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Luciferase

(Bacterial luciferase, EC 1.14.14.3)
Luciferase is useful for enzymatic determination of NAD(P)H by coupling with NADH-FMN oxidoreductase in biochemical analysis as in the following reaction:
                Luciferase
FMNH2 + RCHO + O2 ------> FMN + RCOOH + H2O + hv
R = CH3(CH2)6 ~ CH3(CH2)11




One unit causes the light emission of one micromole of photon per minute at pH 7.0 and 25°C.


  1. Buffer Solution: Mix 50 mM disodium monohydrogen phosphate and 50 mM potassium dihydrogen phophate and adjust pH 7.0 at 25°C.
  2. FMN Solution: 1 mM (4.96 mg FMN-NA-H2O) in 10 ml of Buffer.
  3. Sodium Hydrosulfite Solution: 10 mg/ml in H2O
  4. Aldehyde Solution: 10 µl n-decyladehyde, 0.5g BSA and 50 µl Triton X-100 in 100 ml of buffer.



  1. Into a 3 ml vial pipette 455 µl of Buffer solution and equilibrate for 5 minutes at 30°C.
  2. Add 10 µl of enzyme solution.
  3. Add 25 µl of FMN solution.
  4. Add 10 µl of Sodium hydrosulfite, and then stir until the yellow color is disappeared.
  5. Set the vial into the luminometer
  6. Inject 500 µl of Aldehyde solution as soon as possible by syringe.
  7. Measure the peak height of Light intensity by the luminometer (I test).



Volume Activity (U/mg) = Δlo (l test - l blank)(Vt)(60)(106)(df) X A
(6.02)(1023)(Vs) (l std - l blank)


= Δlo X 9.97 X 10-15 X df X A

(l std - l blank)
Weight Activity (U/mg) = (U/ml) X 1/C


Vt Total Volume (100 Ál)

Vs Sample Volume (10 Ál)

6.02 x 1023 Avogadro's number

60 60 seconds

df Dilution factor

C Enzyme concentration in dissolution (c mg/ml)

I std Light intensity of light standard

A Light standard value (quanta/second)

106 Calculation factor for Ámole
 

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