Reversed-phase chromatography of pentosidine-containing CNBr peptides from collagen

Reversed-phase chromatography with a C4 macroreticular sorbent was elaborated and optimized for the separation of collagen type I and III CNBr peptides. In its final stage the method allows separation of α1(I)CB6 and α2(I)CB3,5 peptides which are the only ones in which pentosidine fluorescence was detected. In both these fractions non-pentosidine fluorescence was observed as well; however, it exhibited a shorter emission maximum (425 nm) as compared to the emission maximum for advanced glycation products (excitation at 370 nm, emission 440 nm). Five fractions were accumulated and evaluated for the presence of fluorescent glycation products. Fraction 1 and 4 were completely devoid of any fluorophores. Fraction 2, however, exhibited a spectrum typical for advanced collagen glycation products (370440 nm), though it did not contain any distinct pentosidine fluorescence peak. The only fractions which exhibited typical pentosidine fluorescence were fractions 3 and 5 (see Fig. 1 for fraction identification). Fluorescence of the peak of ~60000 rel. mol. mass (fraction 5) was attributed to collagen type I fragment (α2(I)CB3,5) present in this fraction as no fluorescence was observed with the contaminating α1(III)CB9 fragment.