Catalog Number: 199800
CAS #: 9014-74-8
Molecular Weight/Structure: 147,000-150,000 heterodimer consisting of 115-135 kD and 35-47 kD subunits. Two disulfide bridges ling the light and heavy chains. The light chain is a trypsin-like serine enzyme, which contains the whole set of amino acids necessary for the formation of both the catalytic center and the tetraaspartyl binding site. However, this is not sufficient for highly effective hydrolysis of the natural substrate, trypsinogen.1
E.C. # 184.108.40.206
- One unit will produce 1.0 nanomole of trypsin from trypsinogen per minute at pH 5.6 at 25°C.
- One unit will cause the activation of one nanomole of trypsinogen per hour under specified conditions.
(One FLAG·BAP unit is equal to 10X the activity of a standard trypsinogen unit (Liepnieks and Light, 1979).)
Description: Enterokinase is a highly specific serine protease. It is a glycoprotein containing 35% carbohydrate. By activating trypsinogen, Enterokinase catalyzes hydrolysis of the polypeptide chain only after an -(Asp)4-Lys- sequence.
It was found that calcium ions regulate the Enterokinase activity.1 A type of intramolecular autolysis resulting from the loss of these ions causes a considerable decrease in the Enterokinase catalytic activity toward trypsinogen, whereas the activity toward synthetic low- and high-molecular-mass substrates containing the tetraaspartyl-lysine linker is retained. The localization of the autolysis degradation sites suggested the presence of a secondary substrate-binding center for trypsinogen in the N-terminal fragment 118-465 of the enzyme heavy chain.1
The high specificity and intricate structure of Enterokinase determine its relations with protein inhibitors. It has been shown that the bovine deudenum contains a natural kunin-like inhibitor of enterokinase (DI-9), which may be involved in the regulation of calcium-dependent autolysis of the heavy enzyme chain. As a result, a truncated form, which is low active toward trypsinogen, forms. It has also been suggested that in the presence of, in certain regions of the heavy chain, one or several calcium binding sites and the regulation of the Enterokinase activity by calcium ions as well as by the inhibitor DI-9.1
Synthetic Substrate: Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide, MP catalog number 157235
* Peptides are resistant to cleavage if proline occupies the X position.
Inhibition: Inhibited by soybean trypsin inhibitor.
- Typically used in protein modification and amino acid sequence determination; in the cleavage of recombinant fusion proteins which contain the EK recognition sequence (Asp)4-Lys, where cleavage occurs on the C-terminal side of the lysine residue.
- Optimal results have been obtained with fusion protein concentrations at approximately 250 ng/ml.
- The pH of the fusion protein solution should be adjusted to a pH between 7.4 and 8.0.
- Add approximately 5.0 units of Enterokinase per ug of fusion protein. Digestion should be carried out in the aqueous buffer used for dilution. Mix and incubate the digestion mixture at 37°C overnight.
- Enterokinase can be removed by gel filtration or affinity chromatography depending on the nature of the fusion protein.
A. 40 mM Succinate Buffer, pH 5.6 at 25°C: Prepare 100 ml in deionized water using succinic acid, free acid, MP catalog number 102972. Adjust to pH 5.6 at 25°C with 1 M NaOH.
B. 1 mM Hydrochloric acid with 5 mM calcium chloride solution: Prepare 100 ml in deionized water using 1 N hydrochloric acid, MP catalog number 1688045, and calcium chloride dihydrate, MP catalog number 193818.
C. 0.1% (w/v) Trypsinogen solution: Immediately before use, prepare 25 ml in cold Reagent B using trypsinogen, MP catalog number 101195.
D. Enterokinase Enzyme Solution: Immediately before use, prepare a solution containing 2-5 units/ml of enterokinase in cold deionized water.
E. 67 mM Sodium Phosphate Buffer, pH 7.6 at 25°C: Prepare 1 liter in deionized water using sodium phosphate monobasic anhydrous, MP catalog number 195500. Adjust to pH 7.6 at 25°C with 1 M NaOH.
F. 0.248 mM N alpha-Benzoyl-L-Arginine Ethyl Ester Solution (BAEE): Prepare 100 ml in Reagent E using BAEE hydrochloride, MP catalog number 100088. Prepare Fresh for each use.
G. 40 mM Hydrochloric acid with 5 mM calcium chloride solution: Prepare 1 liter in deionized water using 1 N hydrochloric acid, MP catalog number 1688045, and calcium chloride dihydrate, MP catalog number 193818.
Immediately mix by inversion and record the increase in A253nm for approximately 5 minutes. Obtain the DA253nm/minute using the maximum linear rate for both the Test and Blank.
5.4 = Volume (in milliliters) of Step 1
df = Dilution factor
0.001 = The change in A253nm/minute per unit of Trypsin as per the unit definition
0.20 = Volume (in milliliter) from step 1 used in step 2
P.A. = Potential activity of trypsinogen (this value will be lot specific)
0.024 = mg trypsin/nanomole trypsin
15 = Time (in minutes) for step 1 as per the unit definition
0.1 = Volume (in milliliter) of Enterokinase used
Final Assay Concentration:
In a 2.40 ml reaction mix, the final concentrations are 30 mM succinate, 1 mM calcium chloride, 0.2 mM hydrochloric acid, 0.5 mg trypsinogen and 0.2 - 0.5 unit Enterokinase.
|199800||Enterokinase from calf intestinal mucosa||1 KU|
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