FIGURE 4.

SDS-PAGE and Western blotting of sulfo-SBED cross-linked MDH and bc₁. A, 10 μg of MDH in 100 μl of 50 mm Na⁺/K⁺ phosphate buffer, pH 7.4, was incubated with sulfo-SBED for 2 h on ice in the dark then dialyzed against 500 ml of a 50 mm Na⁺/K⁺ phosphate buffer, pH 7.4, in the dark to remove any unreacted sulfo-SBED. The chemically labeled MDH was then incubated without and with 30 μg of bc₁ in 200 μl of a 50 mm Na⁺/K⁺ phosphate buffer, pH 7.4, on ice for 1 h. After incubation, the mixtures were photolyzed with a long wavelength UV light (365 nm) at a distance of 5 cm for 15 min. The photolyzed samples were treated with β-mercaptoethanol and subjected to SDS-PAGE and Western blotting. The biotin tags were probed with an avidin-horseradish peroxidase conjugate. B, the interaction partners for MDH were shown in the three-dimensional structure of the bc₁ complex. Protein Data Bank code 1NTM. IMS, intermembrane space; MIM, mitochondrial inner membrane.