Fig. (1A) Analysis of the recombinant bL12 protein on a Superdex 75 column. (A) The column was equilibrated in 50 mM sodium phosphate buffer (pH 7.5) containing 150 mM NaCl. The flow rate was 0.4 ml/min. Elution was monitored by the absorbance at 280 nm (upper peak) and 260 nm (lower peak). The column was calibrated with protein markers of molecular weights 13, 28, 43, 75 and 158 kDa. Analysis on the column of rp (L7/L12)2 with two free NH2-terminus L12 chains and two NH2-terminally acetylated L7 chains, as well as of the fully acetylated (L7)4 form gave rise to superimposable elution profiles. Fractions of 0.4 ml were collected. (B) Analysis in 10% SDS-PAGE of the fractions eluted from the Superdex 75 column. Fractions 15-20 named L12 represent the rp (L7/L12)2 with two free NH2-terminus L12 chains and two NH2-terminally acetylated L7 chains, while fractions 15’-20’ named L7 represent the fully acetylated (L7)4 form. The latter rp (L7)4 was obtained by incubating the (L7/L12)2 form with two free NH2-terminus L12 chains and two NH2-terminally acetylated L7 chains in the presence of purified RIML acetyl-transferase and of N-acetyl coenzyme A (N-Ac-CoA). For details, see Materials and Methods. The SDS-PAGE gels were colored with coomassie blue.