Native Bilirubin Oxidase from Microorganism

Products

Native Bilirubin Oxidase from Microorganism

Product Specifications

Cat#	NATE-1012
Descriptions	Native Bilirubin Oxidase uniformly oxidizes both conjugated and unconjugated bilirubin in serum samples to eliminate inherent bilirubin absorbance interference. It enables accurate direct quantitative bilirubin measurement for liver function diagnostic kits, featuring high catalytic activity and stable performance across icteric patient sample matrices.
Applications	This product can be adopted for enzymatic determination of bilirubin and removal of bilirubin interference in diverse clinical diagnostic detection systems.
Synonyms	Bilirubin: oxygen oxidoreductase
EC Number	EC 1.3.3.5
Catalysis	Bilirubin + 1/2O₂ → Bilverdin + H₂O
Activity	≥ 1.2 U/mg (Bilirubin oxidase activity)
Appearance	Light blue to light bluish green powder, lyophilized
Additive	Saccharose, (NH₄)₂SO₄
Molecular Weight	52,000 (Gel filtration)
Isoelectric Point	4.1
Km	1.9 × 10⁻⁴ M
Optimum pH	6.0-7.0
pH Stability	7.0-11.0 (37°C, 1 h)
Optimum Temperature	40-60°C
Thermal Stability	up to 55°C (pH 7.0, 30 min)
Inhibitors	FeCl₂, NaN₃, KCN
Stabilizers	EDTA, Aspartic acid
Unit Definition	1 unit is defined as the enzyme quantity which oxidizes 1 μ mole of albumin bound bilirubin per minute under the conditions described below.
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 albumin bound bilirubin per minute under the conditions described below.

Reagents

A. 0.05 M Phosphate buffer (Containing EDTA, pH 7.0): Weigh 6.80 g of KH2PO4 and 18.6 mg of EDTA･2Na, dissolve them in approx. 800 mL of deionized water. Adjust the pH to 7.0 with 4N NaOH and fill up to 1000 mL with deionized water.
B. Sodium cholate solution: Weigh 0.50 g of sodium cholate and dissolve in 0.05 M phosphate buffer (A). Fill up to 50 mL with 0.05 M Phosphate buffer (A). (Can be used for 1 month if kept refrigerated)
C. Substrate solution: Weigh approx. 30 mg of Albumin bound bilirubin and dissolve in 1.2 mLof 0.05 M Phosphate buffer (A). Store in a brown bottle. (Can be used for 6hrs after preparation if kept refrigerated) Substrate solution should be prepared so that the absorbance value at 460 nm (see the following method) is in the range of 0.730-0.740.
Pipette 3 mL of Sodium cholate solution (B) and 0.2 mL of Substrate solution (C) into a quartz cell (d = 10 mm), then measure the absorbance at 460 nm (A460). If the value exceeds the above range, Substrate solution should be remade using the weight value calculated by the following formula.: Weight value of Albumin bound bilirubin (mg) = 0.735 × 30 mg ÷ A460
D. Enzyme solution: Weigh out Bilirubin Oxidase and dissolve in chilled 0.05 M Phosphate buffer (A). Enzyme solution should be prepared so that the value of ΔOD/minute is in the range of 0.020±0.002.

Procedure

Pipette 3.0 mL of Sodium cholate solution (B) and 0.20 mL of Substrate solution (C) respectively into a quartz cell (d = 10 mm) and keep at 37±0.5℃ for 10 minutes. Pipette 0.10 mL of Enzyme solution (D) into the quartz cell and mix well immediately. Keep the reaction mixture at 37±0.5℃. Exactly at 1 minute and 3 minutes after the addition of Enzyme solution (D), measure the absorbances of the reaction mixture at 460 nm (A1 and A3). As a blank, pipette 0.05M Phosphate buffer (A) into another quartz cell (d = 10 mm) instead of Enzyme solution (D) and follow the same procedure described above (Ab1 and Ab3).

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.