Magnetic circular dichroism studies XXXIV

ANALYTICAL

65, 1oo- 108 (1975)

BIOCHEMISTRY

Magnetic Improved

Circular

Dichroism

Instrumentation

for

MCD

Studies

XXXIV

Measurements

GRUNTER BARTH, JOHN H. DAWSON, PETER M. DOLINGER, ROBERT E. LINDER, EDWARD BUNNENBERG, AND CARL DJERASSI Department of Chemistry, Stanford University. Stanford, California

94305

Received July 19. 1974: accepted October 10, 1974 The use of a 15-kG electromagnet in conjunction with a circular dichrometer of enhanced sensitivity affords MCD spectra of quality comparable to those obtained using a superconducting magnet and an instrument of lower sensitivity. To demonstrate this, the MCD spectra of zinc octaethylporphyrin, human serum albumin, a microsomal suspension containing cytochromes P-450 and b,, adenine, and benzene in the vapor phase are reported. The convenience, economy of operation, and availability of electromagnets should be a major incentive for the utilization of this spectroscopic technique in a variety of potentially interesting areas in chemistry and biochemistry.

During recent years the measurement of the magnetic circular dischroism (MCD) (2) spectra of biochemically important molecules has proved to be of considerable interest as documented by the rapid increase in the number of publications in this field. This comparatively new spectroscopic technique (3) might well have received even wider acceptance were it not for the fact that many molecules exhibit very weak MCD effects. Since the magnitude of these effects is proportional to the magnetic field strength, adequate measurements often required the use of 50-60 kG fields provided by means of expensive superconducting magnets. Here, we demonstrate with a number of examples that MCD spectra of essentially the same quality can be obtained with an operationally inexpensive 15-kG electromagnet in combination with an instrument of higher sensitivity. INSTRUMENTATION

AND

EXPERIMENTAL

SECTION

MCD spectra were measured using a JASCO model J-40 spectropolarimeter. The main features of this instrument are the following: (1) The sensitivity of the instrument ranges from 0.2 to 100 mdegjcm; (2) The recorder pen time-constant can be varied between 0.25 and 64 set per 6-cm pen deflection; (3) The scanning speed is variable from 0.01 to 100 nm/min; (4) Various split programs with constant resolution 100 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

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of 0.5, I, 2, 4, and 8 nm over the entire spectral region (180-700 nm) can be selected; alternatively the slit width can be set manually between 10 and 2000 @urn. The availability of such wide ranges of recording parameters permits the selection of optimum conditions for spectral recording even under extreme conditions. Because it is often necessary to measure very weak effects, the intensity of the incident light is of crucial importance in determining the optimum sensitivity of the instrument. The optical system of the J-40 instrument contains two major modifications compared to previous models which provide for higher light output: (1) a spherical mirror behind the 450-W Xe high-pressure arc lamp increases the efficiency of light collection; (2) in the double-prism monochomator the second prism is quartz and is oriented with its optical axis perpendicular to the light beam, thereby serving the dual purpose of a disperser and a polarizer. This arrangement avoids the use of an additional polarizer, usually a Rochon prism, which introduces loss of light through partial absorption. An improved electronic design, particularly the use of a higher modulating frequency (368 Hz) for the Pockel’s cell, results in a threefold decrease in spectral noise which permits utilization of a 20-fold faster recorder response time. Aside from the threefold increase in sensitivity over the earlier employed J-5 model, the higher light output of the monochromator has two further important advantages. First, samples with higher optical densities (up to about 2.5) can be measured; second, the instrument can be operated at considerably narrower slit widths and still yield a satisfactory spectrum. This allows full use of the optimal resolving power of the monochromator which is estimated to be 0.1-0.2 nm at a slit width of lo-15 pm. Halfband widths of this magnitude are observed in gas-phase spectra. As an example, the MCD spectrum of the ‘B,, + ‘A,, transition of benzene in the vapor phase is given in Fig. 1. Here, the vibronic components have halfband widths of about 0.25 /

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I. MCD vapor phase spectrum of the ‘B,, +- ‘A,, transition of benzene sensitivity: 1 mdeg/cm, pathlength: 0.5 and 1 cm, time constant: 64 set, scanning rate: 0.025 nmlmin. slit width: 15 Hrn. FIG.

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nm and yet are well resolved. The same spectrum (4) has been previously reported using a superconducting magnet of 53-kG field strength in combination with a Cary 61 spectropolarimeter. An electromagnet capable of providing magnetic fields of up to 15 kG was used, the spectra reported in this study, however, were obtained at 12-kG field strength. The dimensions of this magnet (29 X 29 X 26 cm) are such that it fits into an appropriately designed sample compartment. It is supported by a jack which allows easy removal should CD measurements require the full use of sample compartment space. The width of the pole gap is 1.5 cm, so that standard rectangular sample cells can be used. The field strength was determined with a Bell “ 110” gaussmeter and with a solution of CoSO, for which a molar magnetic ellipticity of [0], = 0.0062 deg cm? dmol-’ gauss-’ at 510 nm has been reported previously (5). Both measurements gave the same value for the field strength with an accuracy of +0.2 kG. The field homogeneity was checked by recording the MCD spectrum of an aqueous CoSO, solution in a 0.1 -cm cell and a 1 : 10 dilution of the same solution in a 1-cm cell. Both measurements gave identical ellipticities indicating that the field inhomogeneity is negligible. The equipment used for low-temperature measurements is shown in Fig. 2 and consists of a holder and jacketed quartz cell (Helma Cells Inc., Jamaica, NY 11424). Cooling is achieved by flushing precooled nitrogen gas through the cell jacket. By regulating the gas flow rate the temperature can be adjusted between -20 and - 196°C with an accuracy of 23°C. The temperature is monitored with an iron-constantan thermocouple, sealed in a glass tube, that reaches directly into the solution. To prevent frosting, the cell windows are flushed with dry nitrogen. Alternatively, the sample space can be evacuated after sealing the bore of the pole pieces with quartz windows. Calibration of the instrument was carried out with an aqueous solution of d-10 camphor sulfonic acid monohydrate (Eastman Organic Chem., Rochester, NY) ( [cY]~~~= 20.4” (c = 4.7, water)) for which Krueger and Pschigoda (6) obtained a molar ellipticity of 7775 at 290 nm using Kronig-Kramers transformation. This value is about 7% higher than the one reported by Cassim and Yang (7) which we previously used for calibration. This discrepancy is due to the fact that camphor sulfonic acid easily forms a stable hydrate when exposed to air. The chemicals used in this study were obtained from the following sources: benzene Spectrograde (M. C. & B., Norwood, OH 45212), human serum albumin, tryst. (Miles Lab. Inc., Kankakee, IL 60901), adenine A grade (Calbiochem, Los Angeles, CA 90054), and CoSO, . 7 H,O (B & A, Morristown, NY). Zinc octaethylporphyrin was prepared from zinc acetate and octaethylporphyrin according to the method of Adler et al. (8) and recrystallized twice from ben-

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FIG. 2. Equipment for low-temperature cell, and thermocouple.

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measurements. Cell holder. jacketed 1-cm quartz

zenelcyclohexane. The method for preparing the suspension of rat liver microsomes has been described in a previous publication (9). RESULTS AND

DISCUSSION

Heme proteins were the first biologically important molecules studied by magnetic optical rotatory dispersion (MORD) and MCD spectroscopy since the intense signals exhibited by the porphyrin ~hromophore made their investigation accessible to instruments using the 3- to 15-kG fields provided by either permanent or electromagnets (10-12). The much weaker signals exhibited by other molecules such as proteins (13-l@, metallo proteins (19-22), nucleic acid bases (23-2% and polymers (26), however, necessitated the use of superconducting magnets. With the advent of more sensitive circular dichrometers such as the one described above, it has become possible to obtain MCD spectra of good quality for these molecules using an electromagnet. A few selected examples will demonstrate this. The MCD spectra of proteins have been investigated recently by sev-

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ET AL.

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FIG. 3. Spectral recordings of human serum albumin in 0.025 M phosphate buffer at pH 7.0 with (1) and without (2) the magnetic field; trace (3) is the solvent blank. Concentration: 8.35 lo-” mole/liter, sensitivity: (a) 1 and (b) 2 mdeg/cm, path length: (a) 0.2 and (b) 0.5 cm, time constant: 16 set, scanning rate 10 nmlmin.

era1 investigators (14- 18). Of particular interest was the observation that the indole chromophore of tryptophan shows a positive B-term at 290 nm and negative B-term at 268 nm. The other aromatic amino acids, tyrosine and phenylalanine, show only weaker negative B-terms at 276 and 275 nm, respectively. The positive B-term of tryptophan at 290 nm is thus not significantly overlapped by contributions from other amino acids and its intensity can be used for quantitatively determining the number of tryptophan residues present in a protein molecule. This method is of particular value when the protein contains a large number of tyrosine but only a few tryptophan residues since in this case tryptophan determinations by absorption spectroscopy cannot be made. The MCD spectrum of human serum albumin, a protein containing 1 tryptophan and 16 tyrosine residues is shown in Fig. 3. The presence of tryptophan is evidenced by the positive B-term at 292 nm. It should be noted at this point that, since the natural CD and MCD are additive effects, the MCD spectrum is the difference between the recordings with and without the magnetic field. Of the naturally occurring nucleosides, adenine (23) shows a particularly interesting MCD spectrum (Fig. 4) since the B,, and B,, bands which overlap to form a single band at 261 nm in the absorption spectrum show oppositely signed B-terms at 270 and 257 nm, respectively. Maestre et al. (26) found that the specific magnitudes of these MCD bands decrease with increasing length of the adenine homopolymer and

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DICHROISM

I

r

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Ah)

FIG. 4. MCD spectrum of adenine in water. Concentration: tivity: 1 mdeg/cm, path length: 0.1 cm, time constant: 16 set,

I

r 300

1.3 lo-” mole/liter, sensiscanning rate: 2.5 nmlmin.

concluded that MCD is sensitive to the degree of stacking of the polymer. Another field of considerable interest where MCD spectroscopy has been successfully applied are the heme proteins (27). The porphyrin chromophore exhibits intense and characteristic MCD bands that are very sensitive to the oxidation state of the heme iron and the type of ligands attached to it. For diamagnetic derivatives A-term MCD bands will be observed whereas in the case of paramagnetic derivatives C-terms will be important (28). Because of the high signal intensity, small quantities are detectable by this technique. An interesting application of this possibility is the identification of the different cytochromes that are present in suspensions of liver microsomes (10,29) since here, because of light-scattering effects, their identification by absorption spectroscopy presents great difficulties. Figure 5a and b shows the MCD spectrum of a microsomal suspension obtained from rat liver in the oxidized form (Fig. 5a) and after reduction with sodium dithionite and complexation with carbon monoxide (Fig. 5b). The presence of cy-

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ET AL.

MO

, h(nm1

t 6co

1

FIG. 5. Spectral recordings of a rat liver microsomal suspension in 0.025 M phosphate buffer at pH 7.0 with (1) and without (2) the magnetic field. Sensitivity: 1 mdeglcm, path length: 1 cm, time constant: 16 set, scanning rate: 10 nmlmin. (a) in the oxidized form and (b) after reduction with sodium dithionite and complexation with carbon monoxide.

0

FIG. 6. MCD spectrum of zinc octaethylporphyrin in EPA (diethylether-isopentaneethano1, 5:s :2 by volume) at -196°C. Concentration: 2.31 . lo+ mole/liter; sensitivity: 100 and 2 mdeg/cm; path length: f cm; time constant: 4 set; scanning rate: 5 and I nmlmin; slit width: 15 pm.

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tochrome P-450 is revealed (Fig. 5b) through the positive A-term at 450 nm whereas the positive A-term at 559 nm is due to cytochrome b,. Therefore, knowing the molar magnetic ellipticities at both bands the two cytochromes can be determined quantitatively. In the present example the concentration of cytochrome P-450 and b, are calculated to be 1.37 . lo+ mole/liter and 1.50 . IO+ mole/liter respectively. The MCD spectrum of zinc octaethylporphyrin at - 196°C in an EPA glass matrix (30) shown in Fig. 6 is typical of the spectra which can be obtained with the low temperature cell shown in Fig. 2. The intense positive A-term at 571 nm is associated with the electronic origin of the Q band, whereas the complicated band pattern at lower wavelengths results from overlapping vibronic components of this transition and is characterized by A-terms of both positive and negative sign. The presence of negative A-terms within the vibronic part of the spectrum has been used to make symmetry assignments (3 l), an area of investigation which is still in progress. It is interesting to note that the vibronic part extends over a wide spectral region as seen from the 50 times expanded spectral recordings in Fig. 6. CONCLUSIONS

Progress in MCD spectroscopy has been directly related to the available instrumentation. With the advent of superconducting magnets which could be fitted into commercially available circular dichrometers the measurement of biochemical interesting molecules became possible and research in this area has lead to a number of interesting applications. However, because of high operating costs of such magnets, work has been carried out only in a few laboratories. We anticipate that the instrumental improvements described in this publication which permit MCD spectra to be obtained with an electromagnet of 15-kG field strength will further stimulate this area of research. ACKNOWLEDGMENTS We thank the National Institutes of Health (Grant GM-20276) and the National Science Foundation for supporting this work. We are furthermore grateful to Dr. L. Vickery for making results of his work available to us prior to publication.

REFERENCES I. For

part XXX111 in this series see: R. E. Linder, G. Barth, E. Bunnenberg, C. Djerassi, L. Seamans and A. Moscowitz (1974) Chem. Phys. Lett. 28, 490. 2. For recent review articles on MCD see: Schatz, P. N., and McCatTery, A. J. (1969) Quart. Reb*. 23, 552; Djerassi. C.. Bunnenberg, E., and Elder, D. L. (1971) Pure Appl. Chem. 25, 57; Caldwell, D., Thorne, J. M.. and Eyring, H. (1971) Ann. Rev. Phys. Chem. 22, 259. 3. For recent publications on instrumental improvements see: Abu-Schumays, A., and

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4. 5. 6. 7. 8. 9. 10.

11. 12. 13.

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Duffield, J. J. (1970) Appl. Spectrosc. 24, 67; Sutherland, J. C., Vickery, L., and Klein, M. D. Rev. Sci. Instrum.. submitted for publication. Douglas, 1. N., Grinter, R., and Thompson, A. J. (1973) Mol. Phys. 26, 1257. McCaffery, A. J.. Stephens, P. J., and Schatz, P. N. (1967) Inorg. Chem. 6, 1614. Krueger, W., and Pschigoda, L. M. (1971) Anul. Chem. 43, 675. Cassim, J. Y., and Yang. J. T. (1969) Biochemistry& 1947. Adler, A. D., Longo. F. R., Kampas, F., and Kim, J. (1970)5. Inorg. NW/. Chem. 32, 2443. Dolinger, P. M., Kielczewski. M., Trudell, J. R., Barth, G.. Linder, R. E., Bunnenberg, E., and Djerassi, C. (1974) Proc. Nut. Acad. Sci. USA 71, 399. Shashoua V. E. (1966) in Hemes and Hemoproteins (B. Chance, R. W. Estabrook, and Y. Yonetani, eds.) p. 93, Academic Press, New York: Shashoua V. E., and R. W. Estabrook, (1966) in Hemes and Hemoproteins B. Chance, R. W. Estabrook, and Y. Yonetani, eds.), p. 427, Academic Press, New York. Morrison, M., and Duffield, J. (I 966) in Hemes and Hemoproteins (B. Chance, R. W. Estabrook. and Yonetani, eds.). p. 43 1. Academic Press, New York. Volkenstein, M. V.. Sharonov, J. A., and Shemelin. A. K. (1966) Nature (London) 209, 709. Barth, G., Records, R., Bunnenberg, E.. Djerassi, C., and Voelter, W. (1971)5. Amer. Chem.

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14. Barth, G.. Voelter, W., Bunnenberg, E., and Djerassi, C. (1972) J. Amer. Chem. Sot. 95, 1293. 15. Barth, G., Bunnenberg, E., and Djerassi, C. (1972) Anal. Biochem. 48, 477. 16. McFarland, M., and Coleman, J. E. (1972) Eur. J. Biochem. 29, 521. 17. Holmquist, B.. and Vallee. B. L. (1973) Biochemisfry 12, 4409. 18. Gabriel, M., Larcher, D., Rinnert, H., and Thirion, C. (1973) Fed. Eur. Biochem. Sot Lett. 35, 148. 19. Bayer, E., Bather. A., Krauss, P.. Voelter. W., Barth, G., Bunnenberg, E., and Djerassi, C. (1971) Eur. J. Biochem. 22, 580. 20. Cheng, J. C.. Osborne, G. A., and Stephens, P. J., (1973) Nature (London) 241, 193. 2 1. Taylor, J. S.. Lau. C. Y.. Meredithe. M. L.. and Coleman, J. E. (1973) J. Bio[. Chem., 248, 62 16. 22. Rotilio, G. R., Calabrese, L., and Coleman, J. E. (1973) J. Biol. Chem. 248, 3855. 23. Voelter, W., Barth, G., Records, R., Bunnenberg, E., and Djerassi, C. (1969)J. Amer. Chem.

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24. Voelter, W., Records, R., Bunnenberg, E.. and Djerassi, C. J. (1968) Amer. Chem. Sot,. 90, 6163. 25. Townsend, L. B., Miles, D. W., Manning, S. J., and Eyring, H. (1973) J. Heterocyclic Chem.

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9.

26. Maestre. M. F., Gray, D. M., and Cook, R. B. (1971) Biopolymers 10, 2537. 27. For most recent work in this area see: Sutherland, J. C. and Klein, M. P. (1972) J. Chem. Phys. 57, 76; Bolard, J., and Garnier, A. (1972) Biochim. Biophys. Acta 263, 535; Sharonova, N. A., Sharonov. Y. A., and Volkenstein, M. V. (1972) Biochim. Biophys. Acta 271, 65; Garnier, A.. Bolard, J., and Danon, J. (1972) Chem. Phys. Lett. 15, 141; Briat, B., Berger, D., and Leliboux, M. (1972) J. Chem. Phys. 57, 5606; Rein, H., Ruckpaul, K., and Haberditzel, W. (1973) Chem. Phys. Left. 20,71. 28. Stephens, P. J., Suetaka, W., and Schatz. P. N. (I 966) J. Chem. Phys. 44, 4592. 29. Vickery, L., Salmon, A., Sauer. K., and Calvin, M. Biochemistry, submitted. 30. Barth, G., Linder. R. E., Bunnenberg, E.. Djerassi, C., Seamans, L., and Moscowitz, J. Chem. Sot., Perkin II, in press. 31. Linder, R. E., Barth, G., Bunnenberg, E., Djerassi, C., Seamans, L., and Moscowitz, A. J. Chem. SOL... Perkin II. in press.