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Chromatography of oxidized and reduced cytochrome c on carboxymethylcellulose
Insights
Chromatography effectively separated oxidized and reduced cytochrome c from horse heart. Higher pH values improved separation, with oxidized cytochrome c showing greater retardation due to molecular interactions.
Area of Science:
- Biochemistry
- Protein Chemistry
- Chromatography
Background:
- Cytochrome c is a crucial protein in cellular respiration, involved in electron transport.
- Understanding the behavior of oxidized and reduced cytochrome c is vital for biochemical studies.
Purpose of the Study:
- To isolate and characterize cytochrome c from horse heart using chromatographic techniques.
- To investigate the chromatographic behavior of oxidized and reduced cytochrome c on CM-cellulose.
- To determine the influence of pH on the separation of cytochrome c forms.
Main Methods:
- Isolation of cytochrome c from horse heart via a chromatographic method.
- Chromatography of oxidized and reduced cytochrome c on CM-cellulose.
- Utilized buffer systems at pH 8.4, 6.75, and 4.9 to assess separation efficiency.
Main Results:
- Improved separation of oxidized and reduced cytochrome c was observed at higher pH values.
- Oxidized cytochrome c exhibited more than twice the retardation of reduced cytochrome c, despite a single charge difference.
- Peak distortion at high protein loads (above 20µM) suggests self-competition among cytochrome molecules.
Conclusions:
- CM-cellulose chromatography is effective for separating oxidized and reduced cytochrome c.
- pH significantly impacts the chromatographic separation of cytochrome c, with higher pH favoring better resolution.
- Molecular self-competition plays a role in chromatographic peak shape for cytochrome c at elevated concentrations.
Abstract:
1. Cytochrome c was isolated from horse heart by a chromatographic method. 2. Oxidized and reduced cytochrome c were chromatographed on CM-cellulose that was in equilibrium with several buffer systems of constant composition at pH values of 8.4, 6.75 and 4.9. 3. Separation was better at the higher pH values; the oxidized form was retarded more than twice as much as the reduced form, though they differed by only a single charge. 4. Self-competition between cytochrome molecules is suggested to account for the peak distortion observed at high loads (above 20mum protein concentration).