Affinity gel titration: quantitative analysis of the binding equilibrium between immobilized protein and free ligand by a continuous titration procedure.
A new method, called affinity gel titration, for analyzing the specific interaction between an immobilized protein and a ligand molecule is presented. Only one or two experimental runs permit the determination of not only the equilibrium constant but also the amount of immobilized protein. A suspension of the immobilized protein on agarose gel beads is confined in a constant volume mixing cell. A solution of a specific ligand molecule of constant concentration is introduced into the cell so that its concentration in the cell increases continuously (as in a mixing chamber for forming a convex gradient). The correlation between the concentration of the ligand in the efflux and the cumulative volume of the efflux can be analyzed either by regression to a theoretical curve or by a graphical method. Specific binding of p-aminobenzamidine to immobilized Streptomyces griseus trypsin was studied by this method. The dissociation constant and the amount of active trypsin were determined. The values obtained were in good agreement with the inhibition constant obtained by a kinetic experiment with free trypsin and with the amount of active site measured by using p-nitrophenyl p'-guanidinobenzoate, respectively. A single run of the titration procedure could be completed within 1 h.
D001322:Autoanalysis; D001550:Benzamidines; D001565:Benzoates; D005782:Gels; D007201:Indicator Dilution Techniques; D007700:Kinetics; D008024:Ligands / Q000032:analysis*; D008433:Mathematics; D008956:Models, Chemical; D011485:Protein Binding; D011506:Proteins / Q000032:analysis*; D012685:Sepharose / Q000032:analysis; D014107:Tosyllysine Chloromethyl Ketone; D014357:Trypsin / Q000032:analysis
D001550:Benzamidines; D001565:Benzoates; D005782:Gels; D008024:Ligands; D011506:Proteins; D014107:Tosyllysine Chloromethyl Ketone; C002238:4-nitrophenyl 4'-guanidinobenzoate; C006500:4-aminobenzamidine; D012685:Sepharose; D014357:Trypsin