Removal of bound Triton X-100 from purified bovine heart cytochrome bc1

Abstract

Cytochrome bc(1) isolated from Triton X-100-solubilized mitochondrial membranes contains up to 120 nmol of Triton X-100 bound per nanomole of the enzyme. Purified cytochrome bc(1) is fully active; however, protein-bound Triton X-100 significantly interferes with structural studies of the enzyme. Removal of Triton X-100 bound to bovine cytochrome bc(1) was accomplished by incubation with Bio-Beads SM-2 in the presence of sodium cholate. Sodium cholate is critical because it does not interfere with the adsorption of protein on the hydrophobic surface of the beads. The resulting Triton X-100-free cytochrome bc(1) retained nearly full activity, absorption spectra, subunit, and phospholipid composition.

Figure 1. Silicic acid HPLC of phospholipids extracted from purified bovine heart cytochrome bc₁

Upper main panel: Separation of phospholipid-TX-100 mixture extracted from 1 nmol of cytochrome bc₁ before treatment with Bio-Beads. Inset: Separation of TX-100 (230 nmol) polyoxyethylene chains by silicic acid HPLC column. Lower panel: HPLC analysis of phospholipids extracted from Bio-Beads treated cytochrome bc₁. Phospholipids were extracted from 1 nmol of Bio-Beads treated cytochrome bc₁. Inset: Separation of mixed phospholipid standards (5 nmol of cardiolipin (CL), 5 nmol of phosphatidylethanolamine (PE), and 5 nmol of phosphatidylcholine (PC)). Detection of phospholipids elution was monitored at 203 nm with a Waters 2487 Dual λ Absorbance Detector.

Figure 2. C₁₈ reversed-phase HPLC purification of bovine heart cytochrome bc₁ subunits

Upper chromatogram: RP-HPLC purification of cytochrome bc₁ subunits before treatment with Bio-Beads. Subunits of cytochrome bc₁ (0.5 nmol) were separated on 10-μm, 300-Å pore size Vydac C₁₈ column (0.46 × 25 cm). The sample preparation and water/acetonitrile gradient were as described previously [9] with minor modifications. Quantification of cytochrome bc₁ bound TX-100 was based upon integration of C₁₈ RP-HPLC peak area since TX-100 elutes as a well-defined single peak at ~44 min during C₁₈ RP-HPLC. Lower chromatogram: RP-HPLC purification of cytochrome bc₁ subunits after treatment with Bio-Beads. Subunits of Cyt bc₁ (0.5 nmol) were purified by RP-HPLC after cytochrome bc₁ was incubated with Bio-Beads as described in the text.