Isolation And Purification Of Urinary Trypsin Inhibitor And The Study Of Its Pharmacodynamics

Urinary Trypsin Inhibitor (UTI),a glycoprotein with 143 amino acid isextracted from human. Relative molecular weight is 67000 Da. UTI is amultipotent inhibitor of serine proteases, including trypsin ,hyaluronidase ,chymotrypsin and plasmin etc. UTI stabilized lysosomalmembrane and inhibited the release of lysosomal enzyme and inflammatorymediator. UTI also inhibited the production of myocardial depressantfactor(MDF) and eliminated the oxygen free radical. Moreover UTI improveda decrease of immunologic function and and nephric functions and thedisorder of proteinic metabolization caused by surgical stimulation. UTI alsoprevented the damage of internal organ and cell caused by surgical operationstimulation and improved the recurrent state of shock. Someone reported thatUTI inhibits tumor cell invasion and metastasis. As the result of above all ,UTIis often used in remedying acute pancreatitis,improving minicirculation,releasing a damage of organ caused by surgical stimulation and releasing renaltoxicity caused by tumor chemotherapy. UTI shows a stronger effect in organprotection. In recent years,people attach importance to the effect of UTI ontumor . 1985,UTI came into the market in Japan. 1999, Urinary TrypsinInhibitor injection of Tianpu Biologic Pharmaceutical Company came intomarket at home. UTI comes from human .Because of its lowhypersusceptibility, low side-effect and higher security , it is of far reachingimportance to study the production process , new form of prepared drugs andthe mechanism of action on tumor.We started studying with mankind urine. At first, adsorbing the interestedprotein with chitin , after eluting with 3N NH3,we gained eluent 30 liter.Salting out with 65% (NH4)2SO4, we gained the crude product of UTI. Thecrude product is ultrafiltrated and concentrated to get rid of small moleculesand pigment. After isolation and purification by Anion-exchangechromatography on DEAE-Sepharose, the total activity of UTI is 6.286×107Uand the specific activity is 1032U/mg protein. The volume of sample is 20 liter.Next, the sample is adsorbed by affinity Chromatography of trypsin-Sepharose 4B and eluted by 0.5M NaCl with pH 2.6 Glycine-HCl buffer.After affinity Chromatography, sample volume is 10 liter and the total activityis 4.815×107U. The specific activity is 2513U/mg protein. Concentrated to 500ml , the sample is isolated by Sephacry-200HR.At last 18.44g purified UTIis isolated and purified from 2000 liter mankind urine and the total activity is4.149×107U. The specific activity is 3026U/mg protein.After isolation and purification, we study the characterization of UTI.UTI is subjected to HPLC .The purity of HPLC is over 95% andmolecular weight determined . is 67KDa. Because of its lower pI, we choose abiochemical reaction method to determine its pI. The pI was 2.76. DilutingUTI to 1mg/ml and scanning by ultraviolet spectrophotometer , the resultshowed the maximum absorb wavelengh is at 278nm. UTI is a glycopeptidecarrying two glycosaminoglycan chains. Carbohydrate content test showed itconsists of 26.5% glycosaminoglycan contrasted with D-mannose. The datafrom amino acid composition analysis by enzyme disintegration isapproximate to documentary reported. The reason of this is that the molecularweight of UTI is big and the method itself isn't accurate enough. TheC-Terminal amino acid fragment is Leu analysed by enzyme disintegration.The N-terminal amino acid sequence wasA-V-L-P-Q-E-E-E-G-S-G-G-G-Q-L.We make an intensive study of pharmacodynamics of UTI .The first is theinhibition effect on trypsin in vitro. The second is the inhibition effect onα-chymotrypsin in vitro and the last is the effect of UTI in remedy rat acutepancreatitis caused by trypsin. In the first test ,the inhibition effect is comparedwith Miraclid and . UTI ,Miraclid and efficiently inhibits the effect of trypsinin a dose-dependant manner and the IC50 of the UTI, Miraclid and is5.11u/ml,4.47u/ml and 4.62 u/ml. In the second test, UTI ,Miraclid andaprotinin efficiently inhibits the effect of α-chymotrypsin in a dose-dependantmanner and the IC50 of the UTI, Miraclid and aprotinin is 9.54u/ml,9.74 u/mland 9.01 u/ml.In the third test ,we made a rat model of acute pancreatitis byinjecting trypsin in a contra-direction on pancreas. The rats were divided intofive groups and injected with different activity UTI , Miraclid of 5×104U/kgacted as masculine contrast and 0.9% NaCl injection acted as negativecontrast .The UTI was injected through caudal vein of the rat thirty minutesbefore trypsin was injected.By observing the survival time and livability of therats ,we demonstrated that UTI can remedy acute pancreatitis efficientlycaused by trypsin and prolong the survival time. The elongation index of UTIis 8.5%~42.6% and there is a obvious difference between varying activity ofUTI. The livability of UTI is 37.5~75% and there is a obvious differencebetween varying activity of UTI.We make an intensive study about the technique of isolation andpurification of UTI. The work above all provide us the basis for produce UTIin industrialized way.