A method for determining binding kinetics applied to thiamine-binding protein.
A rapid and simple method for assaying the binding activity of thiamine-binding protein is described. By this assay method, the binding characteristics of rice bran thiamine-binding protein have been evaluated with [14C]thiamine as ligand. Analysis of these data by Scatchard plot resulted in linear plots giving a dissociation constant (Kd) for thiamine of 0.55 microM and a maximum binding (Bmax) of 14.5 pmol of ligand bound/microgram of protein. Thiamine binding to the binding protein was time dependent and reached equilibrium at approximately 20 min. The Kob was 0.18 min-1 and the k1 was 1.25 X 10(5) min-1 M-1. Reversibility of thiamine binding at equilibrium was completed at 60 min with a k2 value of 0.052 min-1. The Kd calculated from the reverse rate constant was 0.42 microM. These results indicated that this binding assay method was substantially reliable and accurate.
D002352:Carrier Proteins / Q000378:metabolism*; D007700:Kinetics; D012275:Oryza; D010940:Plant Proteins / Q000378:metabolism; D011485:Protein Binding; D013831:Thiamine / Q000378:metabolism*
D002352:Carrier Proteins; D010940:Plant Proteins; C026989:thiamine-binding protein; D013831:Thiamine