Interfacial adsorption and aggregation associated changes in secondary structure of human calcitonin monitored by ATR-FTIR spectroscopy.
The peptide hormone human calcitonin (hCT) has a marked tendency to aggregate in aqueous solutions, resulting in viscous and turbid dispersions consisting of long fibrils approximately 80 A in diameter. Both transmission (T-FTIR) and attenuated total reflection Fourier transform infrared (ATR-FTIR) experiments were applied on hCT adsorption and aggregation kinetics. By means of the surface sensitive ATR-FTIR spectroscopy at a hydrophobic/hydrophilic interface, early adsorption and aggregation steps of hCT could be followed in situ under real time conditions. Since the aggregation of hCT is associated with conformational changes, the secondary structure sensitive amide I'-band (D2O) could be used as a diagnostic marker. ATR-FTIR spectra recorded during the aggregation kinetics of hCT showed an increase of the amide I'-band intensity by a factor of 3.4, interpreted as pronounced adsorption of hCT molecules from bulk solution to the germanium plate. Furthermore, variations in the line shape of the amide I'-band were interpreted. At the beginning, hCT adopted a random coil conformation followed by distinct formations of alpha-helical and intermolecular parallel beta-sheet structures. Finally, the random coil content declined to 63%, whereas alpha and beta contents rose to 8% and 29%, respectively. From our kinetics results the alpha-structures were formed faster than the beta-structures. This was interpreted as an initial induction of amphiphilic helices during the adsorption process of hCT monomers. ATR-FTIR spectroscopy provides a sensitive analytical tool suggested to monitor interfacial adsorption and aggregation phenomena also of other peptides and proteins.
Bauer
H H
HH
Department of Pharmacy, Swiss Federal Institute of Technology, Zurich ETH.
Müller
M
M
Goette
J
J
Merkle
H P
HP
Fringeli
U P
UP
D000327:Adsorption; D002116:Calcitonin / Q000737:chemistry*; D006801:Humans; D016011:Normal Distribution; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared
D002116:Calcitonin