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Influence of Me2SO and incubation time on papain activity studied using fluorogenic substrates
M Szabelski1, K Stachowiak, W Wiczk
1Faculty of Chemistry, University of Gdańsk, Poland.
Acta Biochimica Polonica
|May 9, 2002
Summary
Dimethyl sulfoxide (Me2SO) reduces papain enzyme activity by decreasing active sites and hydrolysis rates. Higher Me2SO concentrations significantly impact substrate binding more than catalytic rates.
Area of Science:
- Biochemistry
- Enzymology
- Proteolysis
Background:
- Papain is a crucial cysteine protease with broad substrate specificity.
- Understanding enzyme kinetics in the presence of organic solvents like dimethyl sulfoxide (Me2SO) is vital for biochemical applications.
Purpose of the Study:
- To investigate the effect of dimethyl sulfoxide (Me2SO) concentration and incubation time on papain activity.
- To elucidate the kinetic parameters (kcat, Km) governing papain-catalyzed hydrolysis in Me2SO.
Main Methods:
- Enzyme kinetics assays using fluorogenic substrates (Z-Phe-Arg-Amc and Dabcyl-Lys-Phe-Gly-Gly-Ala-Ala-Edans).
- Measurement of initial hydrolysis rates and application of the Michaelis-Menten equation.
- Study of papain activity across varying Me2SO concentrations and incubation times.
Main Results:
- Increasing Me2SO concentration decreased the number of active papain sites and the initial hydrolysis rates of both substrates.
- A rapid decline in activity (up to 30%) was observed between 1% and 2% Me2SO.
- Higher Me2SO concentrations led to increased Km (reduced substrate binding) and decreased kcat (reduced catalytic rate), with a more pronounced effect on Km.
Conclusions:
- Dimethyl sulfoxide (Me2SO) inhibits papain activity by reducing active sites and altering kinetic parameters.
- Me2SO primarily affects substrate binding (Km) rather than the catalytic turnover rate (kcat) of papain.
- These findings are crucial for optimizing papain-based reactions in Me2SO-containing buffers.
