Assay Procedure for Glycerokinase

Principle

The appearance of NADH is measured at 340nm by spectrophotometry.

Unit definition

One unit causes the formation of one micromole of NADH per minute under the conditions described below.

Method

Reagents

 

A. Glycine-hydrazine buffer, pH 9.8 :0.2M[Weigh 1.5g of glycine and 20.8g of hydrazine hydrate (MW= 50.06), dissolve in 70ml of H2O, add 0.4ml of 1.0M MgCl2 and after adjusting the pH to 9.8 with 2.0N HCl or 2.0N KOH, fill up to 100ml with H2O]
B. Glycerol solution :0.1M (Should be prepared fresh)
C. ATP solution :0.1M (Should be prepared fresh)
D. NAD+ solution :14mM (Should be prepared fresh)
E. Glycerol-3-phosphate dehydrogenase(G-3-PDH) solution :Crystalline suspension in 3.2M ammonium sulfate solution
(10mg/ml, ca. 60 U/mg from Roche)
F. Enzyme diluent :20mM K-phosphate buffer pH 7.5 containing 0.2% BSA

Procedure

  1. Prepare the following reaction mixture in a cuvette (d=1.0cm) and equilibrate at 25℃ for about 5 minutes.

    3.0 ml 0.2M Glycine-hydrazine buffer, pH 9.8 (A)
    0.10ml Substrate solution                                 (B)
    0.05ml ATP solution                                            (C)
    0.10ml NAD+ solution                                       (D)
    0.03ml G-3-PDH solution                                  (E)

 

Concentration in assay mixture
Glycine buffer 0.18 M
Glycerol 3.0 mM
ATP 1.5 mM
NAD+ 0.42 mM
MgCl2 3.6 mM
G-3-P DH ca.5.4 U/ml
  1. Add 0.05ml of the enzyme solution* and mix by gentle inversion.
  2. Record the increase in optical density at 340nm against water for 3 to 4 minutes in a spectrophotometer thermostated at 25℃ and calculate theΔOD per minute from the initial linear portion of the curve (ΔOD test).

At the same time, measure the blank rate (ΔOD blank) by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution.

* Dissolve the enzyme preparation in ice-cold enzyme diluent (F), dilute to 0.2-0.4U/ml with the same buffer and store on ice.

Calculation

Activity can be calculated by using the following formula:

 

Weight activity (U/mg)=(U/ml)×1/C

 

Vt :Total volume (3.33ml)
Vs :Sample volume (0.05ml)
6.22
:Millimolar extinction coefficient of NADH (F/micromole)
1.0 :Light path length (cm)
df :Dilution factor
C :Enzyme concentration in dissolution (c mg/ml)

 

This procedure is for informational purposes. For a current copy of Sigma’s quality control procedure contact our Technical Service Department.

Materials

     
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