Purification of horse (Equus caballus) serum lecithin:cholesterol acyltransferase.

This research has established a method for purifying the enzyme lecithin:cholesterol acyltransferase from horse serum. This approach implements adsorption, fractionation, 1-butanol treatment, and several chromatographic techniques, leading to a 20,000-fold purified enzyme with 7% yield.

Methodology

• The researchers began this study attempting to find a method to purify a particular enzyme, lecithin:cholesterol acyltransferase, from horse serum (blood fluid).
• The team used different strategies and techniques in their purification method:
  • Adsorption of the enzyme from the diluted horse serum on DEAE-Sephadex A-50, a type of chromatography resin or medium used to separate molecules in a solution.
  • Fractionation with (NH4)2SO4, or ammonium sulfate, a common technique used to selectively precipitate proteins.
  • Treatment with 1-butanol, an organic compound often used to separate and purify proteins.
  • Chromatographic techniques using various mediums including DEAE-Sepharose CL-6B, DEAE-Sephadex A-50, Affi-Gel blue, and hydroxylapatite. Chromatography is a technique used to separate components of a mixture.

Purification results and Enzyme Analysis

• After carrying all the processes, the resulting enzyme preparation essentially formed one main band when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.
  • This indicates that the enzyme was successfully separated from other proteins and impurities.
  • Polyacrylamide gel electrophoresis (PAGE) is a common method used to analyze proteins and their purity.
• The final purification of the enzyme was 20,000-fold with a yield of 7%, reflecting its substantial purification but low overall recovery.
• The apparent molecular weight of the enzyme was found to be 64,000. Knowing the molecular weight helps in identifying and analyzing the protein.
• The activity of the enzyme was determined to remain stable for three days at freezing point (0 degrees Celsius), illustrating its stability under cold conditions.