Purification and Preliminary Characterisation of a Thiamine-binding Protein from Maize Seeds

A thiamine-binding protein was isolated from seeds of maize (Zea mays L.) using a sequence of precipitation techniques, DEAE-cellulose batch adsorption, Sephacryl S-300 gel filtration and EAH-Sepharose chromatography. It was a 130 kDa globulin composed of some combination of 54 kDa and 39 kDa subunits not linked by disulphide bridges. Its isoelectric point was estimated to be 6.8. The protein preparation contained a high percentage of glutamine/glutamic acid, arginine and glycine. Thiamine was bound to a capacity of 13.5 nmoles per mg protein, indicating the 2:1 stoichiometry of thiamine-protein interaction. In optimal 0.05 mol/L sodium phosphate buffer, pH 8.0, the dissociation constant of the thiamineprotein complex was 0.68 μmol/L. This protein also bound several thiamine analogues though with affinity always lower than that to thiamine, discriminating thiamine phosphates by 2–3 orders of affinity constant magnitude.