Native Ascorbate Oxidase from Microorganism

Products

Native Ascorbate Oxidase from Microorganism

Product Specifications

Cat#	NATE-1014
Descriptions	Stable Ascorbate Oxidase rapidly eliminates ascorbic acid interference in clinical serum samples, which otherwise suppress chromogenic signals and lower diagnostic detection accuracy. This enzyme is added as a protective auxiliary reagent to all colorimetric diagnostic kits vulnerable to antioxidant disturbance, greatly improving result reliability for routine biochemical testing.
Applications	This product can be used for enzymatic measurement of ascorbic acid and elimination of ascorbic acid interference in clinical diagnostic biochemical assays.
Synonyms	L-Ascorbate: oxygen oxidoreductase
EC Number	EC 1.10.3.3
Catalysis	L-Ascorbic acid + O₂ → L-Dehydroascorbic acid + H₂O₂
Activity	≥ 200 U/mg (Ascorbate oxidase activity)
Appearance	Light yellowish white to light yellow powder, lyophilized
Additive	Lactose
Molecular Weight	130,000 (Gel filtration)
Isoelectric Point	9.0
Km	4.9 × 10⁻⁴ M (L-Ascorbic acid)
Optimum pH	6.0
pH Stability	3.0-8.0 (37°C, 16 h)
Optimum Temperature	37°C
Thermal Stability	up to 50°C (pH 8.6, 16 h)
Inhibitors	Fe²⁺, Fe³⁺, Monoiodoacetate, N-Ethylmaleimide
Unit Definition	1 unit is defined as the enzyme quantity which oxidizes 1 μ mole of L-Ascorbic acid per minute under the conditions described as follows.
Storage	24 months from the date of analysis when stored at -20°C or below in a dry place under sealed conditions.

General Properties

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Assay Principle

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Activity Assay

Unit Definition

1 unit is defined as the enzyme quantity which oxidizes 1 μ mole of L-Ascorbic acid per minute under the conditions described as follows.

Reagents

A. 0.2 M HCl solution
B. 1 mM HCl solution (containing 1mM EDTA)
C. 10 mM Na2HPO4 solution
D. 0.2 M KH2PO4 solution (containing 1mM EDTA)
E. 10 mM KH2PO4 solution (containing 0.05% BSA): Weigh 1.36 g of KH2PO4 and 0.50 g of bovine serum albumin, then dissolve in approx. 500 mL of deionized water. Fill up to 1000 mL with deionized water. (Can be used for 14 days if kept refrigerated)
F. Substrate stock solution: Weigh 88.0 mg of L-ascorbic acid and dissolve in approx. 40 mL of 1 mM HCl solution (B). Fill up to 50 mL with 1 mM HCl solution (B). (Can be used for 1 month if stored refrigerated and away from light)
G. Substrate solution Accurately measure out 5 mL of Substrate stock solution (F), dilute with 0.2 M KH2PO4 solution (D), and fill up to 50 mL. (Must store refrigerated and away from light, use within 1 hour after preparation)
H. Enzyme solution: Weigh out Ascorbate Oxidase and dissolve in chilled 10 mM KH2PO4 solution (E). Enzyme solution should be prepared so that the value of (A0-A5) becomes in the range of 0.2±0.10. (Use it within 3 hours after preparation)

Procedure

Pipette 0.5 mL of Substrate solution (G) and 0.5 mL of 10 mM Na2HPO4 solution (C) respectively into a test tube and mix, then keep at 37±0.5℃ for 5 minutes in a water bath. After this pre incubation, pipette 0.1 mL of Enzyme solution (H) into the test tube and mix well, then keep at 37±0.5℃. At exactly 5 minutes, pipette 3 mL of 0.2 M HCl (A) and mix well to stop the reaction. Measure the absorbance of the reaction mixture at 245 nm (A5). As a blank, pipette 0.5 mL of Substrate solution (G) and 0.5 mL of 10 mM Na2HPO4 solution (C) into another test tube respectively and mix, keep at 37±0.5℃ for 10 minutes. Then, pipette 3 mL of 0.2 M HCl (A) and mix well. Pipette 0.1 mL of Enzyme solution (H) into the mixture and measure the absorbance at 245 nm (A0). It is recommended that the enzyme reaction and blank process be carried out at the same time to prevent variation in measured values.

Calculation

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Safe Handling Avoid inhalation; in the event of direct skin or eye contact, immediately flush the affected area with abundant water, and consult the SDS for further information.