Diaphorase Assay

ASSAY for Product Code DIL:

Method: The oxidation of NADH is determined by measuring colorimetrically the reduction of 2,6-dichlorophenolindophenol. The assay is based on that described by Edelhoch et al. (1952) and Mahler et al. (1952) with modifications to provide greater linearity of reaction rate and proportionality of initial rate to enzyme concentration. One unit is defined as the amount of enzyme which reduces one micromole of 2,6-dichlorophenolindophenol per minute at 25deg.C and pH 8.5 under the specified conditions.

Reagents

  • 0.2 M Tris⋅HCl buffer, pH 8.5
  • 0.12 M Zinc chloride in 0.005 M HCl
  • 0.006 M NADH. Prepare fresh daily.
  • 0.0024 M Dichlorophenolindophenol (DCPIP) Prepare fresh daily.
  • Enzyme diluent: 0.2 M Tris⋅HCl pH 8.5 containing 0.3 M potassium chloride, 0.025% flavin mononucleotide (FMN), and 0.025% bovine serum albumin. Prepare fresh daily and protect from strong light.

Enzyme

Dissolve lyophilized powders at 10 mg/ml in 0.2 M Tris⋅HCl pH 8.5. Immediately prior to use, dilute further in enzyme diluent to a concentration of 0.1-0.15 mg/ml.

Procedure

Adjust spectrophotometer to 600 nm and 25°C.

Pipette into cuvettes as follows:

  Test Blank
0.2 M Tris⋅HCl, pH 8.5 2.6 ml 2.6 ml
0.12 M ZnCl2 0.1 ml 0.1 ml
0.006 M NADH 0.1 ml 0.1 ml
0.0024 M 2,6-Dichlorophenolindophenol (DCPIP) ----- 0.1 ml
Enzyme diluent ----- -----

Incubate in spectrophotometer at 600 nm and determine A600 of blank cuvette for 8-10 minutes. Determine ΔA600 from initial linear portion of curve. To test cuvette add 0.1 ml of 0.0024 M DCPIP followed immediately by 0.1 ml of diluted enzyme. Record decrease in A600 for 3-4 minutes. Calculate ΔA600 from the initial linear portion of the curve. The reaction is linear for no more than 1-2 minutes.

Calculation

calculation

ASSAY for Product Code DILW:

Method: One unit equals a decrease in absorbance of 1.0 per minute at 25°C at pH 7.5 with 2,6-dichlorophenolindophenol as the chromogen.

Reagents

  • 0.2 M Tris⋅HCl buffer, pH 7.5
  • 0.006 M NADH. Prepare fresh daily.
  • 0.0012 M Dichlorophenolindophenol (DCPIP) Prepare fresh daily.

Stock Enzyme Solution

Prepare a 10 mg/ml solution of enzyme in 0.2 M Tris⋅HCl, pH 7.5.

Dilute further immediately before use to give ΔA/min of 0.15-0.20.

Procedure

Adjust spectrophotometer to 600 nm and 25°C.

Pipette into cuvettes as follows:

  Test Blank
0.2 M Tris⋅HCl, pH 7.5 0.3 ml 0.3 ml
0.006 M NADH 0.1 ml 0.1 ml
Water 2.4 ml 2.5 ml
Mix thoroughly and incubate in spectrophotometer to achieve temperature equilibration and add simultaneously:
Dye 0.1 ml 0.1 ml
Sample 0.1 ml -----

Mix quickly and measure the decrease in absorbance at 600 nm for 2-3 minutes.

Determine the ΔA/min. from the initial linear portion of the curve. (Use portion of curve from t=0 to t=1 minute; the rate is linear for 1/2 to 1 minute.)

Calculation:

calculation

Up: Worthington Enzyme Manual