Protein-protein interaction in low water organic media

Trypsin either modified with polyethylene glycol or as a suspended powder was used to catalyze digestion of protein substrates in benzene in order to get insight into protein–protein interactions in water‐immiscible organic media. Depending on whether suspended or soluble trypsin was used, catalysis...

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Bibliographic Details
Published in:Biotechnology and bioengineering 1990-09, Vol.36 (6), p.601-607
Main Authors: Gaertner, Hubert F., Puigserver, Antoine J.
Format: Article
Language:English
Subjects:
benzene
Biological and medical sciences
Biotechnology
Chemical industry. Chemical engineering
Fundamental and applied biological sciences. Psychology
Industrial applications and implications. Economical aspects
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Summary:Trypsin either modified with polyethylene glycol or as a suspended powder was used to catalyze digestion of protein substrates in benzene in order to get insight into protein–protein interactions in water‐immiscible organic media. Depending on whether suspended or soluble trypsin was used, catalysis was found to proceed differently. In the first case, the amount of water in the reaction mixture (up to 1% v/v) appeared to be critical, and adsorption of water from the reaction medium by the protein substrate allowed it to behave as a hydrophilic support material comparable to that involved in immobilized enzymes. In the latter case, the presence of an additional nucleophile was a prerequisite for catalysis to proceed, and thus both water and nucleophile concentrations had some influence on trypsin activity. Phe–NH2 was the most potent nucleophile for proteolysis catalyzed by polyethylene glycol‐modified trypsin in organic media containing 1–2% water (v/v). The organic solvent‐soluble enzyme was found to bind reversibly to the protein substrate as a function of both extent of hydration of the reaction medium and time of incubation. The overall results strongly suggested that modified trypsin catalyzed peptide bond hydrolysis at the protein substrate‐organic solvent interface. Peptide mapping of bovine insulin digest by reversed‐phase high‐performance liquid chromatography definitely showed that enzyme‐catalyzed proteolysis did occur in organic solvents with a concomitant and significant transpeptidation reaction.
ISSN:0006-3592
1097-0290
DOI:10.1002/bit.260360607