Cite this article as:

Ten G. N., Gerasimenko A. Y., Shcherbakova N. Е., Baranov V. I. Interpretation of IR and Raman Spectra of Albumin. Izvestiya of Saratov University. New series. Series Physics, 2019, vol. 19, iss. 1, pp. 43-57. DOI:


Interpretation of IR and Raman Spectra of Albumin


Object and purpose of work: The subject of the study is bovine serum albumin (BSA). The aim of the work is to give an interpretation of the vibrational spectra of BSA aqueous solution in the region of ~1700–600 cm– 1. Methods: In this regard the experimental measurement of the IR and Raman spectra of BSA and the calculation of vibrational spectra of zwitterionic ion forms 20 amino acids and their dipeptides were carried out. The effect of anharmonicity and intermolecular interaction (IMI) on the vibrational spectra of amino acids was considered. Results: It has been shown that the forms of vibrations of the amino acid side residues forming a polypeptide do not mix with the forms of vibrations of the amide fragment (Amide I, Amide II and Amide III), which allows them to be used for the interpretation of the vibrational IR and Raman spectra of BSA. The comparison of the experimental and calculated spectra of BSA has shown that each of the experimental absorption band of albumin is a superposition of several absorption bands of amino acids side residues, and the influence of IMI between amino acid residues and water molecules leads to a shift of the maximum and the change in the intensity of absorption bands, corresponding to the vibrations of the Amide I, Amide II and Amide III. The calculated energies and vibration frequencies of the bonds involved in the formation of different types of IMI vary within a fairly wide range. If during the formation of a hydrogen bond between the two di-peptides of glycyl-glycine decrease in the frequency of the valence bond vibrational C=O and increase in the intensity of both the absorption band and the Raman line is observed, then for the valence and deformation vibrational of the polar groups of COO – and N+H3 in case of ion-ion and ion-dipole IMI frequency shift is registered, which is 5–80 cm-1, and the intensity varies – by ~3–10 times. It has been shown that the overlap of the absorption bands of amino acid residues with the absorption band of Amide I makes it very sensitive to structural changes, including the manifestation of IMI, whereby the shift in the frequency and intensity of the absorption band of Amide I allows to determine the conformational changes of the protein. Analysis of the intensities of IR and Raman amino acid residues in the region of ~1540 cm-1 has shown that IMI leads to a more significant change in the intensity of the absorption band of Amide II in the IR spectrum as compared to the Raman spectrum. In the area of the Amide III vibrations deformation δ(HE) and δ(NH) vibrations of the side chains of several amino acids involved in the formation of IMI are manifested, resulting in the shift of the values of corresponding frequency of deformation vibrations in the Amide III. In the experimental IR spectrum of BSA in the region of ~660 cm-1, a wide absorption band of medium intensity is manifested. According to the performed calculation, deformation vibrations of the γ(OCO-) angle of amino acid residues Glu and Asp are manifested in this spectral range, whose participation in the IMI with other amino acid residues and water molecules leads to the shift in the vibration frequency of this deformation vibration and broadening of the corresponding absorption band. Conclusion: Thus, a detailed analysis and interpretation of the vibrational IR and Raman spectra of BSA have enabled one to identify and consider in detail one of the main reasons leading to the frequency shift and change in the intensity of Amide I, Amide II and Amide III, which is the formation of various IMI between amino acids and amino acids and solvent molecules. The interpretation of the vibrational spectra of the zwitterionic forms of 20 standard amino acids in different spectral ranges allows to use it not only to determine the conformational changes of proteins, but also to diagnose the interaction with other molecular compounds, leading, for example, to the formation of complexes.


1. Machida K., Izumi M., Kagayama A. Vibrational spectra and intermolecular potential of DL-serine crystal. Spectrochim. Acta, 1979, vol. 35A, pp. 1333-1339. DOI:

2. Ivanov A. A., Korolik E. V., Insarova N. I., Zhbankov R. G., Golubovich V. P. Low-temperature vibrational spectra and molecular structure of l-alanine. J. Appl. Spectr., 1990, vol. 53, no. 2, pp. 265–270. DOI:

3. Ivanov A. A., Korolik E. V., Insarova N. I., Il’ich G. K. Application of low-temperature ir spectroscopy to the analysis of the molecular structure of glycine. J. Appl. Spectr., 1991, vol. 54, no. 3, pp. 464‒468. DOI:

4. Grenie Y., Lassegues J.-C., Garrigou-Lagrange C. Infrared spectrum of matrix-isolated glycine. J. Chem. Phys., 1970, vol. 53, pp. 2980-2982. DOI:

5. Lu W., Liu H. Correlations between Amino Acids at Different Sites in Local Sequences of Protein Fragments with Given Structural Patterns. Chinese J. Chem. Phys., 2007, vol. 20, no. 1, pp. 71-77. DOI:

6. Kakihana M., Akiyama M., Nagumo T., Okamoto M. An empirical potential function of α-glycine derived from infrared spectroscopic data of D-, 13C-, 15N-, and 18O-labeled species. Z. Naturforsch, 1988, vol. 43a, pp. 774-792. DOI:

7. Nobrega G. F., Sambrano J. R., de Souza A. R., Queralt J. J., Longo E. DFT study of α-alanine as a function of the medium polarity. J. Mol. Struct., 2001, vol. 544, pp. 151-157. DOI:

8. Gomez-Zavaglia A., Fausto R. Low-temperature solidstate FTIR study of glycine, sarcosine and N,N-dimethylglycine observation of neutral forms of simple α-amino acids in the solid state. Phys. Chem. Chem. Phys., 2003, vol. 5, pp. 3154-3161. DOI:

9. Nagy P. I., Noszal B. Theoretical study of the tautomeric conformational equilibrium of aspartic acid zwitterions in aqueous solution. J. Phys. Chem. A., 2000, vol. 104, pp. 6834-6843. DOI:

10. Rai A. K., Song C., Lin Z. An exploration of conformational search of leucine molecule and their vibrational spectra in gas phase using ab initio methods. Spectrochim. Acta A, 2009, vol. 73, pp. 865‒870. DOI:

11. Cocinero E. J., Lesarri A., Grabow J-U., Lopez J. C., Alonso J. L. The Shape of Leucine in the Gas Phase. Chem. Phys. Chem., 2007, vol. 8, pp. 599‒604. DOI:

12. Mohamed M. Ali J., Umadevi M., Ramakrishnan V. Vibrational spectral studies of (β-alanine)β-alaninium nitrate. J. Raman Spectr., 2004, vol. 35, pp. 956–960. DOI:

13. Cao X., Fischer G. Infrared spectra of monomeric L-alanine and L-alanine-N-d3 zwitterions isolated in a KBr matrix. J. Chem. Phys., 2000, vol. 255, pp. 195–204. DOI:

14. Silva J. G., Arruda L. M., Pinheiro G. S., Lima C. L., Melo F. E. A., Ayala A. P., Filho J. Mendes, Freire P. T. C. The temperature-dependent single-crystal Raman spectroscopy of a model dipeptide: L-Alanyl-L-alanine. Spectrochim. Acta. Part A: Mol. Biomol. Spectr., 2015, vol. 148, pp. 244–249. DOI:

15. Lima J. A., Freire P. T. C., Melo F. E. A., Mendes Filho J., Fischerb J., Havenithc Remco W. A., Broerc R., Bordallod Heloisa N. Using Raman spectroscopy to understand the origin of the phase transition observed in the crystalline sulfur based amino acid l-methionine. Vibr. Spectr., 2013, vol. 65, pp. 132–141. DOI:

16. Pearson J. F., Slifkin M. A. The infrared spectra of amino acids and dipeptides. Spectrochim. Acta, 1972, vol. 28A, pp. 2403‒2417. DOI:

17. Parker F. S. Application of infrared spectroscopy in biochemistry, biology and medicine. New York, Plenum Press, 1971. 483 p.

18. Kerri P. Primenenie spektroskopii KR i RKR v biohimii [Application of Raman spectroscopy AND RKR in biochemistry]. Мoscow, Mir Publ., 1985. 272 с.

19. Barth A. The infrared absorption of amino acid side chains. Progress in Biophysics & Molecular Biology, 2000, vol. 74, pp. 141–173. DOI:

20. Freire P. T. C. Pressure-Induced Phase Transitions in Crystalline Amino Acids. High-Pressure Crystallography – from Fundamental Phenomena to Technological Applications. Eds. E. Boldyreva, P. Dera. New York, Springer, 2010, pp. 559–572.

21. Wolpert M., Hellwig P. Infrared spectra and molar absorption coefficients of the 20 alpha amino acids in aqueous solutions in the spectral range from 1800 to 500 cm−1. Spectrochim. Acta, Part A: Mol. Biomol. Spectr., 2006, vol. 64, pp. 987–1001. DOI:

22. Shurvell H. F., Bergin F. J. Raman Spectra of L(+)- Glutamic Acid and Related Compounds. J. Raman spectr., 1989, vol. 20, pp. 163-168. DOI:

23. Castro J. L., Montañez M. A., Otero J. C., Marcos J. I. SERS and Vibrational Spectra of Aspartic Acid. J. Mol. Struct., 1995, vol. 349, pp. 113‒116. DOI:

24. Hernández B., Pflüger F., Derbel N., Coninck J., Ghomi M. Vibrational Analysis of Amino Acids and Short Peptides in Hydrated Media. VI. Amino Acids with Positively Charged Side Chains: L-Lysine and L-Arginine. J. Phys. Chem. B., 2010, vol. 114, pp. 1077‒1088. DOI:

25. Ten G. N., Gluhova O. E., Slepchenkov M. M., Shcherbakova N. E., Baranov V. I. Modeling of vibrational spectra of l-tryptophan in condensed States. Izv. Saratov Univ. (N. S.), Ser. Physics, 2017, vol. 17, iss. 1, pp. 20‒32 (in Russian). DOI:

26. Ten G. N., Scherbakova N. E., Baranov V. I. Teoreticheskij analiz struktury i kolebatel’nyh spektrov asparaginovoj i glutaminovoj aminokislot v vode pri raznyh rN [Theoretical analysis of the structure and vibrational spectra of aspartic and glutamic amino acids in water at different pH]. Natural Science, 2017, vol. 60, no. 3, pp. 94–107 (in Russian).

27. Ten G. N., Shcherbakova N. E., Baranov V. I. Modelirovanie struktury i kolebatel'nyh spektrov osnovnyh aminokislot lizina i arginina v vodnom rastvore [Modeling of the structure and vibrational spectra of the major amino acids of lysine and arginine in aqueous solution]. Natural Science, 2017, vol. 60, no. 3, pp. 85-94 (in Russian).

28. Kint S., Tomimatsu Y. A Raman difference spectroscopic investigation of ovalbumin and S-ovalbumin. Biopolimers, 1979, vol. 18, pp. 1073–1079. DOI:

29. Lin V. C., Koenig J. L. Raman studies of bovine serum albumin. Biopolimers, 1976, vol. 15, pp. 203–218. DOI:

30. Chen M. C., Lord R. C. Laser-excited Raman spectroscopy of biomolecules. VIII. Conformational study of bovine serum albumin. J. Am. Chem. Soc., 1976, vol. 98, pp. 990–992. DOI:

31. Yu N.-T. Comparison of protein structure in crystals, in lyophilized state, and in solution by laser Raman scattering alpha.-Lactalbumin. J. Am. Chem. Soc., 1974, vol. 96, pp. 4664–4668. DOI:

32. Bellocq A. M., Lord R. C., Mendelsohn R. Laser-excited Raman Spectroscopy of biomolecules III. Native bovine serum albumin and beta-lactoglublin. Biochim. Biophys. Acta, 1972, vol. 257, pp. 280-287. DOI:

33. Painter P. C., Koenig J. L. Raman spectroscopic study of the proteins of egg white. Biopolymers, 1976, vol. 15, pp. 2155–2162. DOI:

34. Nakamura K., Era S., Osaki Y., Sogami M., Hayashi T., Murakami M. Conformational Changes in Seventeen Cystinedisulfi de Bridges of Bovine Serum Albumin Proved by Raman Spectroscopy. PERS Letters, 1997, vol. 417, pp. 375–383. DOI:

35. Bolton B. A., Sherer J. R. Raman Spectra and Water Absorbtion of Bovin Serum Albumin. J. Phys. Chem., 1989, vol. 93, pp. 7635–7640. DOI:

36. Fazio B., Andrea C. D., Foti A., Messina E., Irrera A., Donato M. G., Villari V., Micali N., Maragò O. M., Gucciardi P. G. SERS detection of Biomolecules at Physiological pH via aggregation of Gold Nanorods mediated by Optical Forces and Plasmonic Heating. Scientifi c Reports, 2016, vol. 6, pp. 26952. DOI:

37. Frisch M. J., Trucks G. W., Schlegel H. B., Scuseria G. E., Robb M. A., Cheeseman J. R., Scalmani G., Barone V., Mennucci B., Petersson G. A., Nakatsuji H., Caricato M., Li X., Hratchian H. P., Izmaylov A. F., Bloino J., Zheng G., Sonnenberg J. L., Hada M., Ehara M., Toyota K., Fukuda R., Hasegawa J., Ishida M., Nakajima T., Honda Y., Kitao O., Nakai H., Vreven T., Montgomery J. A., Jr., Peralta J. E., Ogliaro F., Bearpark M., Heyd J. J., Brothers E., Kudin K. N., Staroverov V. N., Kobayashi R., Normand J., Raghavachari K., Rendell A., Burant J. C., Iyengar S. S., Tomasi J., Cossi M., Rega N., Millam J. M., Klene M., Knox J. E., Cross J. B., Bakken V., Adamo C., Jaramillo J., Gomperts R., Stratmann R. E., Yazyev O., Austin A. J., Cammi R., Pomelli C., Ochterski J. W., Martin R. L., Morokuma K., Zakrzewski V. G., Voth G. A., Salvador P., Dannenberg J. J., Dapprich S., Daniels A. D., Farkas O., Foresman J. B., Ortiz J. V., Cioslowski J., Fox D. J. Gaussian 09. Gaussian Inc., Wallingford CT, 2009. 394 р.

38. Derbel N., Hernández B., Pflüger F., Liquier J., Geinguenaud F., Jaidane N., Lakhdar Y. B., Ghomi M. Vibrational analysis of amino acids and short peptides in hydrated media. I. L-glycine and L-leucine. J. Phys. Chem. B., 2007, vol. 111, pp. 1470–1477. DOI:

39. Chernobay G. B., Chesalov Y. A., Boldyreva E. V. Temperature effects on the IR spectra of crystalline amino acids, dipeptides, and polyamino acids. V. L-serylglycine. J. Struct. Chem., 2008, vol. 49, pp. 1012-1021.

40. Vinogradov S. N. Hydrogen bonds in crystal structures of amino acids, peptides and related molecules. Int. J. Peptide Prot. Res., 1979, vol. 14, no. 4, pp. 281–289. DOI:

41. Petitpas I., Bhattacharya A. A., Twine S., East M., Curry S. Crystal structure analysis of warfarin binding to human serum albumin: anatomy of drug site I. J. Biol. Chem., 2001, vol. 276, no. 25, pp. 22804–22809. DOI:

Short text (in English): 
Full text (in Russian):