Development of a F枚rster resonance energy transfer assay for monitoring bacterial collagenase triple-helical peptidase activity

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• 作者：Michal Tokmina-Roszyk ; Dorota Tokmina-Roszyk ; Manishabrata Bhowmick ; Gregg B. Fields ; ^{gfields@tpims.org" class="auth_mail}
• 关键词：Bacterial collagenase ; Clostripain ; Collagen ; FRET protease assay ; Islet isolation ; Stem cell isolation
• 刊名：Analytical Biochemistry
• 出版年：15 May, 2014
• 年：2014
• 卷：453
• 期：Complete
• 页码：61-69
• 全文大小：1288 K

文摘

Due to their efficiency in the hydrolysis of the collagen triple helix, Clostridium histolyticum collagenases are used for isolation of cells from various tissues, including isolation of the human pancreatic islets. However, the instability of clostridial collagenase I (Col G) results in a degraded Col G that has weak collagenolytic activity and an adverse effect on islet isolation and viability. A F枚rster resonance energy transfer triple-helical peptide substrate (fTHP) has been developed for selective evaluation of bacterial collagenase activity. The fTHP [sequence: Gly-mep-Flp-(Gly-Pro-Hyp)₄-Gly-Lys(Mca)-Thr-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Lys(Dnp)-Ser-(Gly-Pro-Hyp)₄-NH₂] had a melting temperature (T_m) of 36.2 掳C and was hydrolyzed efficiently by bacterial collagenase (k_cat/K_M = 25,000 s^鈭? M^鈭?) but not by clostripain, trypsin, neutral protease, thermolysin, or elastase. The fTHP bacterial collagenase assay allows for rapid and specific assessment of enzyme activity toward triple helices and, thus, potential application for evaluating the efficiency of cell isolation by collagenases.