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Circular dichroism studies
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Circular II. The Far Ultraviolet R. ZAND Biophysics Ann
126, 94-97 (1968)
Dichroism
Studies
Circular Dichroism of Cytochrome AND
c
S. VINOGRADOV’
Research Division,
Institute of Science and Technology, University of Michigan, @iO7; and Department of Biochemistry, School of Medicine, Wayne Slate University, Detroit, Michigan .&32’07
AT~OT, Michigan
ReceivedOctober 5, 1967 The circular dichroism spectra of the ferri- and ferrocytochromes c derived from horse, beef, chicken, and turtle hearts have been measured over the wavelength range of 240-185 w. The spectra in this wavelength region of the ferri and ferro forms appear to be the same within experimental error and indicate that no gross conformational differences appear to exist between the oxidized and reduced forms. The upper
limit of the apparenthelix content hasbeenestimatedto be about 17%with a lower limit in the neighborhoodof 10%. The presenceof circular dichroismbandscharacteristic
of a-helix
conformations
are observed along with bands characteristic
of B
structure. Thesedata strongly suggestthe presenceof a smallbut real helical segment(s)in cytochromec. The utilization of circular dichroism (CD) spectroscopy as a probe in obtaining information on the solution conformation of protkins and polyamino acids is proving a valuable although not unambiguous technique. Several laboratories (l-3) have reported that the presenceof circular dichroism bands at 221-222(-), 207-208(-), and 190192(+) rnp is indicative of the presence of helix in the protein or polyamino acid. The presence of CD bands at 235( -), 217( -), 202( +), and 197(+) rnp indicates the presence of the random conformation (1, 3), while bands at 217( -) and 195(+) denote the anti-parallel ,8 structure (2, 4, 5) and bands at 216( -) and Ml(+) should be characteristic of the parallel P structure (6). The currently available theories of absorption spectra and optical activity of proteins are semi-empirical in their ability to predict the solution conformation of such molecules. These theories are based on assumedmodels 1This work was supportedin part by a grant from the National Institutes of Health, U. S. Public Health Service, No. NB-06306, and the Michigan Heart Association. 94
that can only approximate the true situation, and we must still rely on empirical results that can be correlated by different experimental techniques to adequately explain the spectral data. As part of our studies (7, 8) on the CD properties of a number of cytochromes c, we have extended our measurements into the region from 240-185 rnp to obtain information on the solution conformation of the ferro and ferri forms of cytochrome c derived from horse, beef, chicken, and turtle heart. These studies were of particular inter$st since an X-ray diffraction study at 4 A resolution has been reported for horse heart ferricytochrome c (9), and a correlation of the conformation derived from CD data and from the X-ray studies would be desirable. MATERIALS
AND METHODS
Horse heart cytochrome c, Sigma type VI, was obtained from the Sigma Chemical Company, and was used without additional purification. Beef and chicken heart cytochromes c were prepared according to the procedure of Margoliash
CIRCULAR
DICHROISM
and Walasek (10). Turtle heart cytochrome c was a gift from Dr. E. Margoliash. Solutions were prepared by dissolving the cytochrome c in 0.1 M potassium phosphate buffer at pH 7. Reduction of the ferri to the ferro form was accomplished by adding a minimum quantity of sodium dithionite directly to the solution in the glass-stoppered spectrophotometer cell. The oxidized form was maintained by the addition of a minimum quantity of potassium ferricyanide to the stock solution. Cells used for these studies were of 0.1, 0.5, and 1 mm pathlength and were obtained from Opticell Co., Brentwood, Maryland. The CD spectra were obtained on a Durrum-
6
1
I
95
STUDIES
-
Y .
-5 L
IS0
I 190 WAVELENGTH
I 200 (mc)
-J
210
FIG. 3. Composite CD spectrum of ferro- and ferricytochromes c from horse, beef, chicken, and turtle heart. Solution in 0.1 M potassium phosphate buffer, pH 7.
200
210
220 WAVELENGTH
230
2
J‘40
lm#)
FIG. 1. The CD spectrum of horse (O), beef (+), chicken (O), and turtle (A) heart ferricytochrome c in 0.1 M potassium phosphate buffer, pH 7.
Jasco ORD/UVd instrument with a CD attachment. Ultraviolet and visible spectra were obtained with a Cary model 15 spectrophotometer. The concentrations of cytochrome c were calculated from the absorption spectra and the extinction coefficients of ferri- and ferrocytochromes c reported by Margoliash and Frohwirt (11). The CD curves were obtained by manually setting the desired wavelength at 1-w intervals and allowing the recorder pen to oscillate at this wavelength for 5 minutes. The observed intensity was taken as the difference between the midpoint of the vertical line drawn by the recorder at each wavelength and the baseline value given by the solvent at the same wavelength. All spectra were obtained at room temperature. RESULTS
I 200
I 210
220 WAVELENGTH
230 t m,,
I 240
,
FIG. 2. The CD spectrum of horse (O), beef (f), chicken (0), and turtle (A) heart ferrocytochrome c in 0.1 M potassium phosphate buffer, pH 7.
Figures 1 and 2 show the CD spectra obtained for the ferri- and ferrocytochromes c over the wavelength region of 240-205 rnp. The curves are characterized by a band at 222 rnp( -), a shoulder at 217-218 mH( -), and a band at 207-210 rnp( -). The CD spectra of the four speciesof ferri- and ferrocytochromes c show very little if any differ-
96
ZAND
AND VINOGRADOV
ence in band shape. The slight differences in (15). An alternate calibration value of -6.2 intensity between the different cytochromes X lo4 deg cm2/decimole has been reported is ascribed t.o the experimental uncertainty by Aki et al. (16). Using the former calibrainherent in CD measurementson cytochrome tion value, we calculate an apparent helical c in this wavelength region. Figure 3 shows content of 26 % for ferri- and 28 % for ferrothe results for the wavelength region of 210- cytochrome c. These estimates are in good 190 mp. Positive bands occur at 200-198 agreement with those reported by Flatmark and at 190-192 mp. Crossover points are (14). If the value of - 6.2 X lo4 is used for observed at 202 rnp and 196-197 mp. estimating helix content, we obtainestimates The ultraviolet spectra of both the ferriof 17 % for the ferri- and 18 % for the ferroand ferrocytochromes c exhibit a shoulder cytochrome c. These estimates have negat 230 rnp and a peak at about 200 mp. lected the contribution of the adjacent CD bands to the absorption at 222 rnp. If such a DISCUSSION correction were made, the above estimates The correlation of the solution conforma- would be lowered. Of the estimates made tion of proteins with the position of CD with our data, we would prefer the lower of absorption and the estimation of helical the two estimates, i.e., 17 % as an upper limit contents from the intensity of these bands on the apparent helix content of ferri- and must be made with restraint. Systems which ferrocytochrome c. Assuming a nonnegcontain a preponderance of one conforma- ligible contribution from other relevant contion lend themselves to reasonably unam- formations, we can approximate that a biguous interpretation. Protein systems in realistic lower estimate of the helix content which the molecule may contain segments of cytochrome c lies in the neighborhood of having several different conformations, in 10 %. sufficiently variable concentrations, can give The 4 8 resolution electron density map of rise to quite complex CD spectra, the interhorse heart ferricytochrome c has been inpretation of which cannot be made with the terpreted by Dickerson et al. (9) as showing standard procedures. Schellman and Schell- no a-helix content. We believe that the man (12) have in fact pointed out that the actual situation is intermediate between that percentage of helix calculated from optical suggested by Flatmark of 24% and by the rotatory dispersion (ORD) or CD data is X-ray group of 0 %. The presenceof the CD not necessarily the percentage of helix con- bands at 221-222, 207-210, and at 190-192 tained in the protein molecule but is the is in agreement with the suggested band percentage of helical units which would have assignments for the a-helix. It is difficult to to be mixed with random and /3 conformers rationalize that all of these indicators of to yield the observed spectrum. In this con- helical content can be attributed to the nection one can conceive of instances in proper 4 and $ angles of di- and tripeptide which two or three amino acid residuescould units in the protein chain. At the present have the proper backbone angles 4 (N-0) time we believe that the data may be best and $ (Cm-C’) (13) such that the allowed interpreted by ascribing at least one and electronic transitions would occur in wave- possibly two small helical segments to the length regions close to or ident’ical with the protein moiety in ferri- and ferrocytochromes transitions for the a-helix. The intensities of c. Any additional intensity required to match these bands would then be added to the the observed data could be rationalized as intensity of the absorptions associated with contributions from peptide segments and the a-helix and consequently give rise to overlap from the ,8 structure bands present erroneous estimates of helicity. at 217 and 200-198 rnp. Clearly, the future Flatmark (14) has reported that beef availability of 2 ,& X-ray data will be of heart cytochrome c contains 23-24% helix. This estimate is based on the intensity of the great help in facilitating the correlation of CD band at 222 rnp and a value of the conformation estimated by CD methods mean residue ellipticity for a 100 % helical with conformation established by X-ray structure of -4 X lo4 deg cm*/decimole techniques.
CIRCULAR
DICHROISM
ACKNOWLEDGMENT We thank Dr. E. Margoliash, Abbott Laboratories, Chicago, Illinois, for a gift of turtle heart cytochrome c; and Dr. Y. I’. Myer, SUNY, Albany, New York, for helpful discussions. REFERENCES 1. IIOLZWARTH, C;., AND DOTY, P., J. Am. Chem. Sot. 87, 218 (1965). 2. TOWNEND, R., KUMOSIOSKI, T.F., TIMASHEFF, 8. N., FASM~N, G. D., AND DAVIDSON, B., Biochem. Riophys. Res. Commun. 23, 163 (1966). 3. CARVER, J. P., SHECHTER, E., AND BLOUT, E. R., J. Am. Gem. Sot. 88, 2550 (1966). 4. SARKER, P. K., AND DOTY, I?., Proc. Natl. Acad. Sci. U.S. 66, 981 (1966). T. J., Proc. Natl. 5. IIZUKA, E., AND YANG, Acad. Sci. U.S. 66, 1175 (1966). 6. PYSH, E., Proc. Natl. Acad. Sci. U.S. 66, 825 (1966). 7. ZSND, R., AND VINOGRADOV, S., Biochem. Biophys. Res. Commun. 26, 121 (1967). 8. VINOGRSDOV,S.,~~ND Z.~ND, R., Arch. Riochewl. Biophys., in press.
STUDIES
97
9. DICKERSON, R. W., KOPHA, M. L., WEINZIERL, J., VARNUM, J., EISENBERG, D., AND MARGOLIASH, E., J. Biol. Chem. 242, 3015 (1967). 10. MARGOLIASH, E., AND WALASEK, 0. F., in “Methods in Enzymology,” Vol. 10 (“Oxidation and Phosphorylations,” R. W. Estabrook and M. E. Pullman, eds.), p. 339. Academic Press, New York (1967). 11. M-\RGOLIASH, E., AND FROHWIRT,N., Biochem. J. 71, 570 (1959). 12. SCHELLMAN, J. A., AND SCHELLM.\N, C., in “The Proteins” (H. Neurath, ed.), Vol. 2, p. 80. Academic Press, New York (1964). 13. EDSALL,J. T., FLORY, P. J., KENDHEW,J. C., LIQUORI, A. M., NEMETHY, G., AND 11.4. MACHANDRAN, c;. N., J. I\[o~. &ol. 16, 399 (196G). 14. FLATMARK, T., J. Riol. Chem. 242, 2454 (1967). 15. CASSIM, J. Y., AND YANG, J. T., B&hem. Biophys. Res. Commun. 26, 58 (1967). 16. AKI, K., TAKAGI, T., ISEMCH.~, T., AND YOMANO, T., Riochim. Biophys. Acla 122, 193 (1966).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Circular II. The Far Ultraviolet R. ZAND Biophysics Ann
126, 94-97 (1968)
Dichroism
Studies
Circular Dichroism of Cytochrome AND
c
S. VINOGRADOV’
Research Division,
Institute of Science and Technology, University of Michigan, @iO7; and Department of Biochemistry, School of Medicine, Wayne Slate University, Detroit, Michigan .&32’07
AT~OT, Michigan
ReceivedOctober 5, 1967 The circular dichroism spectra of the ferri- and ferrocytochromes c derived from horse, beef, chicken, and turtle hearts have been measured over the wavelength range of 240-185 w. The spectra in this wavelength region of the ferri and ferro forms appear to be the same within experimental error and indicate that no gross conformational differences appear to exist between the oxidized and reduced forms. The upper
limit of the apparenthelix content hasbeenestimatedto be about 17%with a lower limit in the neighborhoodof 10%. The presenceof circular dichroismbandscharacteristic
of a-helix
conformations
are observed along with bands characteristic
of B
structure. Thesedata strongly suggestthe presenceof a smallbut real helical segment(s)in cytochromec. The utilization of circular dichroism (CD) spectroscopy as a probe in obtaining information on the solution conformation of protkins and polyamino acids is proving a valuable although not unambiguous technique. Several laboratories (l-3) have reported that the presenceof circular dichroism bands at 221-222(-), 207-208(-), and 190192(+) rnp is indicative of the presence of helix in the protein or polyamino acid. The presence of CD bands at 235( -), 217( -), 202( +), and 197(+) rnp indicates the presence of the random conformation (1, 3), while bands at 217( -) and 195(+) denote the anti-parallel ,8 structure (2, 4, 5) and bands at 216( -) and Ml(+) should be characteristic of the parallel P structure (6). The currently available theories of absorption spectra and optical activity of proteins are semi-empirical in their ability to predict the solution conformation of such molecules. These theories are based on assumedmodels 1This work was supportedin part by a grant from the National Institutes of Health, U. S. Public Health Service, No. NB-06306, and the Michigan Heart Association. 94
that can only approximate the true situation, and we must still rely on empirical results that can be correlated by different experimental techniques to adequately explain the spectral data. As part of our studies (7, 8) on the CD properties of a number of cytochromes c, we have extended our measurements into the region from 240-185 rnp to obtain information on the solution conformation of the ferro and ferri forms of cytochrome c derived from horse, beef, chicken, and turtle heart. These studies were of particular inter$st since an X-ray diffraction study at 4 A resolution has been reported for horse heart ferricytochrome c (9), and a correlation of the conformation derived from CD data and from the X-ray studies would be desirable. MATERIALS
AND METHODS
Horse heart cytochrome c, Sigma type VI, was obtained from the Sigma Chemical Company, and was used without additional purification. Beef and chicken heart cytochromes c were prepared according to the procedure of Margoliash
CIRCULAR
DICHROISM
and Walasek (10). Turtle heart cytochrome c was a gift from Dr. E. Margoliash. Solutions were prepared by dissolving the cytochrome c in 0.1 M potassium phosphate buffer at pH 7. Reduction of the ferri to the ferro form was accomplished by adding a minimum quantity of sodium dithionite directly to the solution in the glass-stoppered spectrophotometer cell. The oxidized form was maintained by the addition of a minimum quantity of potassium ferricyanide to the stock solution. Cells used for these studies were of 0.1, 0.5, and 1 mm pathlength and were obtained from Opticell Co., Brentwood, Maryland. The CD spectra were obtained on a Durrum-
6
1
I
95
STUDIES
-
Y .
-5 L
IS0
I 190 WAVELENGTH
I 200 (mc)
-J
210
FIG. 3. Composite CD spectrum of ferro- and ferricytochromes c from horse, beef, chicken, and turtle heart. Solution in 0.1 M potassium phosphate buffer, pH 7.
200
210
220 WAVELENGTH
230
2
J‘40
lm#)
FIG. 1. The CD spectrum of horse (O), beef (+), chicken (O), and turtle (A) heart ferricytochrome c in 0.1 M potassium phosphate buffer, pH 7.
Jasco ORD/UVd instrument with a CD attachment. Ultraviolet and visible spectra were obtained with a Cary model 15 spectrophotometer. The concentrations of cytochrome c were calculated from the absorption spectra and the extinction coefficients of ferri- and ferrocytochromes c reported by Margoliash and Frohwirt (11). The CD curves were obtained by manually setting the desired wavelength at 1-w intervals and allowing the recorder pen to oscillate at this wavelength for 5 minutes. The observed intensity was taken as the difference between the midpoint of the vertical line drawn by the recorder at each wavelength and the baseline value given by the solvent at the same wavelength. All spectra were obtained at room temperature. RESULTS
I 200
I 210
220 WAVELENGTH
230 t m,,
I 240
,
FIG. 2. The CD spectrum of horse (O), beef (f), chicken (0), and turtle (A) heart ferrocytochrome c in 0.1 M potassium phosphate buffer, pH 7.
Figures 1 and 2 show the CD spectra obtained for the ferri- and ferrocytochromes c over the wavelength region of 240-205 rnp. The curves are characterized by a band at 222 rnp( -), a shoulder at 217-218 mH( -), and a band at 207-210 rnp( -). The CD spectra of the four speciesof ferri- and ferrocytochromes c show very little if any differ-
96
ZAND
AND VINOGRADOV
ence in band shape. The slight differences in (15). An alternate calibration value of -6.2 intensity between the different cytochromes X lo4 deg cm2/decimole has been reported is ascribed t.o the experimental uncertainty by Aki et al. (16). Using the former calibrainherent in CD measurementson cytochrome tion value, we calculate an apparent helical c in this wavelength region. Figure 3 shows content of 26 % for ferri- and 28 % for ferrothe results for the wavelength region of 210- cytochrome c. These estimates are in good 190 mp. Positive bands occur at 200-198 agreement with those reported by Flatmark and at 190-192 mp. Crossover points are (14). If the value of - 6.2 X lo4 is used for observed at 202 rnp and 196-197 mp. estimating helix content, we obtainestimates The ultraviolet spectra of both the ferriof 17 % for the ferri- and 18 % for the ferroand ferrocytochromes c exhibit a shoulder cytochrome c. These estimates have negat 230 rnp and a peak at about 200 mp. lected the contribution of the adjacent CD bands to the absorption at 222 rnp. If such a DISCUSSION correction were made, the above estimates The correlation of the solution conforma- would be lowered. Of the estimates made tion of proteins with the position of CD with our data, we would prefer the lower of absorption and the estimation of helical the two estimates, i.e., 17 % as an upper limit contents from the intensity of these bands on the apparent helix content of ferri- and must be made with restraint. Systems which ferrocytochrome c. Assuming a nonnegcontain a preponderance of one conforma- ligible contribution from other relevant contion lend themselves to reasonably unam- formations, we can approximate that a biguous interpretation. Protein systems in realistic lower estimate of the helix content which the molecule may contain segments of cytochrome c lies in the neighborhood of having several different conformations, in 10 %. sufficiently variable concentrations, can give The 4 8 resolution electron density map of rise to quite complex CD spectra, the interhorse heart ferricytochrome c has been inpretation of which cannot be made with the terpreted by Dickerson et al. (9) as showing standard procedures. Schellman and Schell- no a-helix content. We believe that the man (12) have in fact pointed out that the actual situation is intermediate between that percentage of helix calculated from optical suggested by Flatmark of 24% and by the rotatory dispersion (ORD) or CD data is X-ray group of 0 %. The presenceof the CD not necessarily the percentage of helix con- bands at 221-222, 207-210, and at 190-192 tained in the protein molecule but is the is in agreement with the suggested band percentage of helical units which would have assignments for the a-helix. It is difficult to to be mixed with random and /3 conformers rationalize that all of these indicators of to yield the observed spectrum. In this con- helical content can be attributed to the nection one can conceive of instances in proper 4 and $ angles of di- and tripeptide which two or three amino acid residuescould units in the protein chain. At the present have the proper backbone angles 4 (N-0) time we believe that the data may be best and $ (Cm-C’) (13) such that the allowed interpreted by ascribing at least one and electronic transitions would occur in wave- possibly two small helical segments to the length regions close to or ident’ical with the protein moiety in ferri- and ferrocytochromes transitions for the a-helix. The intensities of c. Any additional intensity required to match these bands would then be added to the the observed data could be rationalized as intensity of the absorptions associated with contributions from peptide segments and the a-helix and consequently give rise to overlap from the ,8 structure bands present erroneous estimates of helicity. at 217 and 200-198 rnp. Clearly, the future Flatmark (14) has reported that beef availability of 2 ,& X-ray data will be of heart cytochrome c contains 23-24% helix. This estimate is based on the intensity of the great help in facilitating the correlation of CD band at 222 rnp and a value of the conformation estimated by CD methods mean residue ellipticity for a 100 % helical with conformation established by X-ray structure of -4 X lo4 deg cm*/decimole techniques.
CIRCULAR
DICHROISM
ACKNOWLEDGMENT We thank Dr. E. Margoliash, Abbott Laboratories, Chicago, Illinois, for a gift of turtle heart cytochrome c; and Dr. Y. I’. Myer, SUNY, Albany, New York, for helpful discussions. REFERENCES 1. IIOLZWARTH, C;., AND DOTY, P., J. Am. Chem. Sot. 87, 218 (1965). 2. TOWNEND, R., KUMOSIOSKI, T.F., TIMASHEFF, 8. N., FASM~N, G. D., AND DAVIDSON, B., Biochem. Riophys. Res. Commun. 23, 163 (1966). 3. CARVER, J. P., SHECHTER, E., AND BLOUT, E. R., J. Am. Gem. Sot. 88, 2550 (1966). 4. SARKER, P. K., AND DOTY, I?., Proc. Natl. Acad. Sci. U.S. 66, 981 (1966). T. J., Proc. Natl. 5. IIZUKA, E., AND YANG, Acad. Sci. U.S. 66, 1175 (1966). 6. PYSH, E., Proc. Natl. Acad. Sci. U.S. 66, 825 (1966). 7. ZSND, R., AND VINOGRADOV, S., Biochem. Biophys. Res. Commun. 26, 121 (1967). 8. VINOGRSDOV,S.,~~ND Z.~ND, R., Arch. Riochewl. Biophys., in press.
STUDIES
97
9. DICKERSON, R. W., KOPHA, M. L., WEINZIERL, J., VARNUM, J., EISENBERG, D., AND MARGOLIASH, E., J. Biol. Chem. 242, 3015 (1967). 10. MARGOLIASH, E., AND WALASEK, 0. F., in “Methods in Enzymology,” Vol. 10 (“Oxidation and Phosphorylations,” R. W. Estabrook and M. E. Pullman, eds.), p. 339. Academic Press, New York (1967). 11. M-\RGOLIASH, E., AND FROHWIRT,N., Biochem. J. 71, 570 (1959). 12. SCHELLMAN, J. A., AND SCHELLM.\N, C., in “The Proteins” (H. Neurath, ed.), Vol. 2, p. 80. Academic Press, New York (1964). 13. EDSALL,J. T., FLORY, P. J., KENDHEW,J. C., LIQUORI, A. M., NEMETHY, G., AND 11.4. MACHANDRAN, c;. N., J. I\[o~. &ol. 16, 399 (196G). 14. FLATMARK, T., J. Riol. Chem. 242, 2454 (1967). 15. CASSIM, J. Y., AND YANG, J. T., B&hem. Biophys. Res. Commun. 26, 58 (1967). 16. AKI, K., TAKAGI, T., ISEMCH.~, T., AND YOMANO, T., Riochim. Biophys. Acla 122, 193 (1966).