FIGURE 8.

Binding of 3HB to the reduced enzyme. Experiments were performed using the double-mixing mode of the stopped-flow spectrophotometer. The reactions were performed in 100 mm Tris-H₂SO₄ (pH 8.0) at 4 °C. A, in the first mixing, a solution of the reduced enzyme at 127 μm (concentration before mixing) was mixed with buffer containing various 3HB concentrations, 0.1, 0.2, 0.4, 0.8, 2, 8, and 20 mm (concentrations before mixing) under anaerobic conditions. The mixture of reduced enzyme and 3HB was incubated for 50 s before it was mixed with buffers containing 0.96 mm oxygen and various 3HB concentrations of 0.05, 0.1, 0.2, 0.4, 1, 4, and 10 mm. All concentrations were indicated according to before mixing conditions and the traces of low to high 3HB concentrations correspond to lower to upper traces (indicated by the arrow). The reactions were monitored at 400 nm to detect formation of C4a-hydroperoxyflavin. The increase of amplitude at 400 nm due to formation of C4a-hydroperoxyflavin represents the amount of the reduced enzyme-3HB complex. The dotted line trace is the control reaction without 3HB. Inset in A shows a plot of the absorbance change at 0.003 s versus the concentration of 3HB. B, a solution of the reduced enzyme, 120 μm (concentration before mixing), was mixed with 20 mm 3HB in the first mixing under anaerobic conditions. The reaction was incubated at various age times of 0.05, 0.1, 0.2, 0.5, 1, 5, 10, and 20 s (indicated by arrow) before the mixture was mixed with buffer containing 1.92 mm oxygen and 10 mm 3HB (concentration as before mixing) in the second mixture. The dotted line trace was a control reaction without 3HB. Inset in B shows a plot of the absorbance change at the time of 0.003 s versus age time.