A method for processing 3 litres of human plasma for the purification of phosphatidylcholine-cholesterol acyltransferase is described. The method involves (NH4)2SO4 fractionation, citric acid treatment, and DEAE-cellulose and hydroxyapatite chromatography. At this stage the enzyme preparation is purified approx. 8000-fold. This preparation appears to be free of lipoproteins as determined by immunoelectrophoresis against anti-human serum and is minimally contaminated with albumin (less than 30 mug/mg of enzyme protein) as determined by immunodiffusion. The activity of the enzyme was stable for 4 days, but most of its activity was lost after 20 days On electrophoresis on 5% polyacrylamide gel, a fast-moving band with enzyme activity and a slow-moving band with no enzyme activity was observed. A faint band of albumin was also present. Extracts of enzymically active bands cut from ten gels and then pooled and extracted with 0.15 M-NaC1/4mM-sodium phosphate, pH 7.0, showed a single band on re-electrophoresis on 5% polyacrylamide gel.
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Research Article| June 01 1976
A method for the purification of milligram quantities of stable human phosphatidylcholine-cholesterol acyltransferase
K G Varma ;
Biochem J (1976) 155 (3): 583–588.
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K G Varma, L A Soloff; A method for the purification of milligram quantities of stable human phosphatidylcholine-cholesterol acyltransferase. Biochem J 1 June 1976; 155 (3): 583–588. doi: https://doi.org/10.1042/bj1550583
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