(Diagnostic Reagent Grade) ASAHI KASEI ENZYMES T-207REACH適合品

GLYCEROL-3-PHOSPHATE DEHYDROGENASE [G3PDHⅡ]

from Saccharomyces cerevisiae
(sn-glycerol-3-phosphate:NAD⁺ 2-oxidoreductase, EC 1.1.1.8)

sn -glycerol 3-phosphate + NAD⁺ ↔ glycerone phosphate + NADH + H⁺

Preparation and Specification

Appearance: : White lyophilized powder

Specific activity: : More than 37.0 U/mg solid

Contaminants: : NADH oxidase Less than 0.00015 %(U/U)

Properties

Substrate specificity: : The enzyme is specific for sn-glycerol 3-phosphate and NAD(H)

Molecular weight: : 45.6 kDa (SDS-PAGE)

Isoelectric point: : pH 4.8-5.3

Michaelis constants: : sn-glycerol 3-phosphate 1.4 × 10^-2 M (8.5 mM NAD)

: : NAD 4.6 × 10^-4 M (75 mM sn-glycerol 3-phosphate)

Optimum pH: : 9.0Figure 1

pH stability: : 6.0–7.0 (37℃, 60 min) Figure 2

Optimum temperature: : 40℃Figure3

Thermal stability: : Stable at 45℃ and below (pH 6.5, 30 min)Figure4

Inhibitor¹⁾: : ATP, ADP, fructose 1,6-bisphosphate

Applications for Diagnostic Test

The enzyme is useful for enzymatic determination of triglyceride (TG) coupled with LP (Lipase; T-01, T-63, or T-116) and GK (Glycerol kinase; T-64 or T-223).

	LP
TG + 3H₂O	→	Glycerol + 3 Fatty acid
	GK
Glycerol + ATP	→	G-3-P + ADP
	G3PDHⅡ
G-3-P + NAD⁺	→	DHAP + NADH + H⁺

G-3-P: sn-Glycerol 3-phosphate
DHAP: Dihydroxyacetone phosphate

Fig.1 pH Optimum

〇: TES-NaOH buffer
△: Bicine-NaOH buffer
●: CHES-NaOH buffer

Fig.2 pH Stability

37℃, 60 min
0.1M buffer containing 0.1% BSA
○ : MES-NaOH buffer
▲ : TES-NaOH buffer
◇ : Tris-HCl buffer
△ : CHES-NaOH buffer

Fig.3 Optimum Temperature

Fig.4 Thermal Stability

pH 6.5, 30 min
50mM Phosphate buffer
containing 0.1% BSA

Assay

Principle

This assay is based on the increase in absorbance at 340 nm as the formation of NADH proceeds in the following reactions:

	G3PDH Ⅱ
sn-glycerol 3-phosphate + NAD⁺	→	glycerone phosphate + NADH

NAD: Nicotinamide adenine dinucleotide

Unit definition

One unit is defined as the amount of enzyme which generates 1 μmole of NADH per minute at 37 ℃ under the conditions specified in the assay procedure.

Reagents

Reaction mixture
Combine 1.632 g of Bicine with about 70 ml of distilled water, and adjust pH to about 8.5 at about 25 ℃ with 1N NaOH (Bicine will dissolve after adjusting pH). Add 4.727 g of Disodium Glycerophosphate 5.5-hydrate and 0.564 g (based on pure product) of NAD, dissolve them, add distilled water to make up to about 90 ml, and adjust pH to 8.7 at
25 ℃ with 1N NaOH. Finally add distilled water to make up to 100 ml.
Enzyme dilution buffer
Dissolve 1.211 g of tris (hydroxymethyl) aminomethane with 800 ml of distilled water, and adjust pH to 7.5 at 25 ℃ with 1N HCl. Add 1 g of BSA, dissolve it, check the pH of the solution again, and add distilled water to make 1 L solution.
Reagent
Bicine: Dojindo Laboratories #347‒03282
Disodium Glycerophosphate 5.5-hydrate:
FUJIFILM Wako Pure Chemical Corporation Special grade #192-02055
Tris (hydroxymethyl) aminomethane:Sigma Chemical Co. #T‒1503
BSA: Millipore Fraction V pH 5.2 # 81‒053
NAD: FUJIFILM Wako Pure Chemical Corporation
#304-50443

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 2.00 ml of reaction mixture into a small test tube and preincubate at 37 ℃.
After 5 min. add accurately 20 μl of enzyme solution and mix to start the reaction at 37 ℃.

※ In the case of a test blank, add 20 μl of enzyme dilution buffer in place of enzyme solution.
After starting the reaction, measure the rate of increase per minutes in absorbance at 340 nm. The rate must be measured within the linear portion of the absorbance curve.

Absorbance sample : As/min

blank : Ab/min

ΔA/min =( As/min － Ab/min) ≦ 0.15Abs/min

Calculation

Activity (U/mg of powder) = (ΔA/min)/6.22 × 2.02/0.02 × 1/x

6.22 :	millimolar extinction coefficient of NADH at 340 nm ( cm² /μmole)
2.02 :	final volume (ml)
0.02 :	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

Albertyn J., van Tonder A., Prior BA. (1992) FEBS Lett., 308(2), 130-132.72, 972.
Miura, M., et al., (1989) J. Clin. Lab. Inst. Reag., 12(5),1005-1009.

G3PDH Ⅱ活性測定法 (Japanese)

試薬液

反応試薬混合液
Bicine 1.632 g を精製水約70 ml に混合し、1N　NaOH で約pH8.5 (約25℃) に調整する (pH 調整するとBicine は溶解する) 。さらにグリセロりん酸二ナトリウム5.5 水和物4.727 g とNAD 0.564 gを加えて溶解したあと、精製水を加えて全容を約90 ml とし、1N NaOH でpH8.7 (25℃) に調整する (NAD は純度換算する) 。最後に精製水で全容を100 ml とする。
酵素溶解希釈用液
トリス (ヒドロキシメチル) アミノメタン 1.211 gを精製水800ml に溶解した後、1N HCl でpH7.5 (25℃) に調整し、BSA を1 g 加えて溶解し、再度pH を確認した後、精製水で1 L とする。
試薬
Bicine (ビシン) ：同仁化学製 #347‒03282
グリセロりん酸二ナトリウム5.5 水和物：
富士フイルム和光純薬製特級 #192-02055
トリス (ヒドロキシメチル) アミノメタン：
シグマ社製 #T‒1503
BSA：Millipore 社製 Fraction V pH5.2 #81‒053
NAD (ニコチンアミドアデニンジヌクレオチド･酸化型) ：
富士フイルム和光純薬製 #304-50443

酵素試料液

検品約20 mg を精密に量り、酵素溶解希釈用液で溶解して全容20 ml 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

小試験管に反応試薬混合液 2.00ml を正確に分注し、37°Cで予備加温する。
5分経過後、酵素試料液 20 μlを正確に加えて混和し、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 20μl を加える。
反応開始後、340 nm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についてはAs/min、盲検液についてはAb/min とする。
ΔA/min = (As/min－Ab/min) ≦ 0.15 Abs/min

計算

活性 (U/mg) = (ΔA/min)/6.22 × 2.02/0.02 × 1/x

6.22 :	NADH の340nm におけるミリモル分子吸光係数 (cm² / μmol)
2.02 :	反応総液量 (ml)
0.02 :	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)

Absorbance sample :	As/min
blank :	Ab/min

GLYCEROL-3-PHOSPHATE DEHYDROGENASE [G3PDHⅡ] (T-207)