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 (Knd), 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.

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Keywords Differential scanning fluorimetry, Enzyme kinetics, Enzyme stability, Lactate dehydrogenase, Native denaturation, Urea
Persistent URL dx.doi.org/10.1016/j.ab.2016.05.019
Journal Analytical Biochemistry
Citation
Childers, C.L. (Christine L.), Green, S.R. (Stuart R.), Dawson, N.J. (Neal J.), & Storey, K. (2016). Native denaturation differential scanning fluorimetry: Determining the effect of urea using a quantitative real-time thermocycler. Analytical Biochemistry, 508, 144–117. doi:10.1016/j.ab.2016.05.019