Capillary electrophoresis separation and synchronous fluorescence spectral detection was used off-line to reveal the nature of fluorescent adducts formed in vivo in the collagen molecule and their distribution in the molecule. It was shown that by using the delta lamda in the area of the Stokes shift for the analyzed entities (approximately 10 nm for pentosidine, 4,5(E)-epoxy-2(E)-heptenal and 4,5(E)-epoxy-2(E)-decenal lysine adducts) a distinct profile of spectral bands can be obtained allowing for differentiation of the several entities involved. In combination with capillary electrophoretic separation of the CNBr peptides the location of individual adducts was possible: while pentosidine (and, perhaps, pentosidine related compounds K1-K4) is found in the large alpha 1(I)CB6 and alpha 2(I)CB3.5 peptides along with a complete set of the other fluorescent adducts, low-molecular-mass peptides originating from the terminal region of the molecule are devoid of any fluorescence. All other parts of the molecule possess synchronous fluorescence profiles corresponding to the intact molecule except that they are devoid of pentosidine. The results indicate random distribution of fluorescent adducts in the collagen molecule and, in a broader context, indicate the usefulness of multicomponent analysis by means of combining synchronous luminescence spectra and capillary electrophoresis.

Institute of Physiology Academy of Sciences of the Czech Republic Prague Czech Republic

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