An autosampling differential scanning calorimeter instrument for studying molecular interactions.
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.
Plotnikov
Valerian
V
MicroCal, LLC, Northampton, MA, USA.
Rochalski
Andrew
A
Brandts
Michael
M
Brandts
John F
JF
Williston
Samuel
S
Frasca
Verna
V
Lin
Lung-Nan
LN
D000465:Algorithms; D001322:Autoanalysis; D002152:Calorimetry, Differential Scanning / Q000295:instrumentation*; D003627:Data Interpretation, Statistical; D015195:Drug Design; D007700:Kinetics; D008024:Ligands; D010600:Pharmacology / Q000295:instrumentation; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000494:pharmacology; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D012984:Software; D013594:Syringes; D013816:Thermodynamics
D008024:Ligands; D011506:Proteins; D012259:Ribonuclease, Pancreatic