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Removal of percoll from microsomal vesicles by gel filtration on sephacryl-S-1000 superfine
Abstract:
A microsomal vesicle fraction was prepared from rat liver homogenate by centrifugation in gradients of Percoll. The microsomes were subjected to gel filtration on Sephacryl S-1000 Superfine, which resolved the microsomes from Percoll. The elution pattern of the microsomal marker enzyme NADPH-cytochrome c reductase showed that the main part of the enzyme was present in a peak at Kav about 0.1, while Percoll eluted in a broad peak at Kav about 0.7. The total yield of eluted enzyme activity was 85%. The gel filtration had to be carried out in the presence of 10 mM tris or NaCl. At lower ionic strength or in 0.25 M sucrose alone, anomalous behaviour of the Percoll particles and microsomes on the gel was observed. Electron microscopy of samples from the void volume fraction of the Sephacryl S-1000 Superfine column showed an almost complete removal of Percoll from the microsomes. Furthermore, the vesicle preparation was essentially free of membrane fragments.
Insights
This study developed a method to purify rat liver microsomes from Percoll using Sephacryl S-1000 Superfine gel filtration. The optimized technique effectively removes Percoll, yielding highly pure microsomal vesicles for biochemical analysis.
Area of Science:
- Biochemistry
- Cell Biology
- Analytical Chemistry
Background:
- Microsomal fractions are crucial for studying liver metabolism and drug detoxification.
- Contamination with Percoll, a common density gradient medium, can interfere with biochemical assays.
- Effective purification of microsomes is essential for accurate experimental results.
Purpose of the Study:
- To develop and validate a method for removing Percoll from rat liver microsomal vesicle fractions.
- To optimize gel filtration chromatography for microsomal purification.
- To ensure the integrity and purity of isolated microsomes for further research.
Main Methods:
- Preparation of rat liver microsomal vesicle fraction via Percoll density gradient centrifugation.
- Gel filtration chromatography using Sephacryl S-1000 Superfine to separate microsomes from Percoll.
- Analysis of enzyme activity (NADPH-cytochrome c reductase) and electron microscopy for purity assessment.
Main Results:
- Sephacryl S-1000 Superfine chromatography successfully resolved microsomes (Kav ~0.1) from Percoll (Kav ~0.7).
- The purification method achieved an 85% yield of eluted enzyme activity.
- Electron microscopy confirmed near-complete removal of Percoll and absence of membrane fragments in the purified fraction.
- Optimal purification required the presence of 10 mM Tris or NaCl.
Conclusions:
- Gel filtration on Sephacryl S-1000 Superfine is an effective method for purifying rat liver microsomes from Percoll.
- The developed technique yields a highly pure microsomal vesicle preparation, free from Percoll and membrane debris.
- This purification strategy enhances the reliability of biochemical studies utilizing microsomal fractions.