Early stages of salmon calcitonin aggregation: Effect induced by ageing and oxidation processes in water and in the presence of model membranes

The natural ageing- and hydrogen peroxide-induced aggregation of salmon calcitonin were studied in water and in the presence of dipaimitoylphosphatidyleholine (DPPC) liposomes. The early stages of the aggregation process at low protein concentration were investigated by means of Circular Dichroism spectroscopy (CD) and conventional and inummogold labelling Transmission Electron Microscopy (TEM). In buffered water solution, salmon calcitonin showed a two-stage conformational variation related to fibril formation and phase-separation of larger aggregates. A first stage, characterised by small conformational changes but a decrease in dichroic band intensity, was followed by a second stage, 6 days after, leading to higher conformational variations and aggregations. Salmon calcitonin showed a distinct modification in the secondary structure and aggregate morphology in the presence of hydrogen peroxide with respect to natural ageing, indicating that the two aggregation processes (natural and chemical-induced) followed a distinct mechanism. The oxidised forms of the peptide were separated by liquid chromatography. The same study was performed in the presence of DPPC liposomes. The results obtained by conventional and immunogold labelling TEM evidenced that salmon calcitonin in buffered water solution essentially does not enter the liposomes but forms around them a fibril network characterised by the same conformational changes after 6 days. The oxidised sample in the presence of liposomes showed a "fibrils hank", separated from liposomes. The presence of liposomes did not affect either the aggregation or the conformational. modifications yet observed by TEM and CD in water solution. (c) 2005 Elsevier B.V. All rights reserved.