Trypsin inhibitor

Method 1. Extracting beef lungs as raw materials, salting out, mincing fresh or frozen beef lungs, stirring them with 5 times the amount of water, adjusting pH to 2 with 2.5mol/L sulfuric acid, stirring and extracting for 8 hours, filtering, and then extracting the filter residue in the same way Once, combine the two extracts, add 277g of ammonium sulfate per liter, adjust the pH to 1.5 with 5mol/L sulfuric acid, and leave it overnight. Fresh beef lung (or frozen [water, H2SO4]→[pH2, 8h] extract [(NH4)2SO4]→[pH1.5] salting-out product, removing protein, removing the salting-out product and removing the supernatant, filtering, and using the filtrate Adjust the pH of sodium hydroxide solution to 5.5, then add 255g ammonium sulfate per liter, stir and let stand for 48h, and filter to obtain the salted-out product. Dissolve the salted-out product with 9 times the amount of water, stir for 2h, put it in the refrigerator overnight, filter , Add 1/20 of trichloroacetic acid (50%) to the filtrate, quickly heat to 80℃, then cool to below 30℃, and filter. Salting out [(NH4)2SO4]→[pH5.5, 48h] Salting-out product [Trichloroacetic acid]→[80℃, then 30℃] The filtrate is salted out, dissolved, and the pH of the salted-out filtrate is adjusted to 3 with sodium hydroxide (5mol/L), and 610g of ammonium sulfate is added per liter and stirred to make Dissolve and let stand overnight. Take the precipitate and dissolve it with 4 times the newly prepared 0.1mol/L sodium acetate buffer pH5.5 and 1 times the 0.4mol/L boric acid buffer, and add 1050g magnesium sulfate per liter to dissolve , Overnight, filter the next day, and precipitate the salting out. The filtrate [NaOH, (NH4)2SO4]→[pH3] precipitate [NaAc, boric acid buffer, MgSO4]→[pH5.5] salting out, dialysis, desalting, precipitation Add 4 times the amount of water to dissolve the salt precipitate, dialyze with running tap water below 10°C for 24 hours, add diatomaceous earth to filter, and exchange the filtrate with strong alkaline quaternary ammonium type I anion exchange resin 201×7 with strong acid styrene series cations Resin 001×7 (according to the ratio of 201×7 resin: 001×7 resin=3:1) is stirred and adsorbed for 20 minutes until there is no sulfate ion. The resin is filtered, and the desalting solution (about pH 7) is added with 0.9% sodium chloride and 1.5 times the amount of acetone precipitates out, filtered, and the filtrate is added 4 times amount of acetone to precipitate out overnight. Then the precipitate is washed with acetone and ether, and dried. Salted out product [water] → [below 10°C] filtrate [anion, cation resin] →Desalting solution [NaCl, Acetone]→[pH7] The precipitate is dissolved in 80% methanol solution with 60 times the amount of pyrogen, precipitated, and dried. Stir in an ice bath for 2 hours, filter, and add 30% to 1% of the filtrate volume Sodium chloride solution and 5 times the amount of acetone, precipitate overnight, centrifuge, wash with acetone, and dry to obtain the precipitate. Then dissolve with 60 times the amount of pyrogen-free water, adjust the pH to 11 with 5mol/L sodium hydroxide, and place in the refrigerator 4d, filter, after the filtrate is adjusted to pH 4-5 with glacial acetic acid, add 1% amount of 300g/L sodium chloride solution and 5 times amount of acetone to separate out the precipitate, overnight, wash with acetone, ether, and dry to get the finished product . Precipitate [distilled water, NaOH]→[pH11] pyrogen-free solution [ice HAc, NaCl, acetone]→[pH4-5] finished product method 2. Human urine trypsin inhibitor (UTI) extraction method using human urine as raw material Raw material processing, acidification, take 100L of fresh male urine

Fibrin; non-degradable identification test of fibrinogen