Specialists in the Isolation and Purification of Horseradish Peroxidase and a Select Range of Enzymes

Assay Procedure for Ascorbate Oxidase  (Ref. No. FGAP020)

Based on that of Oberbacher and Vines in which the decrease of absorbance due to oxidation of ascorbate by AO (Ascorbate Oxidase) is measured at 265nm and 25^°C.

	AsOx
2L-ascorbate + O2		2 dehydroascorbate + 2H2O

Unit Definition

That amount of enzyme catalysing the oxidation of 1 micromole ascorbate per minute at 25^°C.

Reagents

A	0.1M Phosphate / EDTA Buffer pH5.6 Dissolve 12.84g KH2PO4 [MM 136.09],0.735g Na2HPO4 [MM 141.96] and 0.186g EDTA. Na2.2H2O [MM372.24] in 800ml distilled H2O, check pH5.6 (1N HCl or 1N NaOH), dilute to 1000ml with distilled H2O and recheck pH.
B	Substrate [0.005M Ascorbic Acid] Dissolve 4.4mg L-ascorbic acid [MM176.13] in 5ml ice-cold distilled H2O. Store on ice. (Prepare fresh daily).
C	Enzyme Dissolve 1-5mg enzyme / ml in buffer [A]. Immediately before assay, dilute to yield 0.185 £ u/ml < 0.278 (0.08 £ DA 265/min £ 0.12).

Procedure

Into 10mm quartz cells pipette the following at 25^°C.

	Blank	Test
Buffer [A]	3.0 ml	2.0 ml
Substrate [B]	0.1 ml	0.1 ml

Place blank cell (Prepare a fresh blank for each assay) in sample compartment, test cell in reference compartment and monitor absorbance over ± 3 minutes.

Enzyme [C] at zero time	-	0.1 ml
	3.1 ml	3.1 ml

Record apparent increase of absorbance at 265nm for ± 4 minutes - check temperature in cells.

Caclulation

	DA265 / min x 3.1 x dilution
Activity (u/ml =
	13.386 x 0.1

and

[e = 13.386;  cuvette volume = 3.1ml;  enzyme volume = 0.1ml]

Reference

Oberbacher M.F. and Vines H.M,: 1963 Nature 197 1203.