SPECIFICATION

PROPERTIES

APPLICATIONS

The enzyme is useful for the determination of lactate in clinical analysis and continuous lactate monitoring sensor.

ASSAY PROCEDURE

Principle

The disappearance of the blue color of DCIP by the reduction is measured spectrophotometrically at 520 nm.

Definition of unit

One unit (U) causes the reduction of one micromole of DCIP per minute under the conditions described below.

Reagents

1. L-Lactate solution, 50 mM: 0.56 g of sodium l-lactate/100 mL of distilled water.
2. Potassium phosphate buffer, 1 M: pH 7.5: mix 1 M KH₂PO₄ solution and 1 M K₂HPO₄ solution to make a pH 7.5 solution.
3. 2, 6-Dichloroindophenol (DCIP) solution, 1.8 mM: 58.7 mg of DCIP/100 mL of distilled water.
4. 5-Methylphenazinium methyl sulfate (PMS) solution, 30 mM: 91.9 mg of PMS/10 mL of distilled water.
5. Enzyme dilution buffer: 10 mM potassium phosphate buffer, pH 7.0, containing 0.15% bovine serum albumin(BSA).

Sample: dissolve the lyophilized enzyme to final concentration about 0.4 μg/mL with enzyme dilution buffer(Reagent E) immediately before measurement.

Procedure

1. Pipette the following reagents into a cuvette (light path: 1 cm).

   600 μL	l-Lactate solution	(Reagent A)
   340 μL	Potassium phosphate buffer pH 7.5	(Reagent B)
   500 μL	DCIP solution	(Reagent C)
   1360 μL	Distilled water

2. Equilibrate at 37℃ for about 3 min.
3. Add 0.1 mL of PMS solution (Reagent D) and mix.
4. Add 0.1 mL of sample and mix.
5. Record the decrease of absorbance at 520 nm against water for 1 min. (30–90 sec) in a spectrophotometer thermostated at 37℃, and calculate the ΔA per min using the linear portion of the curve (ΔA_S).
   The blank solution is prepared by adding Enzyme dilution buffer (Reagent E) instead of sample (ΔA₀).

Calculation

Activity can be calculated by using the following formula:

6.8 : Millimolar extinction coefficient of DCIP under the assay condition (cm²/μmol)
1.0 : Light pass length (cm)
df : Dilution factor

EXPERIMENTAL DATA

REFERENCES