Although there are many methods available for detecting ligand binding to macromolecules (l-5), equilibrium dialysis is still the most commonly used. We report here a simple variation on equilibrium dialysis that greatly shortens the time needed for the assay of the thiaminebinding protein of E. coli.
Materials and Methods. Thiamine-binding protein was prepared by the method previously reported (6). [14C]Thiamine (sp act 2.1 mCi/ mmole) was a gift of Takeda Pharmaceutical Industries. Cellophane tubes of the Visking Company ("18/32" X 8 cm) were used for dialysis experiments after they were boiled for several minutes in dist water.
Dialysis was performed by shaking a lOO-ml polyethylene bottle, which contained 32 ml of 0.1 M potassium phosphate buffer (pH 7.2) containing 0.5 mM P-mercaptoethanol and the cellophane tube, 120 strokes/min. The cellophane tube was hung to the top of the bottle by cotton threads binding the tube. The sample for liquid scintillation counting was prepared as described previously (6).
Results. In the conventional equilibrium dialysis method, it took 20-22 hr at 4ยฐC and 8-10 hr at 37ยฐC to attain complete equilibrium (Fig. 1).
The velocity of the outward flow of thiamine from the dialysis tube was measured. As shown in Fig. 2, it took about 2-3 hr at 37ยฐC and 4-5 hr at 4ยฐC to attain the equilibrium.
Though the reaction time could not be determined accurately, the rate of binding of thiamine to the thiamine-binding protein, when they were mixed directly, was fast enough compared to the dialysis time. It was evident that the reaction time of l&15 min was sufficient for the direct binding when the reaction was stopped by post dialysis.
From those results, a standard procedure for the assay of thiaminebinding protein was determined as follows: to 1.0 ml of sample solution (in, e.g., 0.1 M potassium phosphate buffer, pH 7.2, containing 0.5 mM P-mercaptoethanol) is added 0.1 ml of 1W4 M[W]thiamine (in 0.02 M acetate buffer, pH 4.5), and the mixture is incubated at 37ยฐC for 15 min with gentle shaking. At the end of the incubation period, 0.8 ml of the reaction mixture is transfered into the dialysis tube, and the 282