Table 2.
Reactivity of different SH-reagents towards A/D transition-sensitive SH-group of Complex I in SMP (25 °C)
| SH-reagent | Pseudo-first order rate constant (M−1·min−1·104) |
Relative reactivity (ki/kDTT) | |
|---|---|---|---|
| Inhibition of Complex I (ki) | Reaction with DTTa (kDTT) | ||
| DTNB (standard buffer)b | 0.30 | 6.0 | 0.05 |
| +KCl (100 mM)c | 0.70 | 7.0 | 0.10 |
| +SDS (200 μM) | 0.17 | 6.0 | 0.03 |
| DTNP (standard buffer)b | 50 | 69 | 0.72 |
| +KCl (100 mM)c | 100 | 80 | 1.25 |
| +SDS (200 μM) | 25 | 69 | 0.36 |
| Oxidized glutathione (10 mM, 75 min, pH 8.0)b | no inhibition | not determined | |
a
The reaction was carried out with 1 μM DTT and 10 μM DTNB or DTNP in the standard buffer (see Fig. 2) at pH 7.0. At this pH the reaction proceeded slow enough for reliable rate constant determination.
b
The rate constants were determined as described in Fig. 2. Concentrations of DTNB and DTNP were 200 μM and 1 μM and protein content in the samples was 1 mg/ml and 10 μg/ml, respectively.
c
Other salts (NaCl, RbCl, Sodium acetate) showed almost the same effect on the rate constants.