Carboxylesterases (EC 3.1.1). Purification and titration of chicken, sheep, and horse liver carboxylesterases.

The research article is about the purification and analysis of carboxylesterase enzymes from chicken, sheep, and horse liver, by multiple biochemical procedures.

Purification Process

• To begin with, the researchers extracted liver carboxylesterases from chicken, sheep, and horse. They achieved this by carrying out ammonium sulfate fractionation, a technique that allows soluble proteins to precipitate and be separated from a solution based on their unique solubility characteristics.
• This is followed by ion-exchange chromatography, which separates the components based on the electrostatic interaction between the ion-exchange resin and the proteins. The difference in these interactions allows for separate elution of the components from the column.
• They also used gel filtration, a type of size exclusion chromatography, performed on a packing material called Sephadex. This experimental method assists in the separation of proteins based on their size.

Yields and Composition Checks

• The yields from these purification procedures were recorded as 1g of purified enzyme from 2kg of chicken liver powder, 200mg from 400g of powder in sheep, and 230mg from 800g of powder in horse liver.
• They also verified the purity of the enzyme preparations by performing gel electrophoresis. This tool allows them to visualize the proteins in the preparation, and they confirmed that no non-carboxyl-esterase proteins were present, indicating purity. However, this test also revealed that there were electrophoretic variants, i.e. different forms of the same protein, present in their samples.

Titration and Equivalent Weight

• The researchers performed titration, a procedure to determine the concentration (or in this case, equivalent weight) of the enzyme, with different reagents. The chicken enzyme was titrated using p-nitrophenyl diethyl phosphate or bis(p-nitrophenyl) phosphate, which resulted in an equivalent weight of 67,000.
• Similarly, the sheep and horse enzymes were titrated using p-nitrophenyl dimethylcarbamate, which resulted in an equivalent weight close to 69,500 and 70,000, respectively. These equivalent weights provide insight into the molecular size and, indirectly, the complexity, of the enzyme.