The determination of small amounts of collagen hydroxylysyl glycosides

Glucosylgalactosylhydroxylysine (GlcGalHyl) and galactosylhydroxylysine (GalHyl) in alkaline hydrolysates of insoluble collagens were separated on 130 x 0.6 cm and 45 x 0.6 cm columns of Chromobeads A resin (Technicon) and simultaneous analyses with ninhydrin and orcinol-H2SO4 were performed. Ninhydrin, which is the standard reagent in existing methods, gave erroneously high results with both columns due to overlapping, resistant peptides. A rapid and more accurate procedure employing the smaller column, the orcinol-H2SO4 reagent and a new internal standard was developed. The losses of the glycosides in alkaline hydrolysis, especially in the presence of collagen peptides, were much larger than had been reported previously. A shorter alkaline hydrolysis following digestion of the collagen with papain was effective and allowed more reliable corrections to be made. Purging with nitrogen reduced the losses further. Mature bovine skin collagen contained less GlcGalHyl than embryonic skin collagen whereas the differences in GalHyl were insignificant.