Isolation, characterization, and quantitative analysis of C-reactive protein from horses.

This research article explains the isolation and analysis of C-reactive protein (CRP), a protein found in the blood that increases its level in response to inflammation, from horses, shedding light on its composition, structure, and potential role as an acute-phase reactant protein in these animals.

Method of CRP Isolation

• The researchers used a process known as calcium-dependent affinity chromatography to isolate CRP from equine serum. This involved a conjugated pneumococcal C-polysaccharide, which helps to isolate the C-reactive protein from the rest of the serum.
• Furthermore, anion exchange chromatography and gel filtration were utilised to further purify the isolated CRP.

Identification of Equine CRP

• The isolated CRP was verified to be the genuine article through its immunochemical cross-reactivity with anti-human CRP. This implies that the horse CRP had an immune system response that corresponded with the human CRP, confirming the validity of the extracted protein.
• The amino acid composition and pentameric structure of the equine CRP were found to be homologous to those of human CRP; this identification was achieved via electron microscopy. A pentameric structure means the protein is formed by five identical subunits.

Characteristics of Equine CRP

• The molecular weight of the purified equine CRP was found to be approximately 118,000.
• The protein comprised five identical subunits, each with a molecular weight of approximately 23,000. These subunits were non-glycosylated (not attached to a carbohydrate) and non-covalently associated (not bonding with the sharing of electron pairs).
• In immunoelectrophoresis, the protein migrated between beta- and gamma-globulin, two types of proteins typically present in the blood plasma.
• The isoelectric point, or the pH at which the protein is electrically neutral, of the equine CRP was approximately 7.0.

Role of CRP in Horses

• The study found that an increased concentration of CRP was associated with clinical conditions such as pneumonitis (lung inflammation), enteritis (intestinal inflammation), and arthritis in horses. The increase was benchmarked against values obtained from clinically normal horses using a method called single radial immunodiffusion.
• Experiments involving intramuscular administration of turpentine oil (which is known to cause inflammation) or castration resulted in the serum CRP concentration increasing to six times more than the normal range, indicating the presence of an inflammatory response triggered by these procedures.
• The results from these experiments suggest that CRP may serve as an acute-phase reactant protein in horses. Acute-phase proteins are a class of proteins whose plasma concentrations increase or decrease in response to inflammation.