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Integral cytochrome-c oxidase. Preparation and progress towards a three-dimensional crystallization
1Institut für Biochemie, Rheinisch-Westfälische Technische Hochschule, Aachen, Germany.
European Journal of Biochemistry
|January 15, 1995
Summary
A new, rapid method prepares highly active, monodispersed bovine heart cytochrome-c oxidase. This efficient process yields pure enzyme suitable for structural studies, preserving its native activity and lipid environment.
Area of Science:
- Biochemistry
- Enzymology
- Mitochondrial Respiration
Background:
- Cytochrome-c oxidase is a key enzyme in cellular respiration.
- Previous preparations lacked high activity or native lipid environment.
Purpose of the Study:
- To develop a rapid and efficient method for purifying active cytochrome-c oxidase.
- To characterize the purified enzyme's structural and functional properties.
Main Methods:
- Selective solubilization using non-ionic detergents (Triton X-100 or lauryl beta-D-maltoside).
- Anion-exchange chromatography for enzyme purification.
- Spectroscopic (EPR) and biochemical assays for characterization.
Main Results:
- A two-day preparation yielding 60% of mitochondrial oxidase.
- High specific activity (turnover number ~600 s-1) and correct heme alpha/protein ratio.
- Stable dimeric form (~500 kDa) with a native lipid shell.
Conclusions:
- The new procedure yields highly active, monodispersed cytochrome-c oxidase.
- The enzyme retains its native lipid environment and structural integrity.
- The preparation is suitable for further structural investigations, including X-ray crystallography.