کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10533065 | 961845 | 2005 | 6 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Separation of keratan-sulfate-derived disaccharides by high-performance liquid chromatography and postcolumn derivatization with 2-cyanoacetamide and fluorimetric detection
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
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 â 3)gal, accounting for â95% 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 â 4)glcNAc6s (â50% nmol product) and the disulfated disaccharide gal6sβ(1 â 4)glcNAc6s (â40% 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 â108 pmol, 50 ng, to 2160 pmol, 1000 ng, for the disaccharide galβ(1 â 4)glcNAc6s, and from 92 pmol, 50 ng, to 1840 pmol, 1000 ng, for the disaccharide gal6sβ(1 â 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 â71% nmol of disaccharide galβ(1 â 4)glcNAc6s and 18% nmol of the disulfated disaccharide gal6sβ(1 â 4)glcNAc6s having â1.20 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 (â50 ng) of this glycosaminoglycan and in relation to its biological function and pathological importance.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 342, Issue 2, 15 July 2005, Pages 200-205
Journal: Analytical Biochemistry - Volume 342, Issue 2, 15 July 2005, Pages 200-205
نویسندگان
Nicola Volpi, Francesca Maccari, Stefano Ferrari, Michele De Luca, Graziella Pellegrini,