Fluorescence Spectroscopy of Collagen in Several Solvents
DOI:
https://doi.org/10.12775/MBS.2014.024Keywords
collagen, fluorescence, spectroscopyAbstract
Introduction. Research was carried out into the influence of microenvironment created by 6 solvents on the spectroscopic characteristics of four types of collagen protein. The solvents were selected on the basis of polarity, pH, the presence of a functional group, as well as their application in fixing biological material for histopathological and spectroscopic examinations. As an amino acid with the highest light-absorbing capacity, tryptophan exhibits the spectral properties of whole protein agglomerates. Protein conformation changes occurring in different environments and changes in functional groups surrounding tryptophan allowed to register protein spectra. The aim of the study was to find out whether changing the collagen microenvironment using six selected solvents allows to differentiate four types of collagen by means of stationary fluorescence spectroscopy. Material and methods. The research material consisted of four types of human collagen: I, II, III, and IV. The classification of collagen types was based on the spatial arrangement of α chains of each collagen type as adopted by Bornstein and Traub. The emission spectra were recorded with the use of a Hitachi F-7000 spectrofluorimeter and analysed with Origin 8.0 Pro software. Results. Preliminary tests allowed to determine the most optimal excitation wavelength of polar (λ = 270 nm) and non-polar (λ = 350 nm) solvents. The observed spectra shapes and maxima at a specific excitation wavelength were characteristic for the corresponding solvent. Differences among the fluorescence spectra of the four types of collagen were not observed. Conclusions and discussion. Stationary fluorescence spectroscopy does not allow to differentiate four collagen types using the selected solvents. A major hindrance of this method consists in its high sensitivity to environment changes which may preclude the optimisation of research and in consequence render the approach inferior to techniques using electrophoretic devices, mass spectrometry, or chromatography.References
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