Native 3-Hydroxybutyrate Dehydrogenase from Microorganism

Products

Native 3-Hydroxybutyrate Dehydrogenase from Microorganism

Product Specifications

Cat#	NATE-1011
Descriptions	3-Hydroxybutyrate Dehydrogenase specifically identifies blood β-hydroxybutyrate, the primary marker of diabetic ketosis. The purified microbial enzyme delivers high sensitivity to low ketone body concentrations in plasma samples, supporting early warning diagnosis of diabetic ketoacidosis for emergency and endocrinology clinical diagnostic testing systems.
Applications	This product can be utilized for enzymatic measurement of ketone bodies including D-3-Hydroxybutyrate and Acetoacetate in plasma during clinical diagnosis.
Synonyms	(R)-3-Hydroxybutanoate: NAD⁺ oxidoreductase
EC Number	EC 1.1.1.30
Catalysis	D-3-Hydroxybutyrate + NAD⁺ ⇆ Acetoacetate + NADH + H⁺
Activity	≥ 75 U/mg (HBDH activity)
Appearance	White to light brown powder, lyophilized
Additive	Not added
Molecular Weight	27,000 (SDS-PAGE); 75,000-80,000 (Gel filtration)
Isoelectric Point	4.8
Km	5.0 × 10⁻⁴ M (D-3-Hydroxybutyrate); 4.2 × 10⁻⁵ M (NAD⁺)
Optimum pH	8.5
pH Stability	6.0-10.0 (37°C, 1 h)
Optimum Temperature	55°C
Thermal Stability	up to 37°C (pH 7.0, 1 h)
Inhibitors	Fe²⁺, N-Ethylmaleimide
Unit Definition	1 unit is defined as the enzyme quantity which produces 1 μ mole of NADH per minute under the conditions described below.
Storage	36 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 produces 1 μ mole of NADH per minute under the conditions described below.

Reagents

A. 0.1 M Tris-HCl buffer (pH 8.5)
B. Hydrazine sulfate solution: Weigh 75 mg of Hydrazine Sulfate and dissolve in 0.1 M Tris-HCl Buffer (A), then adjust to pH 8.5 with 4N NaOH. Fill up to 100 mL with 0.1 M Tris-HCl Buffer (A). (Make a fresh solution for each use.)
C. Triton X-100 stock solution (5g/dL deionized water)
D. Substrate solution: Weigh 151.2 mg of (+/-)-3-Hydroxybutyric acid sodium salt and dissolve in 10 mL of deionized water. (Make a fresh solution for each use.)
E. NAD solution: Weigh 86.1 mg of β-NAD+ and dissolve in 10 mL of deionized water. (Make a fresh solution for each use.)
F. Diluent (0.1 M Tris-HCl buffer (pH 8.5) containing 0.1% Triton X-100 and 0.1% BSA): Weigh 12.1 g of Tris (hydroxymethyl) aminomethane and 1.0 g of BSA, dissolved in approx. 800 mL of deionized water. Add 20 mL of Triton X-100 stock solution (C), then adjust to pH 8.5 with 4N HCl. Fill up to 1 L with deionized water. (Can be used for 14 days if kept refrigerated)
G. Enzyme solution: Weigh out 3-Hydroxybutyrate Dehydrogenase and dissolve in chilled Diluent (F). Enzyme Solution should be prepared so that the value of ⊿OD/minute becomes in the range of 0.040±0.010.

Procedure

Pipette 2.0 mL of Hydrazine sulfate solution (B), 0.5 mL of Substrate solution (C) and 0.5 mL of NAD solution (E) respectively into a quartz cell (d =10 mm) and keep at 25±0.5℃ for 5 minutes. Then, pipette 0.05 mL of Enzyme solution (G) into the quartz cell and mix well immediately. Keep the reaction mixture at 25±0.5℃. Exactly at 5 minutes and 8 minutes after the addition of Enzyme solution (G), measure the absorbances of the reaction mixture at 340 nm (A5 and A8). As a blank, pipette Triton X-100 solution (F) into another quartz cell (d =10 mm) instead of Enzyme solution (F) and take the same procedure described above (Ab5 and Ab8).

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.