Purification and characterization of equine complement factor C3.

The research paper discusses a new method to purify equine complement factor C3 (C3), a protein from horse blood plasma. The study also details the biochemical composition and comparison with human complement factor C3.

Method of Purifying Equine C3

• The researchers found a rapid method that allows for the purification of milligram quantities of pure C3 from equine plasma.
• This protein is extracted by selectively precipitating with polyethylene glycol, a water-soluble polymer, which means it can mix well with the plasma and allow for the C3 protein to be isolated.
• Further purification was done by anionic and cationic exchange High-performance liquid chromatography (HPLC), a method used to separate and identify the components of the mixture. This process led to a 12% yield of C3 from the plasma.

Composition of Purified C3

• The researchers examined and characterized the purified C3, finding that it is composed of an alpha chain (118 kD) and a beta chain (68 kD).
• These chains are linked by at least one disulfide bond, which contributes to the protein’s structure and stability.
• The protein has an isoelectric point of 4.7, a scale that measures the pH at which a particular molecule or surface carries no net electrical charge.

Comparison with Human C3

• The amino acid analysis suggested a strong conservation of amino acid usage between equine and human C3, which means the composition of these proteins in both species is very similar.
• The N-terminal sequences, the start of the amino acid chain in the alpha and beta chains of equine C3, were found to be homologous to human, mouse, and rat C3, implying a shared evolutionary history.
• When the researchers triggered the activation of C3, it produced breakdown products similar in molecular weight to C3b and iC3b of other species. C3b and iC3b are two fragments produced when C3 is activated and cleaved. Their presence is an indication of the protein’s functional integrity and normal activity.

Functional Dependency of Equine C3

• It was observed that the function of equine C3 seemed to be reliant on a reactive thiolester bond.
• When fresh equine serum was treated with methylamine, its hemolytic activity was stopped, implying that the serum’s ability to lyse or break down cells was impaired. This chemical basically prevents C3 from functioning as it normally would, reaffirming the importance of the thiolester site for the protein’s activity.