Nattokinase

PRINCIPLE

Nattokinase acts on fibrin, breaking the peptide linkage, and forms low molecular weight acid soluble products, which are measured by spectrophotometer.

One fibrinolityc activity unit (FU) is defined as the amount of the enzyme, which increases the absorbance of the filtrate at 275nm by 0.01 per minute under the conditions specified in the procedure.

REAGENTS Of Nattokinase
1.0.05M Borate Buffer (pH 8.5) - Dissolve 9.54 g of Sodium Borate (Na2B4O710H2O) and 4.5 g of Sodium Chloride (NaCl) in about 200 ml of DI water.  Adjust pH to 8.5 with 1N HCl and add DI water to make 500 ml.
2.0.2N TCA  - Dissolve 8.17 g of Trichloroacetic Acid (CCl3CO2H) in DI water to make 250 ml.
3.Thrombin Solution  - Using a syringe, inject 5.0 ml of 0.05M Borate Buffer in a Thrombin bottle (Sigma, T-6634). Stir it gently to dissolve completely. With the syringe, transfer 1 ml aliquots into plastic vials (100 units/vial) and freeze them for storage.
* At the time of use dilute the content of one vial to 4 ml of 0.05 M Borate Buffer and mix with the pipetor tip (use this same tip when perform the assay) in order to obtain a final concentration of 20 U/ml. Make sure it is completely dissolved.

4.Acetate Solution (pH 6.0) - Dissolve 12.96 g of Sodium Acetate, Anhydrous (CH3CO2Na) in DI water, add 2.38 ml of 2N Acetic Acid solution and dilute to 100 ml with DI water.

10% Triton X-100  - Dissolve 5.0 g of Triton X-100 (t-Octylphenoxypolyethoxyethanol) in DI water with heating and add DI water to make 50 ml. Store in a brown bottle.                     

5.Enzyme Diluent - Dissolve 0.344 g of Calcium Sulfate, Dihydrate (CaSO42H2O) and 0.585 g of Sodium Chloride (NaCl) in DI water, add 2.0 ml of Acetate Solution, and 0.5 ml of 10% Triton Solution, and DI water to make 1 Liter.                                                                           

6.Fibrinogen Substrate - Add 10.0 ml of 0.05 M Borate Buffer to 96.0 mg of Fibrinogen (Sigma, Fraction I type I-S, F8630) little by little with gentle stirring, and dissolve completely in a 50 mL beaker. Prepare fresh daily (*1)      

                                                                 

PREPARATION OF SAMPLE SOLUTION
Dilute the sample with Enzyme Diluent to give a corrected absorbance (∆A) of the filtrate between 0.04 and 0.08 the concentration is normally 0.67 to 1.33 FU/ml

PROCEDURESAMPLE Of Nattokinase

1.Pipet 1.4 ml of Borate Buffer and 0.4 ml of Fibrinogen substrate solution into a suitable centrifuge tube, vortex and incubate in a 37ºC water bath for 5 minutes.
2.Add exactly 0.1 ml of Thrombin Solution and mix properly on the vortex.  
3.After exactly 10 minutes, add 0.1 ml of the Enzyme sample solution, mix for 5 seconds, and incubate at 37ºC.
4.After 20 and 40 minutes from the time the reaction starts, mix for 5 seconds respectively.
5.After exactly 60 minutes, add 2 ml of TCA solution to stop the reaction, mix and incubate at 37ºC for further 20 minutes.
6.Centrifuge at 5,000 rpm for 15 minutes.

7.Pipet 1 ml of supernatant carefully into a QUARTZ cuvette with a Pasteur pipet and read the absorbance (As) at 275 nm.

BLANK
1.Pipet 1.4 ml of Borate Buffer and 0.4 ml of Fibrinogen substrate solution into a centrifuged test tube, and incubate in a 37ºC water bath for 5 minutes.
2.Add exactly 0.1 ml of Thrombin solution and vortex.
3.After exactly 10 minutes, add 2 ml of TCA solution and mix for 5 seconds.
4.Add 0.1 ml of sample into solution, mix for 5 seconds, and incubate at 37ºC for exactly 20 minutes.
5.Centrifuge at 5,000 rpm for 15 minutes.
6.Pipet 1 ml of supernatant carefully into a QUARTZ cuvette with a Pasteur pipette and read the absorbance at (Ab) 275 nm.  (*2)

CALCULATION
                                  (As  -  Ab)              1           1
FU/g =        _____________    x      ________   x      _______   x  D
                             0.01                   60              0.1

D = Dilution factor (1/Weight (g/mL))

CERTIFICATE OF ANALYSIS

Packaging and storage

Solid food packaging bag, 25 kg / barrel.

Nattokinase should be stored in dry, cool and well ventilated places away from the sun, heat.

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