Tryptic hydrolysis at asparagine residues in globin chains

Area of Science:

• Biochemistry
• Enzymology

Background:

• Bovine trypsin (EC 3.4.21.4) is a well-characterized serine protease.
• Its primary function involves cleaving peptide bonds at the C-terminal side of lysine and arginine residues.

Purpose of the Study:

• To investigate an observed minor hydrolysis activity of bovine trypsin at asparagine residues in globin chains.
• To determine if this activity is an inherent property of trypsin or an artifact of contamination or treatment.

Main Methods:

• Enzyme treatment of globin chains with commercially available and purified bovine trypsin.
• Treatment with tosyl-phenylalanyl-chloromethyl ketone (Tos-PheCH2Cl) to inhibit trypsin activity.
• Purification of trypsin using CM-cellulose chromatography and affinity chromatography on trypsin inhibitor columns.

Main Results:

• Commercially available bovine trypsin, both treated and untreated, catalyzed minor hydrolysis at asparagine residues in globin chains.
• This asparagine-specific hydrolysis activity persisted after extensive purification of the enzyme.
• Tos-PheCH2Cl treatment of purified trypsin did not inhibit this minor hydrolysis activity.

Conclusions:

• The observed hydrolysis of globin chains at asparagine residues is likely an inherent feature of the bovine trypsin molecule.
• Standard purification methods and specific inhibitor treatments do not remove this minor proteolytic activity.