[48] Purification of coupling factor B

This chapter describes the coupling factor B activity as assayed by measuring the stimulation of anaerobic ATP-driven NAD reduction by succinate in ammonia ethylenediaminetetraacetic acid (AE) particles at 38°C. The particles exposed to ethylenediaminetetraacetic acid (EDTA) at pH 8.5–8.8 are selectively deficient in factor B, unlike particles depleted at higher pH. Factor B can be isolated equally satisfactorily from heavy, light, or mixed mitochondria. Isolation of mitochondria from beef heart tissue has been revised to give better yields and better coupled preparations. Respiratory control ratios approaching 3.0 are obtained using glutamate plus malate as substrate. The steps of isolation of factor B isolation are preparation of mitochondrial acetone powder, crude extract, diethylaminoethyl-cellulose (DEAE-C) chromatography, purification on CM-cellulose, and molecular sieving, on Sephadex G-75-40. Factor B purified also appears to be homogeneous, showing a single weakly stained band with Coomassie blue in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) corresponding to a molecular weight of approximately 13,000–15,000 daltons.