
⇒D-3-Hydroxybutyrate dehydrogenase (EC 1.1.1.30, from Microorganism)
D-3-Hydroxybutyrate + NAD+ ⇒ Acetoacetate + NADH + H+
PROPERTIES
ASSAY PRINCIPLE
The appearance of NADH is measured at 340nm by spectrophotometry.
UNIT DEFINITION
One unit (U) is defined as the amount of enzyme which produces 1 μmol of NADH per min under the conditions described below.
REAGENTS PREPARATION
Reagent I: 0.1 M pH 8.5 Tris-HCl buffer.
Reagent II: 158mM D-3-Hydroxybutyrate solution, dissolved by Reagent I.
Reagent III: 27.9 mM NAD+, dissolved by Reagent I.
Enzyme diluent: 100 mM Tris-HCl, pH 8.5, contains 0.1% BSA.
Sample: dilute the enzyme to 0.1-0.5 U/ml with enzyme diluent.
PROCEDURE
- Add 2.3ml Reagent I, 0.5ml Reagent II and 0.2ml Reagent III to the 3 mL cuvette (d=1.0 cm) in order, preincubated at 37℃ for 5min.
- Add 0.1 mL the enzyme solution in the reaction mixture and mix to start the reaction, record the ΔAs at 340 nm in 1 minute in a spectrophotometer thermostated at 37 ℃.
At the same time, measure the blank rate ΔAb by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution.
∆A = ∆As – ∆Ab
CALCULATION
Vt: Total volume (3.1mL)
Vs: Enzyme volume (0.1mL)
1.0: Light path length (cm)
df: dilution factor
C: Enzyme concentration (mg/mL)
6.22: Millimolar extinction coefficient of NADH under 340 nm (cm2/μmol)
REFERENCES
(1) F.P. Delafield, K.E. Cooksey and M. Doudoroff; J. Biol. Chem., 240, 4023 (1965).
(2) C.W. Shuster and M. Doudoroff; J. Biol. Chem., 237, 603 (1962).
(3) I. Sekuzu, P. Jurtshuk and D.E. Green; J. Biol. Chem., 238, 975 (1963).
This enzyme is useful for enzymatic determination of ketone bodies (D-3-hydroxybutyrate and acetoacetate) in clinical analysis.
The Certificate Of Analysis (COA) & Material Safety Data Sheet (MSDS) Is A Signed Document That Includes The Storage Temperature,
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