Isolation, Purification Of Heparin And Analysis Of Oligoheparins Degraded By Nitrous Acid

Heparin, a polydisperse, highly sulfated, linear polysaccharide, is comprised of repeating1â†'4linked uronic acid and glucosamine, and rich in mast cells, such as porcine intestinal mucosal and bovine lung. Heparin acts as the most commonly used clinical anticoagulant and antithrombotic agent. Nowadays, China is the largest heparin exporter in the world, but most of them were raw materais with low specific activity, known as USP units per milligram. So, it is of great significance in extensive procession and purification of heparin. In addition to its anticoagulant activity, heparin and oligoheparins present anti-inflammatory and anticancer activity. In this study, the raw heparin, isolated from porcine intestinal mucosal, was processed with various purifying procedure, the results were compared and analysed to seek the desired method. Nitrous acid depolymerization was employed to study controlled depolymerization and prepare oligoheparins, which lay the foundation of analysis of heparin sequence and study of structure and function. All results were given as follows:1.Physicochemical properties of purified heparin from porcine intestinal musical: High-performance gel-permeation chromatography (HPGPC) analysis showed that the heparin molecular mass around23KDa, pre-column PMP derivatization method assay showed that the heparin was composed of GalA, IdoUA and GlcN, Phenol-sulphuric acid analysis indicated that the total sugar content of heparin was63.2%, the sulfate group content of heparin used Barium sulfate turbindimetry was31.0%, the contention of protein in the sample used Bradford analysis was0.5%, the specific activiy of the purified heparin were more than160U.2. Comparation of various purifying process, such as acid-base oxidation, ion exchange chromatography, gel filtration,(3-elimination combined ion exchange chromatography and gel filtration, the (3-elimination combined ion exchange chromatography gives the best results, with higher potency and recovery. 3. The monosaccharide composition analysis-identifying contamination in heparin was established. PMP pre-column derivatization high performance liquid chromatography was established for analysis of heparin. The sample was hydrolysised in8mol/L hydrochloric acid at100℃for30min, and then derivatized with PMP. The derivatization was applied on a reverse phase C18column under the condition of78%ammonium acrtate-22%acetonitrile in isocratic mode. This optium condition was guaranteed by the rate of1.OmL/min at25℃. The HPLC results presented us a well separation profle. This method is an alternative for the determinantion of contamination in heparin with high accuracy and sensitivity, based on differences in monosaccharide composition, such as oversulfated chondroin sulfate.4. The LC-ESI-MS analysis of heparin oligosaccharides derivatized with AEC was built.Purified heparin,100mg/ml in50mmol/L sodium nitrite,was acidified to pH1.5with6mol/L HC1and maintained at this pH for10min at room temperature. The heparin oligosaccharides were derivatied with3-amino-9-ethylcarbazole(AEC),and then applied on HPLC coupling with electrospray ionization ion-trap mass spectrometry.The derivatation was separated and characterized on on a reverse phase C18column in a linear gradient fashion, under the mobile phase of ammonium acrtate-acetonitrile elution system.Various sulfated oligosaccharides derivatized with AEC can be separated and determined using this method,overcoming the incompatibility of the strong polarity sulfated rentained on reverse column. The LC-ESI-MS analysis of heparin oligosaccharides provide a new method for controlled deploymerization and oligoheparin preparation.5. Multi-stage mass spectrometry (MSn) analysis was employed to characterize primary structure of oligoheparins derivatized with AEC. Multi-stage mass spectrometry (MSn) was acquired to confirm the structure of heparin oligosaccharides. The MS" fragments of oligoheparins confirm the modification of sulfate group of heparin,such as3-O and6-O and N-sulfation of glucosamine, and also the sulfation of uronic acid. The MSn analysis could provide a reference for the study of structure and sequence of oligoheparins.