Abstract
Only four intrinsic protein chromophores absorb light significantly in the near-UV region of the spectrum (340-255 nm): the side chains of Trp, Tyr, Phe, and cystine (note: cysteine residues make no significant contribution). The absorption spectra are shown in Fig. 1. Although several amino acid side chains (notably Tyr, Trp, Phe, His, and Met) absorb light strongly in the far-UV region (below 250 nm), the most important contributor here is the peptide bond (amide chromophore), with n ?π * and π ? π * transitions at approx 220 nm and approx 190 nm, respectively. The contribution of any individual chromophore to the total absorbance of the protein will depend, to some extent at least, upon its environment. The experimentally measured parameter, the absorbance A is related to the molar extinction coefficient, εM (M-1cm-1), the path length l (cm), and the protein concentration C (M) by the Beer-Lambert law A = εM.C.l.
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References
Yang, J. T., Wu, C.-S. C., and Martinez, H. M. (1986) Calculation of protein conformation from circular dichroism. Methods Enzymol. 130, 208–269.
Woody, R. W. (1995) Circular dichroism. Methods Enzymol. 246, 34–71.
Woody, R. W. (1996) Theory of circular dichroism of proteins, in Circular Dichroism and the Conformational Analysis of Biomolecules (Fasman, G. D., ed.), Plenum, New York, pp. 25–67.
Johnson, W. C., Jr. (1985) Circular dichroism and its empirical application to biopoly-mers. Methods Biochem. Anal. 31, 61–163.
Johnson, W. C., Jr. (1988) Secondary structure of proteins through circular dichro-ism spectroscopy. Annu. Rev. Biophys. Biochem. 17, 145–166.
Johnson, W. C., Jr. (1990) Protein secondary structure and circular dichroism: a practical guide. Prot. Struct. Funct. Genet. 7, 205–214.
Gill, S. C. and von Hippel, P. H. (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182, 319–326.
Pace, C. N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T. (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4, 2411–2423.
Johnson, W. C., Jr. (1996) Circular dichroism instrumentation, in Circular dichroism and the conformational analysis of biomolecules (Fasman, G. D., ed.), Plenum, New York, pp. 635–652.
Hennessey, J. P., Jr. and Johnson, W. C., Jr. (1982) Experimental errors and their effect on analyzing circular dichroism spectra of proteins. Anal. Biochem. 125, 177–188.
Martin, S. R. (1996) Circular dichroism, in Proteins Labfax (Price, N. C., ed.) BIOS Scientific Publishers Ltd., Oxford, pp. 195–204.
Strickland, E. H. (1974) Aromatic contributions to circular dichroism spectra of proteins. CRC Crit. Rev. Biochem. 2, 113–175.
Woody, R. W. and Dunker, A. K. (1996) Aromatic and cystine side-chain circular dichroism in proteins, in Circular Dichroism and the Conformational Analysis of Biomolecules (Fasman, G. D., ed.), Plenum, New York, pp. 109–157.
Woody, R. W. (1985) Circular dichroism of peptides, in The Peptides, vol. 7 (Hruby, V. J., ed.), Academic, New York, pp. 15–114.
Venyaminov, S. Y. and Yang, J. T. (1996) Determination of protein secondary structure, in Circular Dichroism and the Conformational Analysis of Biomolecules(Fasman, G. D., ed.), Plenum, New York, pp. 69–107.
van Stokkum, I. H. M., Spoelder, H, J. W., Bloemendal, M., van Grondelle, R., and Groen, F. C. A. (1990) Estimation of protein secondary structure and error analysis from circular dichroism spectra. Anal. Biochem. 191, 110–118.
Greenfield, N. J. (1996) Methods to estimate the conformation of proteins and polypeptides from circular dichroism data. Anal. Biochem. 235, 1–10.
Sreerama, N. and Woody, R. W. (1994) Protein secondary structure from circular dichroism spectroscopy. Combining variable selection principle and cluster analy-sis with neural network, ridge regression and self-consistent methods. J. Mol. Biol. 242, 497–507.
Manning, M. C. (1989) Underlying assumptions in the estimation of secondary structure content in proteins by circular dichroism spectroscopy-a critical review. J. Pharm. Biomed. Anal. 7, 1103–1119.
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Martin, S.R., Bayley, P.M. (2002). Absorption and Circular Dichroism Spectroscopy. In: Vogel, H.J. (eds) Calcium-Binding Protein Protocols: Volume 2: Methods and Techniques. Methods in Molecular Biology™, vol 173. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-184-1:043
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DOI: https://doi.org/10.1385/1-59259-184-1:043
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