Practical Protein Chemistry - A. Darbre 1989

Enzymatic fragmentation of the polypeptide chain
General conditions for enzymatic hydrolysis
Buffer solutions

The optimal Hydrolysis conditions are selected by varying four key parameters: buffer composition, Enzyme-to-substrate ratio, Temperature, and duration. The specific conditions required for hydrolysis with a given enzyme are detailed in the sections dedicated to each enzyme. However, a few general Preliminary Remarks should be made first.

Volatile buffers are commonly used, especially when the resulting Peptides are to be separated by Ion-exchange Chromatography or paper Electrophoresis. If subsequent peptide Separation is performed by HPLC, salt concentration is not critical (Chapter 6). For most Enzymes, the optimal pH range is 7.5–8.5. Within this range, 1% (w/v) ammonium bicarbonate (pH 7.9) and 100 mM N-ethylmorpholine acetate are typically employed. Hydrolysis with Pepsin is carried out in 10 mM HCl or 5% acetic acid at pH 2.0. To maintain The activity of certain enzymes, specific Reagents must be added to the reaction mixture, such as sulfhydryl reagents (see Clostripain and Papain) or Ca2+ ions (see Thermolysin).



Last update: 06/08/2026

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