Papain Assay

Method: A titrimetric determinatin of the acid produced during the hydrolysis of benzoyl-L-arginine ethyl ester (BAEE). One unit will hydrolyze one micromole of benzoyl-L-arginine ethyl ester per minute at 25°C and pH 6.2 under the specified conditions.

Reagents

• Enzyme diluent (Activation buffer): Prepare fresh daily by mixing the following:

   

  0.01 M EDTA	10 ml
  0.06 M Mercaptoethanol	0.1 ml
  0.05 M Cysteine⋅HCl	10 ml
  Reagent grade water	70 ml

• Substrate solution: Prepare fresh daily by mixing the following:

   

  0.058 M BAEE	15.0 ml
  0.01 M EDTA	0.8 ml
  0.05 M Cysteine⋅HCl	0.8 ml

• Adjust pH to 6.2 and dilute to a final volume of 21 ml with reagent grade water.
• Titrant: 0.01-0.02 N NaOH, standardized

Enzyme

Activate enzyme by dissolving in enzyme diluent to a concentration of 0.05-0.1 mg/ml. Under these conditions activation is complete within 30 minutes.

Determination of protein concentration

Procedure

The reaction can be measured with either an automatic titrator or a laboratory pH meter. The titration vessel should be maintained at 25°C.

Pipette the following into the titration vessel at 25°C:

Substrate solution	5.0 ml
3.0 M NaCl	5.0 ml
Reagent grade water	5.0 ml

At zero time add 0.1 ml of appropriately diluted enzyme and adjust the pH to 6.2. Record the amount of standardized NaOH added per minute to maintain the pH at 6.2 after a constant rate is achieved.

Calculation

Technical note: Mercuripapain must be activated before use. Mercury is removed from the enzyme in activation buffer. After 30 minutes in this solution, the enzyme is completely activated and the mercury has been chelated. The mercuripapain suspension contains no free mercury. The product has been extensively dialyzed prior to packaging.