TABLE 4.
Effect of eNOSr to cytochrome c ratio on the percentage of fast and slow phase in the reaction The ratio of fully reduced eNOSr versus cytochrome c was varied to 4, 8, and 12 in the absence of NADPH and CaM. A known concentration of fully reduced eNOSr was mixed with a fixed concentration of cytochrome c (2.5 μm) in a stopped-flow instrument under anaerobic conditions at 10 °C. The observed rates and percentages of absorbance change of the fast and slow phase at 550 nm are reported as the mean ± S.D. of 5–6 single mixing experiments and are representative of at least two different enzyme preparations. The relative percentages of absorption change with respect to the full reduction of 1.25 μm cytochrome c are given together with the observed rates, k1 and k2 (s–1) in parentheses. The total absorbance change, ΔA, was measured after 0.5 s.
| Ratio eNOSr/cyt c | % A (k1) | % A (k2) | ΔA (mA)a |
|---|---|---|---|
| 4 | 48 ± 3 (38 ± 2) | 52 ± 3 (3.5 ± 0.2) | 19.3 (77%) |
| 8 | 64 ± 5 (55 ± 3) | 36 ± 4 (9.4 ± 0.5) | 23.1 (93%) |
| 12 | 82 ± 2 (57 ± 1) | 18 ± 2 (3.2 ± 0.2) | 24.4 (98%) |
The expected absorbance (A) chance for 1.25 μm cytochrome c is 25 mA (Δε550 = 21 mm–1 cm–1)