Soybean trypsin inhibitor, enzymatic

Replacement of amino acid residues in a polypeptide chain still is achieved most easily by isolating mutant proteins from bacteria, but Laskowski, Jr. and his colleagues at Purdue University have developed a method for enzymatically removing an essential arginine residue from the reactive site of soybean trypsin inhibitor (Kunitz) and replacing it with a lysine residue (84). 

Incubate with 0.25% (w/v) crystalline trypsin in PBS, 0.25% EDTA for 2-6 min at 37°C to give a suspension of predominandy single cells. Terminate the enzymatic action by adding L-15 plus PCS and 1% (w/v) soybean trypsin inhibitor. 

Soybean trypsin inhibitor, STI the best known plant trypsin inhibitor. AMth bovine trypsin, at pH 8.3, it forms a stoichiometric, enzymatically inactive, stable complex with an association constant of 5 X 10 per mol STI. It also inhibits other vertebrate and invertebrate trypsins and plasmin. Chymotrypsin is inhibited to a small extent, and other endopeptida-ses not at all. STI is a single polypeptide chain, M, 21,100,181 amino acid residues (of known sequence) and two disulfide bridges. The molecule is compact and has a low a-helix content due to the presence of proline residues. It is consequently resistant to proteases and to denaturation. The reactive center contains a specific peptide bond Argj3-Ile(4, which is hydrolysed when the trypsin-STI complex is formed. The ensuing interaction with the active site of trypsin. 

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