and ~Lb, a fact that complicates its interpretation7. Changes in the environment of the aromatic residues produce changes in the fourth-derivative spectra, reflecting the alterations suffered by the electronic energy levels Lg. In order to quantify these~changes, we have used the wavelengths of the maxima and minima and a geometrical parameter R, which is defined as the ratio between the amplitude of the two main peaks 6,7. For example, the main peak of the fourthderivative spectrum of the tyrosine model appears at 282.3 nm in water, and
.
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',
0.12
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.
.
.
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.
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Wavelength (nm) Fig. 2. Absorption ( . . . . ) and fourth-derivative spectra ( ) o f the aromatic models in water, p H 7.0 at 20°C. (a) 1 mM AcPheOEt," (b) O. 7 mM AcTyrOEt; (c) 0.18 mm AcTrpOEt.
pH 3 -0.3MNaCI, pH 7
¢.o
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go -2
.I -4 I
240
I
260
i
i
280
' 3170 ~ 320
Wavelength (nm) Fig. 3. Fourth-derivative spectra ~d" an equimolecular mixture o f non-interacting histones H2A and H 2 B ( . . . . ), and of the dimer (H2A) ( H2B) ( ), at the same concentration.
tophan environment. The value of 294.3 nm for the longest-wavelength minimum of the aqueous sample is the same as that obtained for the model AcTrpOEt in dioxane v. It can be inferred, therefore, that on average the tryptophan residues in bacteriorhodopsin are buried within the molecule. This is in accordance with the known location of tryptophans in bacteriorhodopsinHL To date we have applied this technique to the study of conformational changes in proteins, such as thermal denaturation of histone H1 and cytochrome c 6,7, thermal denaturation of RNase A ~ and SDS denaturation of bacteriorhodopsin 7.11 as well as the study of tryptophan environments in the L550 and M412 intermediates of bacteriorhodopsin ~2. The study of proteinprotein interactions can also be undertaken using the fourth-derivative technique, as indicated by the spectral changes observed on formation of the melittin (bee-venom toxin) tetramer', or the (H2A)(H2B) histone dimer. Figure 3 shows the fourth-derivative spectra of an equimolecular mixture of histones H2A and H2B, under interacting and non-interacting conditions. Notable differences exist between the two spectra, basically in the tyrosine range (270300 nm), although differences are also found in the phenylalanine region (250-265 nm). These changes are interpreted as the consequence of some aromatic residues being buried upon formation of the histone dimer.
510 The applications of the fourth-derivative method can be extended to cases in which broad peaks overlap with those of interest. For example, ionized tyrosine has a broad absorption maximum at 295 nm, which obscures the bands arising from the un-ionized residues. The fourth-derivative operation cancels out the broad band, thus allowing the study of un-ionized tyrosine residues in the alkaline region 6. On the other hand, when there is appreciable non-selective light-scattering, this technique still gives a horizontal baseline, thus permitting the study of moderately turbid samples.
Condusions The examples outlined in this article illustrate that fourth-derivative spectrophotometry can be used to monitor the environment of the aromatic residues in a variety of protein systems. The tech-
TIBS - December 1984
nique offers several advantages over classical absorption or difference spectrophotometry, such as the possibility of studying phenylalanine separately from tyrosine and tryptophan and, under certain conditions, tryptophan separately from tyrosine. Likewise, the method is capable of clearly revealing small peaks not readily apparent in the absorption or difference spectra and it is particularly useful when broad, perturbing bands are present.
Acknowledgement The authors acknowledge the financial support from the Comisi6n Asesora de Investigaci6n Cientifica y T6cnica (grant 403/81).
References 1 Laskowski, M. (1970) in Spectroscopic Approaches to Biomolecular Conformation (Urry, D. W., ed.). pp. 1-21, American Med.
Open Question Cytochrome P-450 and glutathione: what is the significance of their interrelationship in lipid peroxidation?
Ass., USA 2 Butler, W. L. (1979) Methods Enzymol. 56, 501-515 3 Talsky, G., Mayring, U and Kreuzer, H. (1978) Angew. Chem. Int. Ed. Engl. 17,785--799 4 Brandts, J. F. and Kaplan, L. J. (1973) Biochemistry 12, 2011-2024 5 Ichikawa, T. and Terada, H. (1979) Biochim. Biophys. Acta 580, 120-128 6 Padr6s, E., Morros, A., Mafiosa, J. and Dufiach, M. (1982) Eur. J. Biochem. 127, 117122 7 Duhach, M., Sab6s, M. and Padr6s, E. (1983) Eur. J. Biochem. 134, 123-128 8 Horwitz, J., Strikland, E. H. and Billups, C. (1970) J. Am. Chem. Soc. 92, 2119-2129 9 Donovan, J. W. (1969) in Physical Principles and Techniques of Protein Chemistry (Leach, S. J., ed.), part A, pp. 101-170, Academic Press 10 Sherman, W. V. (1981) Photochem. Photobiol. 33, 367-371 ll Padr6s, E., Dufiach, M. and Sab6s, M. (1984) Biochim. Biophys. Acta 769, 1-7 12 Sab6s, M., Dufiach, M., Mafiosa, J., Morros, A. and Padr6s, E. (1984) Photobiochem. Photobiophys. 8, 97-101
atoms or radicals. Malondialdehyde is formed during the propagation phase of both LOOH-independent and LOOHdependent lipid peroxidation. The respective initiation processes differ and are not well understood.
Cytochrome P-450
Enzymically induced lipid peroxidation is frequently studied using NADPH and the microsomal membrane. The enzymes involved are components of the cytochrome P-450 system: NADPH cytochrome P-450 reductase and cytoAalt Bast and Guido R. M. M. Haenen chrome P-450. Stimulation of lipid peroxidation by the reductase can occur via transfer of electrons, not to cytochrome P-450, but to iron or chelated iron (forBoth cytochrorne P-450 and glutathione participate in the metabolism of xenobiotics. Their interrelationship is described here, as well as current findings indicating their mation of reduced (chelated) iron) or to xenobiotics (with subsequent redox cycmutual involvement in lipid peroxidation. ling)2. The role of cytochrome P-450 in lipid peroxidation is more controversial Lipid peroxidation is the process by malondialdehyde (MDA). Propagation and is discussed here. which oxidative degradation of poly- was defined as breakdown of LOOH to High concentrations of the cytounsaturated fatty acids (LH) occurs. give reactive intermediates and products chrome P-450 system are found in the Because of the biomedical implications of lipid peroxidation (e.g. malon- microsomal membrane. The enzyme of lipid peroxidation (its toxicity), the dialdehyde) with reformation of system has attracted researchers in the process has been the subject of stren- LOOH. These definitions have been fields of biochemistry, pharmacology uous research. The effects of both adopted frequently by others, with sub- and toxicology. It plays a pivotal role in enzymic and non-enzymic systems in sequent confusion. For, in general the metabolism of endogenous comchemical terms, initiation means radical pounds (steroids, prostaglandins) and in lipid peroxidation have been studied. Lipid peroxidation was first consid- production and propagation means radi- the biotransformation of xenobiotics. In ered as two distinct sequential phases by cal transfer, m more accurate represen- our western society there is an everSvingen et al.~: initiation and propaga- tation of the reaction is shown in Fig. 1, increasing demand on cytochrome P-450 tion. Initiation was defined as the forma- with classification of LOOH-indepen- to cope with xenobiotics. However, the tion of lipid hydroperoxides (LOOH) dent lipid peroxidation and LOOH- involvement of cytochrome P-450 does accompanied by a minimal formation of dependent lipid peroxidation. Both not guarantee detoxification. It has been types of peroxidation have an initiation hypothesized that cytochrome P-450 A. Bast and G. R. M. M. Haeen are at the Faculty of Pharmacy, Dept. of Pharmacology and Phar- phase and a propagation phase. During played an early role in the detoxification macotherapy, State University of Utrecht, Catharijne- the latter, reactants are converted to of oxygen, during chemical evolution. It products with no net consumption of was probably present in living organisms singel 60, 3511 GH Utrecht, The Netherlands.
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