Thermally induced denaturation and aggregation of BLG-A: effect of the Cu(2+) and Zn (2+) metal ions.
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.
Laboratorio di Biofisica Molecolare, Dipartimento di Fisica e Unità CNISM, Università della Calabria, Ponte P. Bucci - Cubo 31C, 87036, Arcavacata di Rende (CS), Italy.
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Stirpe
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Rizzuti
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Pantusa
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Bartucci
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Sportelli
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Guzzi
D000682:Amyloid / Q000378:metabolism; D052160:Benzothiazoles; D002152:Calorimetry, Differential Scanning; D002413:Cations, Divalent / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology*; D004578:Electron Spin Resonance Spectroscopy; D007782:Lactoglobulins / Q000737:chemistry* / Q000378:metabolism*; D009391:Nephelometry and Turbidimetry; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013844:Thiazoles / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology*
D000682:Amyloid; D052160:Benzothiazoles; D002413:Cations, Divalent; D007782:Lactoglobulins; D013844:Thiazoles; C009462:thioflavin T; D003300:Copper; D014364:Tryptophan; D015032:Zinc