D-3-Hydroxybutyrate Dehydrogenase

⇒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

1. 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.

2. 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.