Plotnikov Valerian, Rochalski Andrew, Brandts Michael, Brandts John F, Williston Samuel, Frasca Verna, Lin Lung-Nan
MicroCal, LLC, Northampton, MA, USA.
Assay Drug Dev Technol. 2002 Nov;1(1 Pt 1):83-90. doi: 10.1089/154065802761001338.
A new ultrasensitive differential scanning calorimeter (DSC) instrument is described, which utilizes autosampling for continuous operation. High scanning rates to 250 deg/h with rapid cooling and equilibration between scans facilitates higher sample throughput up to 50 samples during each 24 h of unattended operation. The instrument is suited for those pharmaceutical applications where higher throughput is important, such as screening drug candidates for binding constant or screening solution conditions for stability of liquid protein formulations. Results are presented on the binding of five different anionic inhibitors to ribonuclease A, which included cytidine 2'-monophosphate (2'CMP), 3'CMP, uridine 3'-monophosphate, pyrophosphate, and phosphate. Binding constants K(B) (or dissociation constants K(d)) are obtained from the shift in the transition temperature T(M) for ribonuclease thermal unfolding in the presence of ligand relative to the transition temperature in the absence of ligand. Measured binding constants ranged from 155 M(-1) (K(d) = 6.45 mM) for the weak-binding phosphate anion to 13100 M(-1) (K(d) = 76.3 microM) for the strongest binding ligand, 2'CMP. The DSC method for measuring binding constants can also be extended to ultratight interactions involving either ligand-protein or protein-protein binding.
本文介绍了一种新型的超灵敏差示扫描量热仪（DSC），该仪器采用自动进样实现连续运行。高达250℃/小时的高扫描速率以及快速冷却和扫描间的平衡，有助于在无人值守操作的每24小时内实现高达50个样品的更高样品通量。该仪器适用于那些对高通量至关重要的药物应用，例如筛选药物候选物的结合常数或筛选液体蛋白质制剂稳定性的溶液条件。文中给出了五种不同阴离子抑制剂与核糖核酸酶A结合的结果，这些抑制剂包括胞苷2'-单磷酸（2'CMP）、3'CMP、尿苷3'-单磷酸、焦磷酸和磷酸。结合常数K(B)（或解离常数K(d)）是通过在有配体存在时核糖核酸酶热解折叠的转变温度T(M)相对于无配体时的转变温度的变化获得的。测得的结合常数范围从弱结合的磷酸根阴离子的155 M(-1)（K(d)=6.45 mM）到最强结合配体2'CMP的13100 M(-1)（K(d)=76.3 microM）。用于测量结合常数的DSC方法也可以扩展到涉及配体-蛋白质或蛋白质-蛋白质结合的超紧密相互作用。
