Thermally induced denaturation and aggregation of BLG-A: effect of the Cu(2+) and Zn (2+) metal ions

Eur Biophys J. 2008 Oct;37(8):1351-60. doi: 10.1007/s00249-008-0346-4. Epub 2008 Jun 17.

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 beta-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 degrees C, corresponding to denaturation; for concentrations >0.13 mM an exothermic peak also appears, above 90 degrees 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 Cu(2+) or Zn(2+) 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 degrees 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amyloid / metabolism
  • Benzothiazoles
  • Calorimetry, Differential Scanning
  • Cations, Divalent / pharmacology
  • Copper / pharmacology*
  • Electron Spin Resonance Spectroscopy
  • Lactoglobulins / chemistry*
  • Lactoglobulins / metabolism*
  • Nephelometry and Turbidimetry
  • Protein Binding / drug effects
  • Protein Conformation / drug effects
  • Protein Denaturation / drug effects
  • Protein Stability / drug effects
  • Spectrometry, Fluorescence
  • Temperature*
  • Thiazoles / metabolism
  • Tryptophan / metabolism
  • Zinc / pharmacology*

Substances

  • Amyloid
  • Benzothiazoles
  • Cations, Divalent
  • Lactoglobulins
  • Thiazoles
  • thioflavin T
  • Copper
  • Tryptophan
  • Zinc