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</o:shapelayout></xml><![endif]--></head><body lang=EN-US link=blue vlink=purple><div class=WordSection1><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New"'>Dear RASMB,<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New"'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New"'>We are working with a controlled substance binding to protein target – I apologize for being vague but disclosure rules limit me from being more specific. The affinity estimate by yeast expression is very approximately 100uM Kd and the real affinity is quite possibly weaker. <o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New"'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New"'>The nature of the ligand and its toxicity preclude its use in the AUC due to the potential to aerosolize the material<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New"'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New"'>Additionally, the controlled drug material interacts with a Series S CM5 chip likewise make quantitation by SPR difficult.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New"'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New"'>For ITC, the given interaction would require 1mM protein in the calorimetric cell, which is not feasible for us. One could design a displacement ITC experiment with a suitable strong endothermic binder but that would also not be practical – at least not yet. <o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New"'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New"'>I have been considering using DSC to monitor the shift in Tm (Brandts et. al. Biochem. Vol.24, 1990) and extract the Kd at the Tm; we would have to correct the Kd to room temperature using the enthalpy of binding determined by a reverse titration using the ITC instrument. I know that this approach has been useful in the measurement of ultra-tight binding, (Gomez et al. JMB vol. 252, 1995), and if my reading of some of reviews in the literature is accurate it should also be useful for the quantitation of weak affinity interactions. However I have not found too many examples of using this approach to quantitate weak affinity interactions. The advantage of this approach seems to me is that it would only require 2uM protein and that the interaction is saturated.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New"'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New"'>I wonder if anyone knows if ligand binding by DSC has been used to quantitate weak interactions and if so if someone might be kind enough to point me at a reference.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New"'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New"'>I very much appreciate any comments and advice that might be provided, and thank you all in advance for your time.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New"'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New"'>Best regards,<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New"'>John Sumida,<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New"'><a href="http://depts.washington.edu/cidb4bio/index.shtml"><span style='color:windowtext'>Analytical Biopharmacy Core Facility</span></a><o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New"'>University of Washington<o:p></o:p></span></p><p class=MsoNormal style='text-autospace:none'><span style='color:black'><o:p> </o:p></span></p></div></body></html>