GLYCEROL KINASE from Microorganism

GYK-311

PREPARATION and SPECIFICATION
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%

PROPERTIES
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)

APPLICATIONS

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.

ASSAY

Principle:

glycerol kinase

Glycerol+ATP ► Glycerol-3-P+ADP

Mg++

G-3-POD

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

Peroxidase

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.

Method:

Reagents
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

Procedure

Concentration in assay mixture
HEPES buffer	95.2 mM
Glycerol	4.76 mM
ATP	3.81 mM
MgCl₂	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.

Calculation

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

13.3×1/2×1.0×Vs

Weight activity (U/mg)=(U/ml)×1/C

Vt: : Total volume (3.15ml)
Vs: : Sample volume (0.1ml)
13.3: : Millimolar extinction coefficient of quinoneimine dye under the assay condition (㎠/micromole)
1/2: : Factor based o the fact that one mole of H₂O₂ produces half a mole of quinoneimine dye
1.0: : Light path length (cm)
df: : Dilution factor
C: : Enzyme concentration in dissolution (c mg/ml)

REFERENCES

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 activity(%)	Chemical	Concn.(mM)	Residual activity(%)
None	−	100	MIA	1.0	101
Metal salt	1.0		NaF	1.0	100
MgCl₂		100	NaN₃	1.0	106
CaCl₂		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
Dithiothreitol	1.0	100	DAC	0.5%	84
PCMB	1.0	0

Ac, CH₃CO; 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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