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(Peptidyl peptide hydrolase; EC

Chymotrypsin occurs in the pancreas of mammals, and has been isolated from bovine pancreas. It is an endopeptidase, which preferentially splits peptide bonds in which the carbonyl group is contributed by aromatic amino acids such as tyrosine, phenylalanine, or tryptophan. In addition, chymotrypsin catalyzes the hydrolysis of the bonds of esters and amides of aromatic amino acids as well as proteins and peptides. Chymotrypsin occurs in different forms which can be isolated by electrophoresis. Different forms of chymotrypsin have different degrees of catalytic activity.

Chymotrypsin catalyzes the following reaction:

N-Benzoyl-L-Tyrosine Ethyl Ester + H2O ------> N-Benzoyl-L-Tyrosine + Ethanol

That amount of enzyme which hydrolyzes 1 micromole of benzoyl-L-tyrosine ethyl ester (BTEE) per minute at 25C and pH 7.8.

The hydrolysis of Benzoyl-L-tyrosine ethyl ester causes an increase in absorbance which is measured at 256 nm. This method is specific for chymotrypsin as trypsin does not hydrolyze BTEE.

  1. 0.08 M Tris/HCl buffer, pH 7.8, containing 0.1M calcium chloride
  2. 0.00107 M BTEE in 50% w/w methanol
  3. 0.001 N HCl
  4. Chymotrypsin (enzyme) solution: Dissolve in 0.001 N HCl to concentration of 1 mg/ml. Dilute in 0.001 N HCl to 0.5-1.0 U/ml, just prior to assay.

  1. Set spectrophotometer (equipped with strip chart recorder and temperature control) to 256 nm at 25C
  2. Into a cuvette, pipette the following reagents in the amounts indicated:
    Tris Buffer 1.5 ml
    BTEE 1.4 ml
  3. Mix and incubate in spectrophotometer at 25C for 4-5 min. to achieve temperature equilibration. Record a blank rate, if any, at 256 nm.
  4. Add 0.1 ml of prepared enzyme dilution to the test cuvette and monitor the reaction at 256 nm for 5 min.
  5. Calculate the (delta)E256nm from the linear portion of the curve.

Activity (U/mg) = (ΔE256nm/min)(1000)
(964)(mg Enz./ml)

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