NIPRO ENZYMESGLYCEROKINASE (GlyK)[EC 2.7.1.30]
from recombinant E. coli
Glycerol + ATP ↔ Glycerol-3-phosphate + ADP
SPECIFICATIONStateSpecific activity
PROPERTIES
Subunit molecular weight
Optimum pH
pH stability
Thermal stability
Optimum temperature
Michaelis constants: Lyophilized: more than 80 U/mg protein: ca. 54,700: 9.8(Fig. 1)
: 5.5 - 9.0(Fig. 2)
: No detectable decrease in activity up to 40 °C.
(Fig. 3): above 50 °C(Fig. 4): (186 mM Glycine-Hydrazine-KOH buffer pH 9.8, at 30 °C)
Glycerol
0.026 mM
ATP
0.025 mM

STORAGE
Stable at -20 °C for at least six months
APPLICATIONThe enzyme is useful for enzymatic determination of glycerol and triglyceride when coupled withglycerol-3-phosphate dehydrogenase

NIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 340 nm according to the following reactions.
Glycerol + ATP

GlyK

Glycerol-3-phosphate + ADP

Glycerol-3-phosphate + NAD+

G3PDH

Dihydroxyacetone phosphate + NADH + H+

Unit Definition
One unit of activity is defined as the amount of GlyK that forms 1 μmol of NADH per minute at30 °C.
Solutions
Ⅰ Buffer solution ; 200 mM Glycine-Hydrazine-KOH, pH 9.8 (Dissolve 1.5 g glycine and 5 mLhydrazine hydrate in 80 mlmL distilled water. After adjusting pH to 9.8 with 1 M KOH, fill up to 100
mL with distilled water.)
Ⅱ MgCl2 solution ; 100 mM (2.03 g MgCl2∙6H2O/100 mL distilled water)
Ⅲ ATP solution ; 100 mM (0.605 g ATP disodium salt∙3H2O/(8.2 mL distilled water + 1.8 mL 1 MNaOH))
Ⅳ NAD+ solution; 100 mM (0.663 g NAD+ free acid/10 mL distilled water)
Ⅴ Glycerol-3-phosphate dehydrogenase ; 1700 U/mL (from rabbit muscle, Roche Diagnostics)
Ⅵ Glycerol solution ; 330 mM (3.04 g Glycerol/100 mL distilled water)
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute the enzyme solution to 0.1 to 1.0U/mL with 50 mM Tris-HCl buffer pH 9.0 containing 0.1 % bovine serum albumin.
Procedure
1. Prepare the following reaction mixture and pipette 2.70 mL of reaction mixture into a cuvette.
SolutionⅠ
27.90 mL
SolutionⅣ 0.15 mL
SolutionⅡ0.57 mLSolutionⅤ 0.30 mL
SolutionⅢ
0.39 mL
SolutionⅥ 0.30 mL
Distilled water
0.39 mL
2. Incubate at 30 °C for about 3 minutes.
3. Add 0.015 mL of enzyme solution into the cuvette and mix.
4. Read absorbance change at 340 nm per minute (∆Abs340) in the linear portion of curve.
Calculation
Volume activity (U/mL) =(∆Abs340) X (2.70 + 0.015)
6.22 X 0.015

Specific activity (U/mg protein) =X d.f.

Volume activity (U/mL)
Protein concentration (mg/mL)*

d.f. ; dilution factor
6.22 ; millimolar extinction coefficient of NADH (cm2/μmol)*Protein concentration ; determined by the absorbance at 280 nm (Abs280),
where 1 Abs280 = 1 mg/mL
REFERENCE
1. Mike J. Comer, Chris J. Bruton, and Tony Atkinson ; J. App. Biochem. 1, 259-270 (1979)

NIPRO ENZYMES

100

Remaining activity (%)

Relative activity (%)

10050045678910115001245678910pH

pHFig. 1 pH profile

Fig. 2 pH stability

1112

treated for 24 hr at 4 ℃ in thefollowing buffer solution (0.1 M);△ MES, ○ phosphate,● Tris-HCl, ■ GlycineHydrazine, ◇ Bicine△ MES, ○ phosphate,● Tris-HCl, ◇ Bicine,▲ Glycine-Hydrazine

120
100

Relative activity (%)

Remaining activity (%)

10050806040200304050607080

Temperature (℃)

Fig. 3 Thermal stability
treated for 15 min in 100 mM
Bicine buffer, pH 8.59001020304050

Temperature (℃)

Fig. 4 Thermal activity60