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Improved method for the purification of biologically active transcobalamin II
Preparative Biochemistry
|January 1, 1976
Summary
Researchers purified Transcobalamin II (TC II), a protein crucial for vitamin B12 (B12) transport. The simplified purification method yielded biologically active TC II, essential for B12 cellular uptake.
Area of Science:
- Biochemistry
- Cell Biology
Background:
- Transcobalamin II (TC II) is vital for vitamin B12 (B12) absorption and transport.
- Efficient purification of TC II is necessary for studying its biological functions.
Purpose of the Study:
- To develop a simplified and efficient method for purifying Transcobalamin II (TC II).
- To confirm the biological activity of the purified TC II in facilitating vitamin B12 (B12) cellular uptake.
Main Methods:
- Modified Allen and Majerus procedure for Transcobalamin II (TC II) purification.
- Incorporation of isoelectric precipitation to streamline the purification process.
- Assessment of vitamin B12 (B12) binding capacity and A280/A361 ratio.
Main Results:
- Achieved a ~300,000-fold purification of TC II from Cohn fraction III.
- The simplified method eliminated two chromatographic steps.
- Purified TC II exhibited high vitamin B12 (B12) binding (26.7 μg/mg protein) and biological activity in facilitating B12 uptake in HeLa cells.
Conclusions:
- A modified, simplified purification protocol for Transcobalamin II (TC II) was successfully established.
- The purified TC II retains biological activity, confirming its role in vitamin B12 (B12) cellular transport.