Diaphorase - Assay

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.)

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.