کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
7556954 | 1491300 | 2018 | 4 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Complete solubilization of cartilage using the heat-stable protease thermolysin for comprehensive GAG analysis
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موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
Articular cartilage comprises collagens, proteoglycans, and glycosaminoglycans (GAGs) together with water, in hyaline matrixes. Articular cartilage is resistant to proteolytic solubilization for comprehensive GAG analyses partly because of assemblies of collagen fibers with thermolabile hydrogen bonds. In this study, we used the heat-stable protease thermolysin to digest collagen in solid articular cartilage at 70â¯Â°C and compared the efficiencies of collagen digestion and GAG extraction to those with collagenase digestion at 50â¯Â°C. Overnight digestion with thermolysin completely solubilized cartilage, whereas collagenase with >10-times higher proteolytic activity digested <20% of collagen. Following thermolysin treatments, almost all GAGs were extracted from the cartilage, whereas only 56% of GAGs were extracted after collagenase digestion. Disaccharide analyses of extracted GAG chains revealed >98% extraction efficiencies of several GAG classes from thermolysin-treated cartilage, compared with <60% extraction efficiencies using collagenase, depending on GAG classes. These results indicate that thermolysin allows complete GAG extraction from solid articular cartilage and that complete solubilization is required for accurate and reproducible analyses of cartilage GAGs. Hence, thermolysin offers a tool for complete solubilization of cartilage prior to comprehensive GAGomic analysis, and is likely applicable to other collagen-rich tissues such as ligaments, skin, and blood vessels.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 548, 1 May 2018, Pages 115-118
Journal: Analytical Biochemistry - Volume 548, 1 May 2018, Pages 115-118
نویسندگان
Harumi Osago, Mikiko Kobayashi-Miura, Yoshifumi Hamasaki, Nobumasa Hara, Mineyoshi Hiyoshi, Mikako Tsuchiya,