Characterization of a trypsin inhibitor from equine urine.

This research paper explores the characterization of a trypsin inhibitor identified in the urine of pregnant horses. The inhibitor, which is a kind of glycoprotein, was isolated using various methods, and its properties were identified.

Research Methodology

• The trypsin inhibitor was first isolated from the urine of pregnant mares. Adsorption on bentonite and elution with aqueous pyridine was used to achieve this.
• Thereafter, the substance went through batch processing with DEAE-Cellulose. This improved the quality of the extracted inhibitor by removing unnecessary elements and contaminants.
• The sample isolation process was concluded by using column chromatography, which separated the compound further based on its chemical properties.
• The final purification step to an electrophoretically homogenous glycoprotein was performed using gel permeation chromatography. This ensured that only the trypsin inhibitor was present in the sample, offering a clear sample for examination.

Characteristics of the Trypsin Inhibitor

• The equine urinary trypsin inhibitor (E-UTI) was found to be acid- and heat-stable. This means that it retains its structure and function regardless of the pH level or temperature to which it’s exposed.
• The molecular weight of the E-UTI was determined to be between 22 to 23 kDa.
• The isoelectric point of the E-UTI was found at 4.55. This is the pH at which the molecule is electrically neutral.
• The researchers found that E-UTI forms a 1:1 molar complex with trypsin, indicating that the inhibitor binds to trypsin in a 1-to-1 ratio.
• Serine was identified as the N-terminal amino acid of the E-UTI. N-terminal refers to the end of a protein or polypeptide terminated by an amino acid with a free amine group (-NH2).

Comparison with Other Trypsin Inhibitors

• The N-terminal amino acid sequence of the E-UTI was found to be almost identical to that of EI-14, a trypsin inhibitor obtained from horse serum.
• However, the E-UTI has two additional amino acid residues, Ser-Lys- on its N-terminal end, compared to the EI-14.
• In addition, the researchers noted that the E-UTI had a different isoelectric point from EI-14 and an inhibitor obtained from human urine.