Purification And Partial Characteristics Of Pig Aprotinin

Aprotinin was a kind of non-special,multifunctional proteinase inhibitor, wildlyapplied to clinic. The method of extracting aprotinin at present was "two stepsmethod", which was contaminative,high-cost,long-period. Then, the crosslinkedchitosan microsphere was used to immobilize trypsin to preparation the affinitychromatogram sorbent, which was applied to purify aprotinin. The present material ofpreparing aprotinin was cattle lung,human urine,trypsin crystal solution. But it's noreport that extracting aprotinin from pig lung. so we try to prepare aprotinin from piglung. The results show:1. The symmetric crosslinked chitosan microsphere was prepared throughinverse suspersion crosslinking, which was used to immobilize trypsin. Theoptimization condition of immobilizing trypsin: The crosslinked chitosanmicrosphere react with glutaraldehyde whose final concentration is 0.6% in 50℃forsix hours, after then adding trypsin 12mg/g, reacting 24h at 0～5℃. The optimumtemperature and pH of immobilized trypsin is 80℃and 7 separately. The apparentMichcalis Constant of immobilized trypsin K_m is 3.16mmol/L. Compared with thechitosan, the crosslinked chitosan microsphere hold the advantage of good strengthsymmetrical-granularity,strong-acid-resistance,less no-special absorption of BSA.2. The new immobilized trypsin was used to purify cattle lung aprotinin, and theactivity of aprotinin reach 4308U/mg,the purification multiple reach 350. The trypsinimmobilized by Sepharose 4B is also used to purify cattle lung aprotinin, and theactivity of aprotinin reach 4593U/mg,the purification multiple reach 373. The newimmobilized trypsin's extraction efficiency is little lower than Sepharose 4B. Butcomparing to Sepharose 4B, the new immobilized trypsin have the advantage of highspeed eluting,good strength and can be used for several times.3. The result of purifying pig lung solution by affinity chromatogram show that thecollection solution eluting by 0.5 mol/L, pH 1.5NaCl—HCl has inhibition activity. itshows that the pig lung contain trypsin inhibitor. The optimal condition of purifying pig aprotinin by affinity chromatogram: the activity of immobilized trypsin is1526.7U/g., static absoption.4. The pig aprotinin is a kind of single purified protein according to electrophoresisexperiment. The activity is invariable when it was treated at the pH from 1to 11 for12h. But the activity start to drop when pH reach 12, and the activity is 89.7%compared to the activity at the pH8.0 when pH reach 13. The effection of different pHon pig aprotinin is reversible. The activity is invariable when it was treated at thetemperature from 40 to 90 for 15min. when it was treated at 100℃for 15min, therelative activity was 96% compared to it was at 30℃. The pig aprotinin containsaromatic amino acid residues and had special absoption at 280nm. Inhibition kinetictests of the inhibitor show that pig aprotinin is competitive inhibitor. The K_m of trypsinis 1.94×10^-3. The K_i is more smaller than K_m, so the pig aprotinin had strong inhibitionto trypsin.