Purchase this article with an account.
Y. Zhang, A. H. Conrad, Y. Hiromasa, J. M. Tomich, G. W. Conrad; Biochemical Mechanism of Collagen Cross-Linking in a Model Reaction System. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4994.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Devise a model reaction system to study biochemical mechanisms of collagen cross-linking.
Purified collagen types I, III and IV were used to assess the mechanisms of cross-linking induced by the photosensitizer riboflavin (RF) and ultraviolet A (UVA) under conditions which resemble those for the clinical treatment of progressive keratoconus. The mixture of collagen (1 µg/µL in PBS) and RF (0.1% in PBS) was irradiated with UVA of 370 nm for 30 minutes at a distance of 5 cm from the light source.
For collagen type I, the α1 and α2 monomer bands and the β1 and β2 dimer bands that are visible on SDS-polyacrylamide gel electrophoretograms of untreated collagen type I almost disappeared after cross-linking. Intensity of the γ band at approximately 300 kDa heavily increased. After cross-linking, one new polymer band was found to stack at the top of SDS gels, just below the bottom of the sample well. For collagen type III and IV, all bands less than 300 kDa on SDS gels almost disappeared after cross-linking, whereas, simultaneously, polymer bands appeared at the top of SDS gels, just below the bottom of the sample wells of such gels. Compared to native collagen, methionine (Met) and hydroxylysine (Hylys) decreased 60% and 20%, respectively, and both tyrosine (Tyr) and histidine (His) were no longer detected at all in cross-linked collagen type I. After cross-linking, collagen was less well digested into peptides by cyanogen bromide, but was more easily digested by collagenase than was native collagen.
These results indicate that exposure to RF + UVA causes collagen cross-linking of α1, α2, β1 and β2 chains, producing polymers of approximately 300 kDa and larger. Modifications of Met, Hylys, Tyr and His contribute to this cross-linking of collagen.
This PDF is available to Subscribers Only