Analytical and Bioanalytical Chemistry

, Volume 400, Issue 9, pp 2921–2931 | Cite as

Use of Raman spectroscopy for the identification of radical-mediated damages in human serum albumin

Original Paper


Damages induced by free radicals on human serum albumin (HSA), the most prominent protein in plasma, were investigated by Raman spectroscopy. HSA underwent oxidative and reductive radical stress. Gamma-irradiation was used to simulate the endogenous formation of reactive radical species such as hydrogen atoms (H), solvated electrons (eaq) and hydroxyl radicals (OH). Raman spectroscopy was shown to be a useful tool in identifying conformational changes of the protein structure and specific damages occurring at sensitive amino acid sites. In particular, the analysis of the S–S stretching region suggested the radical species caused modifications in the 17 disulphide bridges of HSA. The concomitant action of eaq and H atoms caused the formation of cyclic disulphide bridges, showing how cystine pairs act as efficient interceptors of reducing species, by direct scavenging and electron transfer reactions within the protein. This conclusion was further confirmed by the modifications visible in the Raman bands due to Phe and Tyr residues. As regards to protein folding, both oxidative and reductive radical stresses were able to cause a loss in α-helix content, although the latter remains the most abundant secondary structure component. β-turns motifs significantly increased as a consequence of the synergic action of eaq and H atoms, whereas a larger increase in the β-sheet content was found following the exposure to OH and/or H attack.

Schematic presentation of the procedure followed for the identification of damaging mechanisms on proteins undergone to oxidative and reductive radical stress. Step 1: generation of free radicals by gamma-radiolysis of water, mimicking an endogenous radical stress on human serum albumin (HSA); step 2: analysis of the Raman and IR spectra of the samples before and after radical stress exposure and step 3: identification of the main targets of the free radical attack and hypothesis of damaging radical-mediated mechanisms


Human serum albumin Raman spectroscopy Gamma-irradiation Radical-induced damage Free radicals 

Supplementary material

216_2011_4970_MOESM1_ESM.pdf (36 kb)
Fig. S1Raman spectra of HSA samples in the 3600–1500 cm-1 region before (a) and after radical stress exposure: (b) Ar-flushed (Meth. A1: HO, eaq and H) and (b) N2O-saturated aqueous solutions (Meth. A2: HO and H) following 300 Gy irradiation dose. Although the laser power on the samples was around 100 mW, the focused part of the samples did not tend to be carbonised as shown by this figure. (PDF 35 kb)


  1. 1.
    Stadtman ER (1995) Methods Enzymol 258:379–393CrossRefGoogle Scholar
  2. 2.
    Hawkins CL, Davies MJ (2001) Biochim Biophys Acta Bioenerg 1504:196–219CrossRefGoogle Scholar
  3. 3.
    Vince GS, Dean RT (1987) FEBS Lett 216:253–256CrossRefGoogle Scholar
  4. 4.
    Dean RT, Pollak JK (1985) Biochem Biophys Res Commun 126:1082–1089CrossRefGoogle Scholar
  5. 5.
    Davies KJA (1986) J Free Radic Biol Med 2:155–173CrossRefGoogle Scholar
  6. 6.
    Perry G, Raina AK, Nunomura A, Wataya T, Sayre LM, Smith MA (2000) Free Radic Biol Med 28:831–834CrossRefGoogle Scholar
  7. 7.
    Sohal RS (2002) Free Radical Biol Med 33:573–574CrossRefGoogle Scholar
  8. 8.
    Peters TJ (ed) (1996) All about albumin. Academic, San DiegoGoogle Scholar
  9. 9.
    Robertson A, Brodersen R (1991) Dev Pharmacol Ther 17:95–99Google Scholar
  10. 10.
    Monti S, Manet I, Manoli F, Capobianco ML, Marconi G (2008) J Phys Chem B 112:5742–5754CrossRefGoogle Scholar
  11. 11.
    Jurasekova Z, Marconi G, Sanchez-Cortes S, Torreggiani A (2009) Biopolymers 91:917–927CrossRefGoogle Scholar
  12. 12.
    Gutteridge JMC (1986) Biochim Biophys Acta 869:119–127CrossRefGoogle Scholar
  13. 13.
    Halliwell B (1988) Biochem Pharmacol 37:569–571CrossRefGoogle Scholar
  14. 14.
    Rasheed Z, Ahmad R, Rasheed N, Ali R (2007) J Exp Clin Cancer Res 26:395–404Google Scholar
  15. 15.
    Musante L, Candiano G, Petretto A, Bruschi M, Dimasi N, Caridi G, Pavone B, Del Boccio P, Galliano M, Urbani A, Scolari F, Vincenti F, Ghiggeri GM (2007) J Am Soc Nephrol 18:799–810CrossRefGoogle Scholar
  16. 16.
    Oettl K, Stadlbauer V, Petter F, Greilberger J, Putz-Bankuti C, Hallstrom S, Lackner C, Stauber RE (2008) Biochim Biophys Acta Mol Basis Dis 1782:469–473Google Scholar
  17. 17.
    Cohen MP (2003) Arch Biochem Biophys 419:25–30CrossRefGoogle Scholar
  18. 18.
    Rasheed Z, Ali R (2006) Life Sci 79:2320–2328CrossRefGoogle Scholar
  19. 19.
    Garrison WM (1987) Chem Rev 87:381–398CrossRefGoogle Scholar
  20. 20.
    Torreggiani A, Tamba M, Manco I, Faraone-Mennella MR, Ferreri C, Chatgilialoglu C (2005) J Mol Struct 744:767–773CrossRefGoogle Scholar
  21. 21.
    Torreggiani A, Tamba M, Manco I, Faraone-Mennella MR, Ferreri C, Chatgilialoglu C (2006) Biopolymers 81:39–50CrossRefGoogle Scholar
  22. 22.
    Ferreri C, Manco I, Faraone-Mennella MR, Torreggiani A, Tamba M, Manara S, Chatgilialoglu C (2006) Chembiochem 7:1738–1744CrossRefGoogle Scholar
  23. 23.
    Ferreri C, Chatgilialoglu C, Torreggiani A, Salzamo AM, Renzone G, Scaloni A (2008) J Proteome Res 7:2007–2015CrossRefGoogle Scholar
  24. 24.
    Yeh CC, Hou MF, Wu SH, Tsai SM, Lin SK, Hou LA, Ma H, Tsai LY (2006) Cell Biochem Funct 24:555–559CrossRefGoogle Scholar
  25. 25.
    Rietsch A, Beckwith J (1998) Annu Rev Genet 32:163–184CrossRefGoogle Scholar
  26. 26.
    Hawkins CL, Morgan PE, Davies MJ (2009) Free Radical Biol Med 46:965–988CrossRefGoogle Scholar
  27. 27.
    Davies MJ, Fu SL, Wang HJ, Dean RT (1999) Free Radical Biol Med 27:1151–1163CrossRefGoogle Scholar
  28. 28.
    Schoneich C, Sharov VS (2006) Free Radical Biol Med 41(10):1507–1520CrossRefGoogle Scholar
  29. 29.
    Onorato JM, Thorpe SR, Baynes JW (1998) In: Harman D, Holliday R, Meydani M (eds) Towards prolongation of the healthy life span—practical approaches to intervention. Ann N Y Acad Sci 854:277–290Google Scholar
  30. 30.
    Carey PR (1999) J Biol Chem 274:26625–26628CrossRefGoogle Scholar
  31. 31.
    Tuma R, Thomas GJ (2002) In: Chalmers JM, Griffiths PR (eds) Handbook of vibrational spectroscopy, vol 23. Wiley, Chichester, p 215Google Scholar
  32. 32.
    Torreggiani A (2009) In: Kozyrev D, Slutsky V (eds) Handbook of free radicals: formation, types and effects. Nova Science Publisher, Inc., New York, pp 377–419Google Scholar
  33. 33.
    Buxton GV, Greenstock CL, Helman WP, Ross AB (1988) J Phys Chem Ref Data 17:513–886Google Scholar
  34. 34.
    Ross ABMW, Helman WP, Buxton GV, Huie RE, Neta P (eds) (1998) NDRL-NIST solution kinetic database—version 3. Notre Dame Radiation Laboratory, Notre DameGoogle Scholar
  35. 35.
    Spinks JWT, Woods RJ (1990) An introduction to radiation chemistry, 3rd edn. Wiley, New York, pp 243–313Google Scholar
  36. 36.
    Salzano AM, Renzone G, Scaloni A, Torreggiani A, Ferreri C, Chatgilialoglu C (2010) Mol Biosyst 7:889–898CrossRefGoogle Scholar
  37. 37.
    Winterbourn CC (1995) Toxicol Lett 82–3:969–974CrossRefGoogle Scholar
  38. 38.
    Steinberg D, Witztum JL (2002) Circulation 106:E195–E195CrossRefGoogle Scholar
  39. 39.
    Tu AT (1986) Spectroscopy of biological systems. In: Clark RJH, Hester RE (eds) Advances in spectroscopy, vol 13. Wiley, Chichester, pp 47–112Google Scholar
  40. 40.
    Tuma R (2005) J Raman Spectrosc 36:307–319CrossRefGoogle Scholar
  41. 41.
    Williams RW (1986) Meth Enzymol 130:311–331CrossRefGoogle Scholar
  42. 42.
    Bandekar J (1993) Vibr Spectrosc 5:143–173CrossRefGoogle Scholar
  43. 43.
    Prestrelski SJ, Byler DM, Liebman MN (1991) Biochemistry 30:133–143CrossRefGoogle Scholar
  44. 44.
    Miura T, Thomas GJ Jr (1995) In: Biswas BB, Roy S (eds) Subcellular biochemistry; proteins: structure, function, and engineering. Plenum Publishing, New York, pp 55–99Google Scholar
  45. 45.
    Vass E, Hollosi M, Besson F, Buchet R (2003) Chem Rev 103:1917–1954CrossRefGoogle Scholar
  46. 46.
    Torreggiani A, Domenech J, Tinti A (2009) J Raman Spectrosc 40:1687–1693CrossRefGoogle Scholar
  47. 47.
    Navarra G, Tinti A, Leone M, Militello V, Torreggiani A (2009) J Inorg Biochem 103:1729–1738CrossRefGoogle Scholar
  48. 48.
    Pelton JT, McLean LR (2000) Anal Biochem 277:167–176CrossRefGoogle Scholar
  49. 49.
    Byler DM, Susi H (1986) Biopolymers 25:469–487CrossRefGoogle Scholar
  50. 50.
    Torreggiani A, Tamba M, Ferreri C (2007) Prot Pept Letters 14:716–722CrossRefGoogle Scholar
  51. 51.
    Alix AJP, Pedanou G, Berjot M (1988) J Mol Struct 174:159–164CrossRefGoogle Scholar
  52. 52.
    Fagnano C, Fini G, Torreggiani A (1995) J Raman Spectrosc 26:991–995CrossRefGoogle Scholar
  53. 53.
    Torreggiani A (2008) Spectrosc Int J 22:279–286Google Scholar
  54. 54.
    Vanwart HE, Scheraga HA (1976) J Phys Chem 80:1823–1832CrossRefGoogle Scholar
  55. 55.
    Armstrong DA (1990) In: Chatgilialoglu C, Asmus KD (eds) Sulfur-centered reactive intermediates in chemistry and biology, vol. 197. Nato Advanced Science Institutes Series, Series A, Life Sciences, pp 121–134Google Scholar
  56. 56.
    Asmus K-D, Bonifacic M (1999) In: Alfassi ZB (ed) S-Centered radicals. Wiley, Chichester, pp 141–191Google Scholar
  57. 57.
    Vogt W (1995) Free Radic Biol Med 18:93–105CrossRefGoogle Scholar
  58. 58.
    Prutz WA, Butler J, Land EJ (1985) Int J Radiat Biol 47:149–156CrossRefGoogle Scholar
  59. 59.
    Atrian S, Bobrowski K, Capdevila M, Chatgilialoglu C, Ferreri C, Houee-Levin C, Salzano AM, Scaloni A, Torreggiani A (2008) Chimia 62:721–727CrossRefGoogle Scholar
  60. 60.
    Torreggiani A, Domenech J, Orihuela R, Ferreri C, Atrian S, Capdevila M, Chatgilialoglu C (2009) Chem Eur J 15:6015–6024CrossRefGoogle Scholar
  61. 61.
    Takeuchi H, Watanabe N, Satoh Y, Harada I (1989) J Raman Spectrosc 20:233–237CrossRefGoogle Scholar
  62. 62.
    Liddle WK, Tu AT (1981) Appl Spectrosc 35:444–446CrossRefGoogle Scholar
  63. 63.
    Ploder M, Neurauter G, Spittler A, Schroecksnadel K, Roth E, Fuchs D (2008) Amino Acids 35:303–307CrossRefGoogle Scholar
  64. 64.
    Overman SA, Thomas GJ (1999) Biochemistry 38:018–4027CrossRefGoogle Scholar
  65. 65.
    Seredynski J, Soylemez T, Baumeister W, Herbertz LM (1981) Z Nat Forsch C J Biosci 36:310–318Google Scholar
  66. 66.
    Torreggiani A, Fagnano C, Fini G (1997) J Raman Spectrosc 28:23–27CrossRefGoogle Scholar
  67. 67.
    Chou PY, Fasman GD (1974) Biochemistry 13:222–245CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  1. 1.Istituto I.S.O.F.Consiglio Nazionale delle RicercheBolognaItaly
  2. 2.Dipartimento di BiochimicaUniversità di BolognaBolognaItaly

Personalised recommendations