(Diagnostic Reagent Grade) ASAHI KASEI ENZYMES T-64REACH compliant product

GLYCEROL KINASE [GKZ]

from Flavobacterium meningosepticum
(ATP: Glycerol–3–phosphotransferase, EC 2.7.1.30)

Glycerol + ATP → L–α–Glycerolphosphate + ADP

★	Advantage
	①	Thermal stability
	②	Stability in solution
	③	Antiseptic stability

Preparation and Specification

Appearance: : White to light grayish amorphous powder, lyophilized

Specific activity: : More than 70 U/mg solid

Properties

Substrate specificity: : See Table 1

Molecular weight: : 150 kDa (TSK G3000SWXL)
50 kDa (SDS–PAGE)

Isoelectric point: : pH 4.3

Michaelis constants: : Glycerol 8.8 × 10^-5M
ATP 3.0 × 10^-5M

Optimum pH: : 8.0Figure 1

pH stability: : 5.0–11.0 (37℃, 60 min) Figure 2

Optimum temperature: : 80℃ (Tris–HCl buffer) Figure3

Thermal stability: : Stable at 60℃ and below (pH 6.5, 10 min)
Comparison data between GKZ and GKFigure4

Antiseptic stability: : See Figure 5

Reactivity after long incubation: : See Figure 6

Applications for Diagnostic Test

This enzyme is useful for enzymatic determination of triglyceride.

	LPBP
TG + 3 H₂O	→	Glycerol + 3 Fatty acid
	GKZ
Glycerol + ATP	→	G - 3 - P + ADP
	GPOSP
G - 3 - P + O₂	→	DHAP + H₂O₂
	POD
H₂O₂ + 4-AA + Phenol	→	Quinoneimine dye + 4 H₂O

TG: Triglyceride, DHAP: Dihydroxyacetone phosphate

Table 1. Substrate Specificity

Substrate	Relative activity (%)
Glycerol	100
Glycerol–α–monochlorohydrin	0
Ethylene glycol	0
1,3–Propanediol	0
1,3–Butanediol	0
1,4–Butanediol	0
1,2–Butanediol	0
d–Mannitol	0
d–Sorbitol	0
d–Glucose	0
Robitol	0

Fig.1 pH Optimum

●: Phosphate buffer
〇: Tris-HCl buffer
▲: Glycine-NaOH buffer

Fig.2 pH Stability

37℃, 60min
■: 3,3-Dimethylglutarate-NaOH buffer
〇: Tris-HCl buffer
▲: Glycine-NaOH buffer

Fig.3 Optimum temperature

pH 8.0
100 mM Tris-HCl buffer

Fig.4 Thermal stability of GKZ

pH 6.5, 10min.
50 mM PIPES
●: GKZ(T-64)
▲: GK

Fig.5 Resistance of GKZ against antiseptics

1：NaN₃ (0.03%)
2：Benzalkonium chloride (0.001%)
3：Gentamicin (0.0005%)

4：Chloramphenicol (0.0005%)
5：Sulphamethizole (0.0005%)
6：Dihydrostreptomycin (0.0005%)

Fig.6 Reactivity of GKZ after long-term incubation

■:	initial
●:	After 8 days incubation
▲:	After 28 days incubation

Storage conditions:

25℃, 1.2 U/ml GKZ or 0.4 U/ml GK(existing product)

50 mM PIPES pH 6.5, 3 mM ATP, 2 mM MgCl2, 0.05% antliseptic

Autoanalyzer: HITACHI 7150

Sample: Glycerol solution

Notes:

The reason for the high absorbance number comes from co-existence of TOOS and 4-amino antipyrine in the first reagents.

Assay

Principle

The assay is based on the increase in absorbance at 366 nm as NADH is produced in the following reactions:

	GKZ
Glycerol+ATP	→	Glycerol–3–P+ADP
	G3PDH
Glycerol–3–P+NAD⁺	→	Dihydroxyacetone phosphate+NADH+H⁺

ATP : Adenosine triphosphate
NAD : Nicotineamido adenine dinucleotide
G3PDH : Glycerol–3–phosphate dehydrogenase

Unit definition

One unit is defined as the amount of enzyme which converts 1 μmole of glycerol to glycerol–3–phosphate per minute at 37℃ under the conditions specified in the assay procedure.

Reagents

Substrate–coenzyme mixture

80 mM ATP solution
pH 7.0

0.1 M NAD solution 2.0 ml

0.1 M Glycerol solution 2.0 ml
Buffer solution

Glycine–hydrazine buffer containing 1.5mM MgCl ₂ pH 9.8

Dissolve 12.5g hydrazine hydrate and 1.5g glycine with about 80ml distilled water. Add 1.5ml 0.1M MgCl₂ solution and adjust pH to 9.8 with 1N HCl at 25℃. Add distilled water to make a total of 100ml.
G3PDH solution
Use G3PDH (NH₄) ₂SO₄ suspension (undiluted)
Enzyme dilution buffer
10 mM KH₂PO₄–NaOH buffer pH 7.0 containing
10 mM glycerol
Reagents
ATP: Kyowa Hakko Co., Ltd. Hydrazine hydrate:
Tokyo Kasei Kogyo Co., Ltd. Purity 79% #H0204
NAD: NACALAI TESQUE, INC. #24334–84
G3PDH: Roche Diagnostics GmbH
(NH4) 2SO4 suspension #10127752001

Enzyme solution

Accurately weigh about 20 mg of the sample and add enzyme dilution buffer to make a total of 20 ml. Dilute it with enzyme dilution buffer to adjust the concentration as required.

Procedure

Pipette accurately 1.80 ml of buffer into a small test tube, add 0.15 ml of substrate/coenzyme mixture and 20 μl of G3PDH, mix immediately.
After mixing, preincubate at 37℃.
After 5 min, add 30 μl of enzyme solution and mix to start the reaction at 37℃.

※ In the case of a test blank, add 30 μl of enzyme dilution buffer in place of enzyme solution.
After starting the reaction, measure the rate of increase per minute in absorbance at 366 nm. The rate must be measured within the linear portion of the absorbance curve.

Absorbance sample : As/min

blank : Ab/min

△A/min = (As/min−Ab/min) ≦ 0.050 Abs/min

Calculation

Activity (U/mg of powder) = {(△A/min)/3.3} × 2.00/0.03 × 1/x

3.3 :	milimolar extinction coefficient of NADH at 366 nm (cm²/μmole)
2.00 :	final volume (ml)
0.03 :	volume of enzyme solution (ml)
X :	concentration of the sample in enzyme solution ( mg/ml)

Storage

Storage at −20℃ in the presence of a desiccant is recommended.

References

Sakasegawa, S., Yoshioka, I., Koga, S., Takahashi, M., Matsumoto, K., Misaki, H. and Ohshima, T. (1998) Biosci, Biotechnol, Biochem., 62, 2388-2395

GKZ 活性測定法 (Japanese)

試薬液

基質、補酵素混合液

80mM ATP 溶液 pH7.0
2.0 ml

0.1M NAD 溶液
2.0 ml

0.1M グリセロール溶液
2.0 ml

緩衝液 (1.5mM MgCl ₂ を含むグリシン–ヒドラジン緩衝液　pH9.8)

	ヒドラジン一水和物 (純度79%) 12.5g とグリシン1.5g および0.1M 塩化マグネシウム溶液 1) 1.5ml を精製水80ml に溶解した後、1N HCl でpH9.8 (25℃) に調整し、精製水で全容100ml とする。
1) ：	塩化マグネシウム溶液塩化マグネシウム203mg を精製水で溶解して全容10ml とする。
	又はヒドラジン一水和物 (純度79%) 12.5g とグリシン1.50g 及び0.1M 塩化マグネシウム溶液1) 1.50ml を精製水で溶解して全容100ml とし、pH が9.8 ± 0.05 (25℃) であればそのまま使用する。

G3PDH 溶液
ロシュ製のG3PDH 懸濁液をそのまま使用する。
酵素溶解希釈用液
10mM グリセロールを含む10mM KH₂PO₄–NaOH
緩衝液 pH7.0
試薬
ATP (アデノシン三リン酸・2Na・3H₂O) ：
協和発酵製
ヒドラジン一水和物：
東京化成工業製　純度79%　#H0204
NAD (ニコチンアミドアデニンジヌクレオチド) ：
ナカライテスク製　#24334–84

G3PDH (グリセロ–3–リン酸脱水素酵素) ：
ロシュ製　硫安懸濁液

酵素試料液

検品約20mg を精密に量り、酵素溶解希釈用液で溶解して全容20ml とする。
その液を酵素溶解希釈用液で適宜希釈する。

測定操作法

小試験管に緩衝液1.80ml を正確に分注して、加温する直前に基質、補酵素混合液0.15ml とG3PDH 溶液20 μl を分注して混和した後、直ちに37℃で予備加温する。
5 分経過後、酵素試料液30 μl を正確に加えて混和し、直ちに37℃で反応を開始する。

※ 盲検は酵素試料液の代わりに酵素溶解希釈用液30μl を加える。
反応開始後、366nm における吸光度を測定して直線的に反応している1 分間当たりの吸光度変化を求める。
求められた吸光度変化を試料液はAs/min、盲検液は
Ab/min とする。
ΔA/min = (As/min−Ab/min) ≦ 0.050 Abs/min

計算

活性 (U/mg) = {(△ A/min)/3.3} × 2.00/0.03 × 1/x

3.3 :	NADH の366nm におけるミリモル分子吸光係数 (cm² / μmole)
2.00 :	反応総液量 (ml)
0.03 :	反応に供した酵素試料液量 (ml)
X :	酵素試料液中の検品濃度 (mg/ml)

80mM ATP 溶液 pH7.0	2.0 ml
0.1M NAD 溶液	2.0 ml
0.1M グリセロール溶液	2.0 ml

Absorbance sample :	As/min
blank :	Ab/min

GLYCEROL KINASE [GKZ] (T-64)