Equilibrium constants for nonspecific binding of proteins to DNA may be obtained by quantitative zonal DNA affinity chromatography.
Quantitative zonal DNA affinity chromatography may be used to determine accurate equilibrium constants for the binding of proteins nonspecifically to DNA. Zonal quantitative affinity chromatography has not previously been applied to the determination of binding constants of proteins to DNA, although its use is quite commonplace for determination of affinity constants for protein-protein or protein-small ligand interactions. Equilibrium constants were measured for the nonspecific binding of bovine pancreatic ribonuclease A and Escherichia coli lac repressor to double-stranded DNA immobilized on cellulose. The equilibrium constants determined agree with literature values evaluated using other techniques. The experimental advantages of the zonal technique, when it can be applied, are that collection of data is fast and data analysis is simple. Detection of the protein elution profile by absorbance at 220 nm with an in-line detector can provide adequate sensitivity when binding constants are in the range 10(2)-10(4) M-1.
Jenuwine
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ES
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA.
Shaner
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SL
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D002417:Cattle; D002623:Chemistry Techniques, Analytical / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods*; D004247:DNA / Q000302:isolation & purification* / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D007700:Kinetics; D056968:Lac Repressors; D010179:Pancreas / Q000201:enzymology; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D012097:Repressor Proteins; D004247:DNA; D012260:Ribonucleases