Experiments for the determination of trypsin

Summary

This presentation describes proteases that preferentially cleave Arg and Lys peptide bonds. Source: Laboratory Manual of Enzymology

Operation method

basic program

Materials and Instruments

Trypsin
Potassium phosphate N'-benzoyl-L-argininyl-p-nitroanilide
Spectrophotometer

Move

The "reagents" required for the experiment are specified in "Others".

Measure the absorbance at 405 nm at 25°C. _ε405 = 9620 l/(mol-cm).

Common Problems

Reagents:

0.3 mol/L potassium phosphate, pH 8.0

20 mool/L N'-benzoyl-L-argininyl-p-nitroanilide (BAPNA, Mr=434.9; 174 mg in 20 ml DMSO)

1 mmol trypsin, 23.4 mg dissolved in 1 ml 1 mmol/L HCl (diluted 50-fold with 0.05 mol/L potassium phosphate pH 8.0 immediately prior to assay)

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