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. 1995 Nov;4(11):2411–2423. doi: 10.1002/pro.5560041120

How to measure and predict the molar absorption coefficient of a protein.

C N Pace 1, F Vajdos 1, L Fee 1, G Grimsley 1, T Gray 1
PMCID: PMC2143013  PMID: 8563639

Abstract

The molar absorption coefficient, epsilon, of a protein is usually based on concentrations measured by dry weight, nitrogen, or amino acid analysis. The studies reported here suggest that the Edelhoch method is the best method for measuring epsilon for a protein. (This method is described by Gill and von Hippel [1989, Anal Biochem 182:319-326] and is based on data from Edelhoch [1967, Biochemistry 6:1948-1954]). The absorbance of a protein at 280 nm depends on the content of Trp, Tyr, and cystine (disulfide bonds). The average epsilon values for these chromophores in a sample of 18 well-characterized proteins have been estimated, and the epsilon values in water, propanol, 6 M guanidine hydrochloride (GdnHCl), and 8 M urea have been measured. For Trp, the average epsilon values for the proteins are less than the epsilon values measured in any of the solvents. For Tyr, the average epsilon values for the proteins are intermediate between those measured in 6 M GdnHCl and those measured in propanol. Based on a sample of 116 measured epsilon values for 80 proteins, the epsilon at 280 nm of a folded protein in water, epsilon (280), can best be predicted with this equation: epsilon (280) (M-1 cm-1) = (#Trp)(5,500) + (#Tyr)(1,490) + (#cystine)(125) These epsilon (280) values are quite reliable for proteins containing Trp residues, and less reliable for proteins that do not. However, the Edelhoch method is convenient and accurate, and the best approach is to measure rather than predict epsilon.

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Selected References

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  1. ASCHAFFENBURG R., DREWRY J. Improved method for the preparation of crystalline beta-lactoglobulin and alpha-lactalbumin from cow's milk. Biochem J. 1957 Feb;65(2):273–277. doi: 10.1042/bj0650273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adams B., Burgess R. J., Pain R. H. The folding and mutual interaction of the domains of yeast 3-phosphoglycerate kinase. Eur J Biochem. 1985 Nov 4;152(3):715–720. doi: 10.1111/j.1432-1033.1985.tb09252.x. [DOI] [PubMed] [Google Scholar]
  3. Amaratunga M., Lohman T. M. Escherichia coli rep helicase unwinds DNA by an active mechanism. Biochemistry. 1993 Jul 13;32(27):6815–6820. doi: 10.1021/bi00078a003. [DOI] [PubMed] [Google Scholar]
  4. Benson A. M., Suruda A. J., Talalay P. Concentration-dependent association of delta5-3-ketosteroid isomerase of Pseudomonas testosteroni. J Biol Chem. 1975 Jan 10;250(1):276–280. [PubMed] [Google Scholar]
  5. Brandts J. F., Kaplan L. J. Derivative sspectroscopy applied to tyrosyl chromophores. Studies on ribonuclease, lima bean inhibitors, insulin, and pancreatic trypsin inhibitor. Biochemistry. 1973 May 8;12(10):2011–2024. doi: 10.1021/bi00734a027. [DOI] [PubMed] [Google Scholar]
  6. Damaschun G., Damaschun H., Gast K., Misselwitz R., Zirwer D., Gührs K. H., Hartmann M., Schlott B., Triebel H., Behnke D. Physical and conformational properties of staphylokinase in solution. Biochim Biophys Acta. 1993 Feb 13;1161(2-3):244–248. doi: 10.1016/0167-4838(93)90220-l. [DOI] [PubMed] [Google Scholar]
  7. Grunert H. P., Zouni A., Beineke M., Quaas R., Georgalis Y., Saenger W., Hahn U. Studies on RNase T1 mutants affecting enzyme catalysis. Eur J Biochem. 1991 Apr 10;197(1):203–207. doi: 10.1111/j.1432-1033.1991.tb15900.x. [DOI] [PubMed] [Google Scholar]
  8. Hu C. Q., Sturtevant J. M., Thomson J. A., Erickson R. E., Pace C. N. Thermodynamics of ribonuclease T1 denaturation. Biochemistry. 1992 May 26;31(20):4876–4882. doi: 10.1021/bi00135a019. [DOI] [PubMed] [Google Scholar]
  9. Jaenicke L. A rapid micromethod for the determination of nitrogen and phosphate in biological material. Anal Biochem. 1974 Oct;61(2):623–627. doi: 10.1016/0003-2697(74)90429-1. [DOI] [PubMed] [Google Scholar]
  10. KAY C. M., MARSH M. M. Some optical properties of fetuin and glucagon. Biochim Biophys Acta. 1959 May;33(1):251–253. doi: 10.1016/0006-3002(59)90526-8. [DOI] [PubMed] [Google Scholar]
  11. Kalnin N. N., Baikalov I. A., Venyaminov SYu Quantitative IR spectrophotometry of peptide compounds in water (H2O) solutions. III. Estimation of the protein secondary structure. Biopolymers. 1990;30(13-14):1273–1280. doi: 10.1002/bip.360301311. [DOI] [PubMed] [Google Scholar]
  12. Kirschenbaum D. M. Molar absorptivity and A 1 per cent 1 cm values for proteins at selected wavelengths of the ultraviolet and visible regions. XVI. Anal Biochem. 1978 Oct 1;90(1):309–330. doi: 10.1016/0003-2697(78)90035-0. [DOI] [PubMed] [Google Scholar]
  13. Kuliopulos A., Mildvan A. S., Shortle D., Talalay P. Kinetic and ultraviolet spectroscopic studies of active-site mutants of delta 5-3-ketosteroid isomerase. Biochemistry. 1989 Jan 10;28(1):149–159. doi: 10.1021/bi00427a022. [DOI] [PubMed] [Google Scholar]
  14. Kupke D. W., Dorrier T. E. Protein concentration measurements: the dry weight. Methods Enzymol. 1978;48:155–162. doi: 10.1016/s0076-6879(78)48008-5. [DOI] [PubMed] [Google Scholar]
  15. Lee J. C., Timasheff S. N. Partial specific volumes and interactions with solvent components of proteins in guanidine hydrochloride. Biochemistry. 1974 Jan 15;13(2):257–265. doi: 10.1021/bi00699a005. [DOI] [PubMed] [Google Scholar]
  16. Lesser G. J., Rose G. D. Hydrophobicity of amino acid subgroups in proteins. Proteins. 1990;8(1):6–13. doi: 10.1002/prot.340080104. [DOI] [PubMed] [Google Scholar]
  17. Li Y. K., Kuliopulos A., Mildvan A. S., Talalay P. Environments and mechanistic roles of the tyrosine residues of delta 5-3-ketosteroid isomerase. Biochemistry. 1993 Feb 23;32(7):1816–1824. doi: 10.1021/bi00058a016. [DOI] [PubMed] [Google Scholar]
  18. Loewenthal R., Sancho J., Fersht A. R. Fluorescence spectrum of barnase: contributions of three tryptophan residues and a histidine-related pH dependence. Biochemistry. 1991 Jul 9;30(27):6775–6779. doi: 10.1021/bi00241a021. [DOI] [PubMed] [Google Scholar]
  19. Mach H., Middaugh C. R., Lewis R. V. Statistical determination of the average values of the extinction coefficients of tryptophan and tyrosine in native proteins. Anal Biochem. 1992 Jan;200(1):74–80. doi: 10.1016/0003-2697(92)90279-g. [DOI] [PubMed] [Google Scholar]
  20. Martinez-Oyanedel J., Choe H. W., Heinemann U., Saenger W. Ribonuclease T1 with free recognition and catalytic site: crystal structure analysis at 1.5 A resolution. J Mol Biol. 1991 Nov 20;222(2):335–352. doi: 10.1016/0022-2836(91)90215-r. [DOI] [PubMed] [Google Scholar]
  21. Minato S., Tagawa T., Nakanishi K. Crystallization of ribonuclease T1. J Biochem. 1966 May;59(5):443–448. doi: 10.1093/oxfordjournals.jbchem.a128325. [DOI] [PubMed] [Google Scholar]
  22. Nozaki Y. Determination of the concentration of protein by dry weight--a comparison with spectrophotometric methods. Arch Biochem Biophys. 1986 Sep;249(2):437–446. doi: 10.1016/0003-9861(86)90020-2. [DOI] [PubMed] [Google Scholar]
  23. Okajima T., Kawata Y., Hamaguchi K. Chemical modification of tryptophan residues and stability changes in proteins. Biochemistry. 1990 Oct 2;29(39):9168–9175. doi: 10.1021/bi00491a010. [DOI] [PubMed] [Google Scholar]
  24. Pace C. N., Grimsley G. R., Barnett B. J. Purification of ribonuclease T1. Anal Biochem. 1987 Dec;167(2):418–422. doi: 10.1016/0003-2697(87)90186-2. [DOI] [PubMed] [Google Scholar]
  25. Perkins S. J. Protein volumes and hydration effects. The calculations of partial specific volumes, neutron scattering matchpoints and 280-nm absorption coefficients for proteins and glycoproteins from amino acid sequences. Eur J Biochem. 1986 May 15;157(1):169–180. doi: 10.1111/j.1432-1033.1986.tb09653.x. [DOI] [PubMed] [Google Scholar]
  26. Pettigrew D. W., Ma D. P., Conrad C. A., Johnson J. R. Escherichia coli glycerol kinase. Cloning and sequencing of the glpK gene and the primary structure of the enzyme. J Biol Chem. 1988 Jan 5;263(1):135–139. [PubMed] [Google Scholar]
  27. Prakash V., Loucheux C., Scheufele S., Gorbunoff M. J., Timasheff S. N. Interactions of proteins with solvent components in 8 M urea. Arch Biochem Biophys. 1981 Sep;210(2):455–464. doi: 10.1016/0003-9861(81)90209-5. [DOI] [PubMed] [Google Scholar]
  28. Privalov P. L., Tiktopulo E. I., Venyaminov SYu, Griko YuV, Makhatadze G. I., Khechinashvili N. N. Heat capacity and conformation of proteins in the denatured state. J Mol Biol. 1989 Feb 20;205(4):737–750. doi: 10.1016/0022-2836(89)90318-5. [DOI] [PubMed] [Google Scholar]
  29. Richards F. M. Areas, volumes, packing and protein structure. Annu Rev Biophys Bioeng. 1977;6:151–176. doi: 10.1146/annurev.bb.06.060177.001055. [DOI] [PubMed] [Google Scholar]
  30. Robinson G. W. Isolation and characterization of papaya peptidase A from commercial chymopapain. Biochemistry. 1975 Aug 12;14(16):3695–3700. doi: 10.1021/bi00687a028. [DOI] [PubMed] [Google Scholar]
  31. Runyon G. T., Lohman T. M. Escherichia coli helicase II (uvrD) protein can completely unwind fully duplex linear and nicked circular DNA. J Biol Chem. 1989 Oct 15;264(29):17502–17512. [PubMed] [Google Scholar]
  32. SELA M., ANFINSEN C. B., HARRINGTON W. F. The correlation of ribonuclease activity with specific aspects of tertiary structure. Biochim Biophys Acta. 1957 Dec;26(3):502–512. doi: 10.1016/0006-3002(57)90096-3. [DOI] [PubMed] [Google Scholar]
  33. Scopes R. K. Measurement of protein by spectrophotometry at 205 nm. Anal Biochem. 1974 May;59(1):277–282. doi: 10.1016/0003-2697(74)90034-7. [DOI] [PubMed] [Google Scholar]
  34. Shirley B. A., Laurents D. V. Purification of recombinant ribonuclease T1 expressed in Escherichia coli. J Biochem Biophys Methods. 1990 Mar;20(3):181–188. doi: 10.1016/0165-022x(90)90076-o. [DOI] [PubMed] [Google Scholar]
  35. Solli N. J., Herskovits T. T. Solvent perturbation studies and analysis of protein and model compound data in denaturing organic solvents. Anal Biochem. 1973 Aug;54(2):370–378. doi: 10.1016/0003-2697(73)90365-5. [DOI] [PubMed] [Google Scholar]
  36. Span J., Lenarcic S., Lapanje S. Solvation of lysozyme and beta-lactoglobulin in aqueous guanidine hydrochloride solutions. Biochim Biophys Acta. 1974 Aug 8;359(2):311–319. doi: 10.1016/0005-2795(74)90230-x. [DOI] [PubMed] [Google Scholar]
  37. Voordouw G., Roche R. S. The cooperative binding of two calcium ions to the double site of apothermolysin. Biochemistry. 1974 Nov 19;13(24):5017–5021. doi: 10.1021/bi00721a024. [DOI] [PubMed] [Google Scholar]
  38. WHITE F. H., Jr Regeneration of native secondary and tertiary structures by air oxidation of reduced ribonuclease. J Biol Chem. 1961 May;236:1353–1360. [PubMed] [Google Scholar]
  39. YANARI S., BOVEY F. A. Interpretation of the ultraviolet spectral changes of proteins. J Biol Chem. 1960 Oct;235:2818–2826. [PubMed] [Google Scholar]
  40. Yu Y., Makhatadze G. I., Pace C. N., Privalov P. L. Energetics of ribonuclease T1 structure. Biochemistry. 1994 Mar 22;33(11):3312–3319. doi: 10.1021/bi00177a023. [DOI] [PubMed] [Google Scholar]

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