Presence of a thiamine-binding protein in rice bran.

potassium phosphate buffer (pH 7.0) containing 2 mM 2-mercaptoethanol and 1 NaCl by stirring for 3 hr at 4•Ž, and the solution was centrifuged at 13,000g for 15 min. The resulting supernatant fluid (crude extract) was used for the assay of thiamine-binding activity according to the method described previously (2). The crude extract of rice bran bound 393 pmoles of thiamine per mg protein by equilibrium dialysis in the presence of 1ƒÊM [14C]thiamine ([thiazole-2-14C]thiamine hydrochloride, 2.43 Ci/mole) at pH 7.5. The binding activity was proportional at least up to 5.0mg protein and was completely lost by heating at 100•Ž for 10 min. The crude extract (710ml) was then fractionated with ammonium sulfate. The active fraction precipitated at between 55 and 85% saturation, and the precipitate was dissolved in 96ml of 0.05 M potassium phosphate buffer (pH 7.0) containing 2mM 2-mercaptoethanol. The solution was dialyzed against 6 liters of the same buffer. After the dialyzed protein was centrifuged, 91ml of the supernatant were applied to a column (3•~18cm) of DEAE-cellulose equilibrated with the buffer