Worthington -- Specification of Worthington trypsin inhibitor

AI Meijie Worthington  Classification of trypsin inhibitors ：

1、 Lima bean inhibitor ： Lima bean trypsin inhibitor can inhibit bovine and human trypsin and plasmin , It acts on trypsin and chymotrypsin by forming equimolar complexes . Lima bean inhibitors can be chromatographed into up to six variants .Jones et al., Biochem., 2 , 66, (1963)  Characterized four of them . Both have similar but different amino acid compositions , Contains six or seven disulfide bonds , Lack of methionine and tryptophan . The molecular weight is  8 kDa  and  10 kDa  Change between .

stability / Store ： Lima bean inhibitors in  2-8°C  Stable under  1-2  year .

Egg mucin ： Egg mucin is a glycoprotein protease inhibitor of avian egg white . There are several protease inhibitors in egg white .Worthington  Trypsin inhibitors are  Lineweaver  and  Murray JBC, 171 , 565 (1947)  Described as , Its molecular weight is about  28 kDa.

stability / Store ： Egg mucin in  2-8°C  Stable under  1-2  year .

2、 Pancreatic inhibitors ：Worthington  Pancreatic inhibitors are often called  Kunitz  Inhibitors , from  Kunitz  and  Northrop J. Gen. Physiol., 19 , 991 (1936)  First crystallization . It is associated with  pH  value  3  and  10  The formation of bovine trypsin and human trypsin is very stable  1:1  compound . Chymotrypsin is also inhibited by this product , But the combination is not so close . On the other hand , Ester hydrolysis of porcine elastase 、 Protein hydrolysis and elastic hydrolysis activities were not inhibited .Kunitz  The inhibitor is  58  A single polypeptide chain of amino acids , The molecular weight is  6.5 kDa. When it comes from lung tissue , The inhibitor is called aprotinin .

stability / Store ： Pancreatic inhibitors are  2-8°C  Stable when stored  1-2  year .

3、 Soybean inhibitor ： Soybean trypsin inhibitor in  1945  Year by year  Kunitz shou Secondary crystallization , It is one of several such inhibitors found in soybeans . Its molecular weight is  21,500 ± 800  Dalton , Most suitable  pH  The value is  7.0. Soybean inhibitors inhibit trypsin by mole , And inhibit chymotrypsin to a lesser extent .

stability / Store ： Soybean inhibitors are  2-8°C  It can be stored stably under  1-2  year .

AI Meijie Worthington Definition of trypsin inhibitor unit ： The activity of the inhibitor is expressed as twice crystallized trypsin inhibited by each mg of inhibitor （Worthington Code：TRL） The amount of .1mg TRL ≥180 TAME  Company 、10,350 BAEE  Company 、3,450 USP/NF  Company .

 

 

AI Meijie Worthington Determination of trypsin inhibitor ：

Various trypsin inhibitors were measured by spectrophotometry to prevent benzoyl -L- Ability of Trypsin Hydrolysis of arginine ethyl ester . No active units are currently assigned . The activity of the inhibitor is expressed as twice crystallized trypsin inhibited by a milligram of inhibitor （TRL） The amount of .

Reagents ：

1. Trypsin reagent ：0.25 mM  Benzoyl -L- Arginine ethyl ester in  0.067 M  In phosphate buffer ,pH 7.0

2.0.001 N  hydrochloric acid

3.0.5 M  Sodium potassium phosphate buffer ,pH 6.5

4. Trypsin solution ： stay  0.001 N HCl  Prepared in  0.5 mg/ml  Trypsin （Worthington  Code ：TRL）.

5. Inhibitors ： Dissolve various inhibitors as follows ：

Lima bean inhibitor ： 1% NaCl  in  1 mg/ml

Egg mucin ： 1 mg/ml  stay  0.001 N HCl  in

Trypsin inhibitor ： 1 mg/ml  stay  0.001 N HCl  in

Soybean inhibitor ： 1 mg/ml  stay  0.01 M  Phosphate

In buffer ,pH 6.5, contain  0.15M NaCl