Early stage aggregation of human serum albumin in the presence of metal ions

The heat induced aggregation of human serum albumin (HSA) with and without an equimolar amount of Cu(II) and Zn(II) was investigated by using optical absorption, fluorescence, AFM and EPR spectroscopy. Turbidity experiments as a function of temperature indicate that the protein aggregation occurs after the melting of the protein. The kinetic of HSA aggregation, investigated between 60 and 70 °C by monitoring the optical density changes at 400 nm on a 180 min time window, shows an exponential growth with a rate that increases with the temperature. Fluorescence of the thioflavin T evidences a significant increase of the intensity at 480 nm at increasing incubation time. These results combined with AFM experiments show that the protein aggregates are elongated oligomers with fibrillar-like features. The absence of a lag-phase suggests that the early stage aggregation of HSA follows a downhill pathway that does not require the formation of an organized nucleus. The presence of Cu(II) and Zn(II) ions does not affect the thermally induced aggregation process and the morphology of HSA aggregates. The result is compatible with the binding of the metal ions to the protein in the native state and with the high conformational stability of HSA.