REVERSED-PHASE CHROMATOGRAPHY OF PENTOSIDINE-CONTAINING CNBR PEPTIDESFROM COLLAGEN

Reversed-phase chromatography with a C-4 macroreticular sorbent was el aborated and optimized for the separation of collagen type I and III C NBr peptides. In its final stage the method allows separation of alpha (1)(I)CB6 and alpha(2)(I)CB3,5 peptides which are the only ones in whi ch 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 n m). Five fractions were accumulated and evaluated for the presence of fluorescent glycation products. Fraction 1 and 4 were completely devoi d of any fluorophores. Fraction 2, however, exhibited a spectrum typic al for advanced collagen glycation products (370/440 nm), though it di d not contain any distinct pentosidine fluorescence peak. The only fra ctions which exhibited typical pentosidine fluorescence were fractions 3 and 5 (see Fig. 1 for fraction identification). Fluorescence of the peak of similar to 60000 rel. mel. mass (fraction 5) was attributed t o collagen type I fragment (alpha(2)(I)CB3,5) present in this fraction as no fluorescence was observed with the contaminating alpha(1)(III)C B9 fragment. (C) 1997 Elsevier Science B.V.