The existence of two different forms of apo-electron-transferring flavoprotein

The association process of FAD and apo-electron-transferring flavoprotein (apoETF) from hog kidney was investigated. The reaction schemes which involve the association-dissociation of the protein species could be excluded by the light scattering data, which indicated that the molecular weights of apoETF and holoETF are identical. The binding reaction between FAD and a large excess of apoETF was monophasic and obeyed pseudo-first order kinetics. On the other hand, the reaction between apoETF and a large excess of FAD was biphasic: the fast phase obeyed a pseudo-first order reaction, and the rate of the slow phase was almost independent of FAD concentration. These results suggest the existence of two different forms of apoETF, as represented in the following reaction scheme: formula: see text] where "F" is FAD, "H" is holoETF, and "A" and "A" are the different forms of apoETF. The kinetic parameters were determined as k-1 = 3.9 x 10(4) M-1.s-1, k-1 approximately 10(-5) s-1, k+2 = 1.0 x 10(-3) s-1, and k-2 = 3.1 x 10(-3) s-1, in 50 mM potassium phosphate buffer, pH 7.6, containing 0.3 mM EDTA, and 5% v/v glycerol, at 7 degrees C. The elution patterns of apoETF on molecular sieve chromatography were very different from that of holoETF although the true molecular weights were identical. This result suggests that the structure of apoETF differs greatly from that of holoETF.