Figure 7.

Anaerobic size exclusion chromatography of Av1 on a 1 cm × 30 cm column of Superdex S-200. The flow rate was 0.5 mL/min at 22 °C. (A) Reaction mixtures (1.0 mL) containing 1.0 mg of Av1, with or without Av2 at a CR of 3.4 and with or without the ATP regenerating system, were incubated at pH 9.5 in tribuffer and 50 mM NaCl at 30 °C for 3.5 h; 0.8 mL of the reaction mixture was injected from the reaction mixture onto the column that was equilibrated with 50 mM Tris buffer (pH 7.5) containing 200 mM NaCl and 5 mM sodium dithionite: elution line 1 (red), Av1 and Av2 incubated without the ATP regenerating system; elution line 2 (blue), Av1 alone; elution line 3 (black), complete turnover conditions, Av1 and Av2 with the ATP regenerating system. Elution was monitored at 410 nm. (B) Same column as in panel A equilibrated with 50 mM MOPS buffer (pH 7.3) containing 100 mM NaCl, 1 mM MgCl₂, 5 mM NaF, 0.25 mM AlCl₃, and 5 mM sodium dithionite. Av1-mod was prepared as described for line 3 of panel A and re-isolated in MOPS buffer (pH 7.3) without AlCl₃ or NaF. Av1-mod (∼0.2 mg) was incubated with Av2 (CR = 4.6) in 2.4 mL of MOPS buffer containing 5 mM NaF, 0.25 mM AlCl₃, and either no nucleotide [elution line 1 (black)], 1 mM ATP regenerating system [elution line 2 (purple)], or 1 mM ADP [elution line 3 (green)]. After incubation at 25 °C for 30 min to form the complex, 0.9 mL of each reaction mixture was applied to the column. The absorption of the eluate was monitored at 390 nm.