ASSAY METHOD OF NATTOKINASE
Nattokinase activity can be obtained by the spectrophotometric measurement of the amount of acid-soluble low-molecular products, which is increased owing to hydrolysis of the peptide linkages when nattokinase acts on fibrin.
DEFINITION OF ACTIVITY UNIT
One unit ( 1 FU ) is defined as the amount of the enzyme which increases the absorbance of the filtrate at 275 nm by 0.01 per minute under the conditions specified in the procedure.
(1) Water bath ： 37±0.3 ℃
(2) Test tubes ： 15×150 mm
(3) Mixer ： YAMATO SCIENTIFIC MT－31
(4) Micro test tubes ： EPPENDORF , safe lock 2.0 mL
(5) High-speed microcentrifuge ( The Rotor with the radius of 4 cm )
(6) Pastuer pipettes ： for example IWAKI GLASS , 9 inches length
(8) Cuvette ： 10 mm in a path length , 4 mm in a path width
(1) 0.05 mol／L Borate buffer ( pH 8.5 , containing NaCl )
Dissolve 19.07 g of Na2B4O7･10H2O and 9.0 g of NaCl in about 900 mL of distilled water.
Adjusted pH to 8.5 with concentrated HCl and add distilled water to make 1000 mL.
(2) 0.2 mol／L Trichloroacetic acid ( TCA ) solution
Dissolve 32.68 g of TCA in distilled water to make 1000 mL.
(3) 0.72 % Fibrinogen solution
Pipette the 10 mL of 0.05 mol／L Borate buffer into an Erlenmeyer flask, add 96 mg of fibrinogen ( SIGMA , FIBRINOGEN ( FractionⅠ) TypeⅠ-S : From Bovine Plasma , PRODUCT NUMBER F8630 ), and keep standing for some time. Grind the undisolved with glass rod to dissolve completely. Filtrate the mixture through a filter paper ( ADVANTEC , No .6 , 7 cm ), to remove insoluble materials. Prepare before use. 〔Note Ⅰ〕
(4) Thrombin solution
Dissolve thrombin ( SIGMA , From Bovine Plasma , PRODUCT NUMBER T6634 ) in 0.05 mol／L Borate buffer to make the 1000 U／mL solution. Dispense it little by little into bottles and make them freeze for storage. Dilute one of them 50-fold with 0.05 mol／L Borate buffer before use.
(5) 1 mol／L Acetic acid solution
Dissolve 60.1 g of CH3COOH in distilled water to make 1000 mL.
(6) 1 mol／L Acetate buffer ( pH 6.0 )
Dissolve 129.6 g of CH3COONa･3H2O in distilled water. Add 47.6 mL of 1mol/L Acetic acid solution and add distilled water to make 1000 mL.
(7) 10 % Triton X-100 solution
Dissolve 10 g of Triton X-100 in distilled water by heating and add distilled water to make 100 mL.
Dissolve 0.334 g ( final conc. 2 mmol／L ) of CaSO4･2H2O and 0.585 g( final conc. 10 mmol／L ) of NaCl in distilled water. Add 2.0 mL of 1 mol／L Acetate buffer ( pH 6.0 ), 0.5 mL ( final conc. 0.005 % ) of 10 % Triton X-100 solution, and distilled water to make 1000 mL. 〔Note Ⅱ〕
PREPARATION OF SAMPLE SOLUTION
Dilute the sample with diluent to give corrected absorbance ( Δ A ) of the filtrate between 0.04 and 0.08. The concentration is normally 0.67 to 1.33 FU／g.
( SAMPLE )
1. Dispense the 1.4 mL of 0.05 mol／L Borate buffer and 0.4 mL of 0.72 % Fibrinogen solution into a test tube, and preincubate it in a 37 ± 0.3 ℃ water bath for 5 minutes.
2. Add 0.1 mL of Thrombin solution and mix. 〔Note Ⅲ〕
3. After exactly 10 minutes, add 0.1 mL of the sample solution, mix for 5 seconds, and incubate at 37 ± 0.3 ℃.
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 0.2 mol／L TCA solution to stop the reaction, mix, and incubate at 37 ± 0.3 ℃ for further 20 minutes.
6. Transfer the mixture into a micro test tube and centrifuge at 15000 rpm for 5minutes.
7. Transfer the 1 mL of supernatant carefully into a cuvette by Pasteur pipette and read the absorbance ( AT ) at 275 nm.
( BLANK )
8. Dispense the 1.4 mL of 0.05 mol／L Borate buffer and 0.4 mL of 0.72 % Fibrinogen solution into a test tube, and preincubate it in a 37 ± 0.3 ℃ water bath for 5 minutes.
9. Add 0.1 mL of Thrombin solution and mix.
10. After exactly 10 minutes, add 2 mL of 0.2 mol／L TCA solution and mix for 5 seconds.
11. Add 0.1 mL of sample into solution, mix for 5 seconds, and incubate at 37 ± 0.3 ℃ for 20 minutes.
12. Transfer the mixture into a micro test tube and centrifuge at 15000 rpm for 5 minutes.
13. Transfer the 1 mL of supernatant carefully into a cuvette by Pasteur pipette and read the absorbance ( AB ) at 275 nm. (4)
Nattokinase activity（FU/g）＝（ＡＴ－ＡＢ）／0.01 × 1／60 × 1／0.1 × Ｄ
Ｄ ： Dilution rate of sample
Ⅰ. The measured activity can vary with the lot of fibrinogen. Accordingly, when fibrinogen’s lot is changed, correct the measured activity by multiplying the correction factor if necessary.
Ⅱ. This diluent can be kept 15 days at the room temperature.
Ⅲ. Use mixer for every mixing.
Ⅳ. The standard sample should be assayed simultaneously at every test batch to confirm the accuracy of the test result.