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Enterokinase

(Enteropeptidase; EC 3.4.21.9)

Trypsinogen is transformed into trypsin as a result of the cleavage of a single peptide bond by Enterokinase near the N-terminus of the zymogen (E.W. Davie and H. Neurath, J.Biol Chem., 212, 515, 1955) and the appearance of activity is accompanied by conformation changes. The latter autocatalytic process is accelerated by calcium ions which bind to the N-terminal region of the zymogen and promote the specific bond cleavage (T.M. Radhakrishnan, et al, Biochemistry, 8, 4020, 1969 and J.P. Abita, et al, European J. Biochem, 8, 314, 1969).





The amount of enzyme that causes the activation of 1 micromole of Trypsinogen under the following conditions.


  1. 0.07 M sodium succinate buffer, pH 5.6: Dissolve 5.8 g of succinic acid, disodium salt (hexahydrate) into 400 ml of distilled water. Adjust to pH 5.6 with 1 M NaOH then add distilled water to make the total volume 500 ml.
  2. 0.07 M potassium phosphate buffer, pH 7.6
  3. 0.005 M CaCl2 in 0.001 M HCl solution
  4. 0.00025 M N-a-benzoyl-L-arginine ethyl ester (BAEE) in potassium phosphate buffer, pH 7.6
  5. 2.0 M HCl
  6. Trysinogen solution (1 mg/ml) in Reagent #3.
  7. Enzyme solution (5 mg/ml) in Reagent #1.



  1. Heat water bath to 25C.
  2. Into six test tube pipette the following:

    Sodium succinate 1.0 ml
    Distilled Water 1.0 ml
    Trypsinogen sol'n 0.5 ml

  3. Mix and incubate in water bath to obtain the temperature equilibrium and then initiate reaction by adding to three test tubes 0.1 ml of enzyme solution and keep the other test tubes only as water blanks.
  4. Incubate the enzyme at 25C for 15 minutes, 30 minutes and 45 minutes and stop the reaction by adding 0.1 ml of 2 M HCl.
  5. Measure the Trypsin activity according to the method described in this catalog and compare to the blank.
  6. Calculate (delta)E253 for every 15 minutes and average values.


Activity (U/mg) = (ΔE253nm/15min)(Total Vol.)(Enz. Diln.)(4)
(0.001)(Trypsin Act.)(Enz. Vol.)(mg Enz./ml)
 

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