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

Abstract

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

Keywords

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)

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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

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