Separation of keratan-sulfate-derived disaccharides by high-performance liquid chromatography and postcolumn derivatization with 2-cyanoacetamide and fluorimetric detection

In this paper, we report a rapid, sensitive, and quantitative procedure to conduct disaccharide compositional analyses of keratan sulfates (KS) by means of high-performance liquid chromatography (HPLC) separation and postcolumn derivatization with 2-cyanoacetamide and fluorimetric detection of products generated by hydrolysis of this glycosaminoglycan with Bacillus sp. keratanase II or Escherichia freundii endo-β-galactosidase. Following E. freundii endo-β-galactosidase digestion of bovine corneal KS, the monosulfated disaccharide glcNAc6sβ(1 → 3)gal, accounting for ≈95% nmol and 50% yield products, is produced. On the contrary, bovine corneal KS treated with endo-β-N-acetylglucosaminidase (keratanase II) from Bacillus sp. generates two major products, the monosulfated disaccharide galβ(1 → 4)glcNAc6s (≈50% nmol product) and the disulfated disaccharide gal6sβ(1 → 4)glcNAc6s (≈40% nmol product) for over 90% nmol products. These disaccharides are separated and readily determined within 30 min by using a linear-gradient strong anion-exchange separation. A linear relationship was found for the two purified disaccharides over a wide range of concentrations, from ≈108 pmol, 50 ng, to 2160 pmol, 1000 ng, for the disaccharide galβ(1 → 4)glcNAc6s, and from 92 pmol, 50 ng, to 1840 pmol, 1000 ng, for the disaccharide gal6sβ(1 → 4)glcNAc6s. HPLC analysis was applied to the quantitative and qualitative determination of KS produced by 3T3-J2 murine fibroblasts in the cell medium. The amount of KS was found to be 2.80 ± 0.34 μg/ml/106 cells and composed of ≈71% nmol of disaccharide galβ(1 → 4)glcNAc6s and 18% nmol of the disulfated disaccharide gal6sβ(1 → 4)glcNAc6s having ≈1.20 sulfate groups/disaccharide. Our data illustrate that the HPLC procedure reported represents an improved approach for the quantitative and compositional microanalyses of KS, especially applicable to experimentation involving small amounts (≈50 ng) of this glycosaminoglycan and in relation to its biological function and pathological importance.