This work evaluates the use of a competitive binding assay using flow-through partial-filling affinity capillary electrophoresis (FTPFACE) to estimate binding constants of neutral ligands to a receptor. We demonstrate this technique using, as a model system, carbonic anhydrase B (CAB, EC 4.2.1.1) and arylsulfonamides. In this technique, the capillary is first partially filled with a negatively charged ligand, a sample containing CAB and two noninteracting standards, and a neutral ligand, then electrophoresed. Upon application of a voltage the sample plug migrates into the plug of negatively charged ligand (L(-)) resulting in the formation of a CAB-L(-) complex. Continued electrophoresis results in mixing between the neutral ligand (L(0)) and the CAB-L(-) complex. L(0) successfully competes out L(-) to form the new CAB-L(0) complex. Analysis of the change in the relative migration time ratio (RMTR) of CAB relative to the noninteracting standards, as a function of neutral ligand concentration, yields a value for the binding constant. These values are in agreement with those estimated using other binding and ACE techniques. Data demonstrating the quantitative potential of this method is presented.
SummaryA new approach for estimating binding constants of ligands to receptors, flow-through partial-filling affinity capillary electrophoresis (FTPFACE), is introduced for studying the interaction of carbonic anhydrase B (CAB, EC 4.2.1.1) with arylsulfonamides and vancomycin from Streptomyces orientalis with D-Ala-D-Ala peptides. In this technique the capillary is first partiallyfilled with ligand followed by a sample of receptor and non-interacting standards. Upon application of a voltage the receptor and standards flow into the ligand plug where equilibrium is achieved between the receptor and ligand. Continued electrophoresis results in the receptor and standards flowing through the domain of the ligand plug. Analysis of the change in the relative migration time ratio of the receptor, relative to the non-interacting standards, as a function of the concentration of ligand, yields a value for the binding constant. These values are comparable to those estimated using other binding and ACE techniques. Data demonstrating the quantitative potential of this method is presented.
Defective DNA replication can result in substantial increases in the level of genome instability. In the yeast Saccharomyces cerevisiae, the pol3-t allele confers a defect in the catalytic subunit of replicative DNA polymerase d that results in increased rates of mutagenesis, recombination, and chromosome loss, perhaps by increasing the rate of replicative polymerase failure. The translesion polymerases Pol h, Pol z, and Rev1 are part of a suite of factors in yeast that can act at sites of replicative polymerase failure. While mutants defective in the translesion polymerases alone displayed few defects, loss of Rev1 was found to suppress the increased rates of spontaneous mutation, recombination, and chromosome loss observed in pol3-t mutants. These results suggest that Rev1 may be involved in facilitating mutagenic and recombinagenic responses to the failure of Pol d. Genome stability, therefore, may reflect a dynamic relationship between primary and auxiliary DNA polymerases.
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