Native denaturation differential scanning fluorimetry: Determining the effect of urea using a quantitative real-time thermocycler

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• 作者：Christine L. Childers ; Stuart R. Green ; Neal J. Dawson ; Kenneth B. Storey ; ^{kenneth_storey@carleton.ca" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Differential scanning fluorimetry ; Enzyme kinetics ; Enzyme stability ; Urea ; Native denaturation ; Lactate dehydrogenase
• 刊名：Analytical Biochemistry
• 出版年：2016
• 出版时间：1 September 2016
• 年：2016
• 卷：508
• 期：Complete
• 页码：114-117
• 全文大小：592 K

文摘

The effect of protein stability on kinetic function is monitored with many techniques that often require large amounts of expensive substrates and specialized equipment not universally available. We present differential scanning fluorimetry (DSF), a simple high-throughput assay performed in real-time thermocyclers, as a technique for analysis of protein unfolding. Furthermore, we demonstrate a correlation between the half-maximal rate of protein unfolding (K_nd), and protein unfolding by urea (I₅₀). This demonstrates that DSF methods can determine the structural stability of an enzyme's active site and can compare the relative structural stability of homologous enzymes with a high degree of sequence similarity.