GLYCEROL KINASE from Microorganism


Appearance White amorphous powder, lyophilized
Activity GradeⅢ 30 U/mg-solid or more
Contaminants Catalase ≤1.0×10⁻¹%
NADH oxidase ≤1.0×10⁻³%
Adenosine triphosphatase ≦1.0×10⁻³%
Stability Stable at -20°C for at least One year (Fig.1)
Molecular weight approx. 220,000 (by gel filtration)
Structure Four subunits of approx. 58,000
Isoelectric point 4.3
Michaelis constants 9.4×10⁻⁵M (Glycerol), 1.3×10⁻⁵M (ATP),
2.1×10⁻³M (Dihydroxyacetone)
Inhibitors p-Chloromercuribenzoate, Hg⁺⁺, Ag⁺
Optimum pH 10.0(Fig.2)
Optimum temperature 70°(Fig.3)
pH Stability pH 5.5-10.0 (25°C, 20hr)(Fig.4)
Thermal stability below 65°C (pH 7.5, 30min)(Fig.5)
Substrate specificity (Table 1)
Effect of various chemicals (Table 2)


This enzyme is useful for enzymatic determination of glycerol and triglyceride when coupled with glycerol-3-phosphate oxidase (=G-3-P oxidase, G3O-321) or pyruvate kinase and lactate dehydrogenase (LCD-209, LCD-211, LCD-221), lipoprotein lipase (LPL-311, LPL-314) in clinical analysis.




glycerol kinase

Glycerol+ATP                                                  Glycerol-3-P+ADP



Glycerol-3-P+O₂                                           Dihydroxyacetone-P+H₂O₂


2H₂O₂+4-Aminoantipyrine+Phenol                                          Quinoneimine dye+4H₂O

The appearance of quinoneimine dye is measured at 500nm by spectrophotometry.

Unit definition:

One unit causes the formation of one micromole of hydrogen peroxide (half a micromole of quinoneimine dye) per
minute under the conditions described below.


A. Glycerol solution 0.3M (Should be prepared fresh)
B. 4-AA solution 0.1 % (100mg of 4-aminoantipyrine / 100ml of H₂O)
C. Phenol solution 0.1 % (100mg of phenol / 100ml of H₂O)
D. Peroxidase solution 20mg Peroxidase (110 purpurogallin units/mg)/100ml of H₂O
E. G-3-POD solution 20U/ml (dissolve in 200 mM HEPES buffer, pH 7.9)
F. Buffer solution 200mM HEPES, pH 7.9 contg. 20mM MgCl₂ and 40mM ATP (should be prepared freshly)
G. Enzyme diluent 20mM K-phosphate buffer, pH 7.5


Concentration in assay mixture
HEPES buffer 95.2 mM
Glycerol 4.76 mM
ATP 3.81 mM
1.90 mM
4-AA 0.469 mM
Phenol 2.02 mM
Peroxidase ca.5.2 U/ml
G-3-POD ca.7.6 U/ml

1. Prepare the following working solution in a brownish bottle and store on ice.

10 ml 4-AA solution (B)
20 ml Phenol solution (C)
20 ml Peroxidase solution (D)
40 ml G-3-POD solution (E)
10 ml Buffer solution (F)

2. Pipette 3.0 ml of working solution in a cuvette (d=1.0cm).

3. Add 0.1ml of enzyme solution*, mix by gently inversion and equilibrate at 37°C for about 5 minutes.

4. Add 0.05ml of glycerol solution (A) and mix by gentle inversion.

5. Record the optical density at 500nm against water for 3 to 4 minutes in a spectrophotometer thermostated
at 37°C, and calculate ΔOD per minute from the initial portion of the curve (ΔOD test).
At the same time, measure the blank rate (ΔOD blank) by the same method as test except the enzyme
diluent is added instead of the enzyme solution.

* Dissolve the enzyme preparation in ice-cold enzyme diluent (G) and dilute to 0.2-0.4U/ml with the same buffer, immediately before assay.


Activity can be calculated by using the following formula :

ΔOD/min (OD test−OD blank ) ×Vt × df

Volume activity (U/ml) =                                                          =ΔOD/min ×4.74×df


Weight activity (U/mg)=(U/ml)×1/C

: Total volume (3.15ml)
: Sample volume (0.1ml)
: Millimolar extinction coefficient of quinoneimine dye under the assay condition (㎠/micromole)
: Factor based o the fact that one mole of H₂O₂ produces half a mole of quinoneimine dye
: Light path length (cm)
: Dilution factor
: Enzyme concentration in dissolution (c mg/ml)


  1. H.-S.Huang, T.Yoshida, Y.Meng, T.Kabashima, K.Ito, Y.Nishiya, Y.Kawamura, and T.Yoshimoto; J.Ferment.Bioeng., 83, 328 (1997).
Table 1. Substrate Specificity of Glycerol kinase
[Pyruvate kinase-Lactate dehydrogenase system with 50mM HEPES buffer, pH 7.9]
Substrate (4.5mM) Relative activity(%) Substrate (4.5mM) Relative activity(%)
Glycerol 100 2,3-Butanediol 0.2
Glycerol-α-monochlorohydrin 0.1 D-Mannitol
Ethylene glycol D-Sorbitol
1,2-Propanediol D-Glucose
1,3-Propanediol 0.2 Ribitol
1,3-Butanediol Methanol
1,4-Butanediol 0.1 Ethanol
Table 2. Effect of Various Chemicals on Glycerol kinase
[The enzyme dissolved in 20mM K-phosphate buffer, pH 7.5 (100U/ml) was incubated with each chemical at 25°C for 1hr.]
Chemical Concn.(mM) Residual
Chemical Concn.(mM) Residual
None 100 MIA 1.0 101
Metal salt 1.0   NaF 1.0 100
MgCl₂   100
NaN 1.0 106
  102 EDTA 5.0 100
Ba(OAc)₂   101
o-Phenanthroline 1.0 102
FeSO₄   98 α,α′-Dipyridyl 1.0 101
FeCl₃   89 Borate 50 103
CoCl₂ 104 IAA 1.0 99
MnCl₂   99 NEM 1.0 100
ZnCl₂   103 Hydroxylamine 1.0 99
Cd(OAc)₂   101 Triton X-100 1.0% 103
NiCl₂   98 Brij 35 0.1% 104
CuSO₄   99 Tween 20 0.1% 103
Pb(OAc)₂   100 Span 20 0.1% 102
AgNO₃   10 Na-cholate 0.5% 105
HgCl₂   2 SDS 0.5% 1
1.0 100 DAC 0.5% 84
PCMB 1.0 0      

Ac, CHCO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate; IAA, Iodoacetamide; NEM, N-Ethylmaleimide; SDS, Sodium dodecyl sulfate; DAC, Dimethyl-benzyl-alkyl-ammonium chloride.

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