Isolation, Purification And Sequence Of A Novel Bioactive Peptide From Canine Intestine

The gastrointestinal tract has been recognized as the largest bodily endocrine organ. Due to investigating of the gastrointestinal hormones, a new science, gastrointestinal endocrinology, came in being. As the development of protein chemistry, A strenuous and extensive study has been made in this area of research. Most of intestinal hormones are thermo-stable peptides, and the molecular weights rang between 2kD-5kD.Since secretin was discovered by bayliss in 1902, many bioactive peptide have been isolated from the gastrointestinal tract and characterized chemically and biologically. In mammals, so far, the following groups of peptides have been isolated from, or associated with the gut system: the secretin/glucagon family(the biggest group); the cholecystokinin/gastrin group; the pancreatic polypeptide group; the tachykinins; the somatostatin/cortistatin group; the opioids, and the gastrin-releasing peptide/bombesin group; the trefoil peptides existing in the gut and the pancreas; and the anti-bacterial peptides of different families. There are also many peptides that appear to lack significant sequence similarity to previously known. Proteins (peptides) play key roles in virtually all biological processes by sophisticated regulatory system, have related with disease. The purification of a protein (or peptide) is a first and essential step in the study of its physical and chemical properties, or in the elucidation of its structure and function(s). In this thesis, A novel bioactive peptide was purified from the dog small intestine and part of its amino acid sequence has been analyzed. With the boiling of the intestine tissue, acetic acid extraction, alginic acid adsorption, hydrochloric acid elution, sodium chloride salting out and the ethanol precipitation, followed by Sephadex G-25 (fine) column chromatography and a two-step reverse-phase HPLC purification, a thermo-stable peptide has been purified and its molecular size has been determined about 4KD by using the Tris-Tricine-SDS-PAGE. After the digestion of the peptide with trypsin and the separation of the trypsin-peptide, one of the fragments subjected to sequence with CapLC-ESI-MS-MS ( Q-TOF2 ) and a clear amino acid sequence was obtained as following :T-E-Y-T-A-L-N-V-L-A-T-T-E-E-N-G. By searching the sequence from Protein Bank, it has been found that this peptide is a novel peptide. We have found that the peptide can enhance current of beta cell by patch clamp. The physiology function of the peptide deserves to be further investigated.