Non-specific binding in the affinity chromatography of chymotrypsin

Area of Science:

• Biochemistry
• Chromatography
• Enzyme kinetics

Background:

• Chymotrypsin binds strongly to N-sigma-amino caproyl-D-tryptophan methyl ester agarose columns.
• This binding was previously assumed to be purely biospecific.

Purpose of the Study:

• To investigate the nature of chymotrypsin binding to the modified agarose column.
• To determine the role of enzyme surface residues in chromatographic interactions.

Main Methods:

• Acetylation of chymotrypsin's surface amino groups using acetic anhydride.
• Comparison of binding affinity and catalytic activity of native and acetylated chymotrypsin.
• Analysis of interactions between the enzyme and the chromatography support.

Main Results:

• Acetylation abolished chymotrypsin binding to the column.
• Catalytic properties of acetylated chymotrypsin remained unchanged.
• Biospecific binding alone was weak, indicating a role for non-specific interactions.

Conclusions:

• Chymotrypsin's high affinity for the column relies on both weak biospecific and non-specific interactions.
• Electrostatic interactions with the agarose matrix and lysyl residues are postulated.
• Enzyme surface residues outside the active site significantly influence chromatographic behavior.