Self purification of two serine endopeptidases

Area of Science:

• Biochemistry
• Enzymology

Background:

• A serine protease from Pronase, similar to bovine chymotrypsin, is active and stable in 6 M guanidinium chloride.
• This unique property suggests potential for self-purification by cleaving other proteins.

Purpose of the Study:

• To investigate if the protease can purify itself by cleaving companion proteins.
• To develop a rapid isolation technique for specific serine proteases.

Main Methods:

• Incubation of Pronase in 6 M guanidinium chloride at pH 8.0 for 1 week.
• Analysis of enzyme activity against Nalpha-acetyl-L-tyrosine ethyl ester.
• Separation and purification of resulting protease components.

Main Results:

• Only esterase activity against Nalpha-acetyl-L-tyrosine ethyl ester remained after prolonged incubation.
• Two distinct serine proteases were isolated: a subtilisin-like and a chymotrypsin-like enzyme.
• The subtilisin-like enzyme was purified to homogeneity for the first time, with no demonstrable free amino-terminal group.

Conclusions:

• A unique and rapid method for isolating serine proteases from Pronase using guanidinium chloride was developed.
• This method yields highly pure subtilisin-like and chymotrypsin-like enzymes.
• These purified enzymes can serve as probes for protein conformation and aid in preparing enzyme-free macromolecules.