Qualitative and quantitative analysis of the secondary structure of cytochrome C Langmuir-Blodgett films.
A qualitative and quantitative analysis of the conformation of Langmuir-Blodgett (LB) dried films of cytochrome C on silicon wafers was performed by Fourier transform ir (FTIR) spectroscopy. A deconvolution procedure was applied to the amide I band analysis, in order to determine the percentage of the different secondary structures. Qualitative analysis was performed by examining difference spectra. Films obtained by spreading protein solutions at pH 7.4 and 1, dried at 25 and 100 degrees C, on silicon wafers were also examined in order to detect spectral components associated with denatured protein domains, and to compare them with cytochrome C LB films. FTIR spectroscopy showed that the following important changes characterise LB film spectra: (a) the alpha-helix component is higher (its percentage is 57 and 54%) than the one estimated in dried film obtained by spreading the solutions at pH 7.4 on a silicon substrate (43%), (b) there is an increase in the intensity of bands attributed to protonated carboxy group bands, involved and not involved in the formation of hydrogen bonds, and a decrease in those attributed to deprotonated carboxy groups, (c) the intensity of several bands attributed to aromatic amino acids and aliphatic chains increases, and (d) bands due to O-H stretching vibrations of crystallization water are present. These conformational changes could be induced by protein-protein interaction caused by the close packing of molecules that occurs during LB film formation; it cannot be excluded that they may be accompanied by partial changes in the tertiary structure of the protein. A preferential orientation of protein molecules in LB films is also a possibility.
Bramanti
E
E
Institute of Instrumental Analytical Chemistry, CNR, Pisa, Italy.
Benedetti
E
E
Nicolini
C
C
Berzina
T
T
Erokhin
V
V
D'Alessio
A
A
Benedetti
E
E
D000818:Animals; D003574:Cytochrome c Group / Q000737:chemistry*; D006736:Horses; D009206:Myocardium; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012825:Silicon; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods
D003574:Cytochrome c Group; D012825:Silicon