European Biophysics Journal

, Volume 37, Issue 8, pp 1351–1360 | Cite as

Thermally induced denaturation and aggregation of BLG-A: effect of the Cu2+ and Zn2+ metal ions

  • A. Stirpe
  • B. Rizzuti
  • M. Pantusa
  • R. Bartucci
  • L. Sportelli
  • R. Guzzi
Original Paper
First Online:
Received:
Accepted:

Abstract

There is growing evidence that metal ions can accelerate the aggregation process of several proteins. This process, associated with several neuro-degenerative diseases, has been reported also for non-pathological proteins. In the present work, the effects of copper and zinc ions on the denaturation and aggregation processes of β-lactoglobulin A (BLG-A) are investigated by differential scanning calorimetry (DSC), fluorescence, electron paramagnetic resonance (EPR) and optical density. The DSC profiles reveal that the thermal behaviour of BLG-A is a complex process, strongly dependent on the protein concentration. For concentrations ≤0.13 mM, the thermogram shows an endothermic peak at 84.3°C, corresponding to denaturation; for concentrations >0.13 mM an exothermic peak also appears, above 90°C, related to the aggregation of the denaturated BLG-A molecules. The thioflavin T fluorescence indicates that the thermally induced aggregates show fibrillar features. The presence of either equimolar Cu2+ or Zn2+ ions in the protein solution has different effects. In particular, copper binds to the protein in the native state, as evidenced by EPR experiments, and destabilizes BLG-A by decreasing the denaturation temperature by about 10°C, whereas zinc ions probably perturb the partially denaturated state of the protein. The kinetics of BLG-A aggregation shows that both metal ions abolish the lag phase before the aggregation starts. Moreover, the rate of the process is 4.6-fold higher in the presence of copper, whereas the effect of zinc is negligible. The increase of the aggregation rate, induced by copper, may be due to a site-specific binding of the metal ion on the protein.

Keywords

β-Lactoglobulin Thermal denaturation Aggregation Copper Zinc 

Abbreviations

BLG-A

β-Lactoglobulin A

DSC

Differential scanning calorimetry

EPR

Electron paramagnetic resonance

PBS

Phosphate buffer solution

ThT

Thioflavin T

Trp

Tryptophan

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Notes

Acknowledgement

This work was supported by a national project (PRIN2005: Research grant 2005023002_003) of the Italian Ministry of University Research.

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Copyright information

© European Biophysical Societies' Association 2008

Authors and Affiliations

  • A. Stirpe
    • 1
  • B. Rizzuti
    • 2
  • M. Pantusa
    • 1
  • R. Bartucci
    • 1
  • L. Sportelli
    • 1
  • R. Guzzi
    • 1
  1. 1.Laboratorio di Biofisica Molecolare, Dipartimento di Fisica e Unità CNISMUniversità della CalabriaArcavacata di Rende (CS)Italy
  2. 2.Laboratorio Licryl CNR-INFM, Dipartimento di FisicaUniversità della CalabriaArcavacata di Rende (CS)Italy

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