1,5 – ANHYDROGLUCITOL – 6 – PHOSPHATE DEHYDROGENASE [AG6PDHⅡ] (T-95)

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(Diagnostic Reagent Grade) Nagase Diagnostics ENZYMES T-95REACH適合品

1,5 – ANHYDROGLUCITOL – 6 – PHOSPHATE DEHYDROGENASE [AG6PDHⅡ]

from Escherichia coli

1,5 – Anhydroglucitol – 6 – phosphate + NADP+C6H11O8P+NADPH + H+

Preparation and Specification

Appearance
: White amorphous powder, lyophilized
Specific activity
: More than 20 U/mg solid

Properties

Substrate specificity
: See Table 1
Molecular weight
: 78 kDa (TSK gel G 3000 SWXL gel filtration) 40 kDa (SDS–PAGE)
Isoelectric point
: pH 4.7
Michaelis constants
: 1,5–Anhydroglucitol–6–phosphate 25 mM (pH 10.0) NADP 0.09 mM (pH 10.0)
Optimum pH
: 9.0–10.0Figure 1
pH stability
: 7.0–9.0 (50℃, 30 min) Figure 2
Optimum temperature
: 37–50℃Figure 3
Thermal stability
: Stable at 42℃ and belowFigure 4

Applications for Diagnostic Test

This enzyme is useful for enzymatic determination of 1,5–AG.

ADP-HKT Ⅱ
ADP + 1, 5-AG→AMP + 1, 5-AG-6-P
AG6PDH Ⅱ
1, 5-AG-6-P + NADP+→C6H11O8P + NADPH + H+

Table 1. Substrate specificity

Substrate (5mM)Relative activity
(%)
1,5–Anhydroglucitol–6–phosphate100
Glucose–6–phosphate8
Fructose–6–phosphate0
Galactose–6–phosphate0
Mannose–6–phosphate0
Sorbitol–6–phosphate0
Glucose–1–phosphate0
Glucose–1, 6–diphosphate0
1,5–Anhydroglucitol0
Glucose0
Sorbitol0
myo–Inositol0

Table 2. Effect of various chemicals on AG6PDH activity

AdditivesConcentrationRelative activity
(%)
None–100
KCl100mM142
NaCl250mM113
CaCl21mM114
MgCl21mM118
MnCl21mM138
NH4Cl1mM97
MgSO41mM113
EDTA1mM111
Triton X–1000.1%99
Sodium Deoxycholate0.01%107

Fig.1 pH Optimum


50 mM buffer
□: Tris buffer
■: CAPS buffer

Fig.2 pH Stability


50℃, 30 min.
50 mM buffer
〇: Citrate buffer
●: Bis Tris buffer
□: Tris buffer
■: CAPS buffer

Fig.3 Optimum Temperature


pH 9.5
50 mM CAPS buffer

Fig.4 Thermal Stability


pH 9.5, 30min.
50 mM CAPS buffer

Assay

Principle

  1. The assay is based on the increase in absorbance at 340 nm as the formation of NADPH proceeds in the following reactions:

ADP–HKT Ⅱ
1,5–A+ADP→1,5–AG–6–P+AMP
 Mg2+
＊ 1,5–AG: 1,5–Anhydro–D–Glucitol
 
AG6PDH Ⅱ
1,5–AG–6–P+NADP +→C6H11O8P+NADPH+H +
 
DIP
NADPH+H ++NTB→NADP+NTBH2
Unit definition

  1. One unit is defined as the amount of enzyme which converts 1 μmole of 1,5–AG–6–phosphate to C6H11O8P per minute at 37 ℃ under the conditions specified in the assay procedure.

Reagents
  1. Reaction mixture–Ⅰ
    0.2M Tris–HCl buffer pH7.50.10 ml
    0.4M ADP solution (pH7.5)0.05 ml
    0.4M MgCl2 solution0.05 ml
    200U/ml ADP–HKT Ⅱ solution0.10 ml
    Distilled water0.15 ml
  1. Substrate solution (400mM 1,5–AG Solution)
    Dissolve 65.6mg of 1,5–AG with 1ml of distilled water.
  2. Reaction mixture–Ⅱ
    0.2M EDTA・2Na (pH10.0) solution0.05 ml
    1.0M Glycine–NaOH buffer pH10.00.10 ml
    2.0% Triton X–100 solution0.05 ml
    0.5% NTB solution0.05 ml
    100U/ml DIP solution0.05 ml
    20mM NADP solution0.10 ml
    Distilled water0.10 ml
  3. Enzyme dilution buffer
    10mM Tris–HCl Buffer pH9.0 (25℃)
  4. Reaction Stopper
    0.1N HCl
  5. Reagents
    Tris (hydroxymethyl) aminomethane:
    Sigma Chemical Co. #T–1503
    ADP (Adenosine diphosphate・2Na) : Oriental Yeast Co.Ltd.
    ADP–HKT Ⅱ: Nagase Diagnostics Co., Ltd. #T–93
    1,5–Anhydro–D–Sorbitol ( 1,5–Anhydro–D–Glucitol)
    FUJIFILM Wako Pure Chemical Corporation
    #012–13533
    EDTA・2Na: KISHIDA CHEMICAL Co., Ltd.
    #060–29133
    Glycine: FUJIFILM Wako Pure Chemical Corporation
    #077–00735
    Triton X–100: The Dow Chemical Company
    NTB (Nitrotetrazorium blue) : Dohjindo Laboratories
    #344–02033
  1. DIP (Diaphorase) : Nagase Diagnostics Co., Ltd.
    #T–10
    NADP (Nicotinamide adenine dinucleotide phosphate, oxidized form) :
    FUJIFILM Wako Pure Chemical Corporation
    #308–50463
Enzyme solution

  1. Accurately weigh about 10 mg of the sample and add enzyme dilution buffer to make a total of 10 ml.

    Dilute it with enzyme dilution buffer to adjust the concentration as required.

Procedure
  1. Pipette accurately 0.45 ml of reaction mixture–Ⅰ into a small test tube.
  2. Add 0.05 ml of substrate solution.
    ※In the case of a test blank, add 0.05 ml of distilled water in place of substrate solution.
    ※above the mixed solution keep on ice until use.
  3. Above the mixed solution incubate at 37 ℃ and after 15 min. add acculately 0.50 ml of reaction mixture–Ⅱ at 37 ℃.
  4. After 5 min. add accurately 0.05 ml of enzyme solution and mix to start the reaction at 37 ℃.
  5. At 10 min. after starting the reaction, add 2.0 ml of the reaction stopper to stop the reaction.
  1. Measure the absorbance at 550 nm.
    Absorbance sample :As
    blank :Ab
    △ A = (As−Ab) = 0.2–0.5 Abs.
Calculation
  1. Activity (U/mg of powder) = △ A/17.0 × 3.05/0.05 × 1/x
    17.0 :millimolar extinction coefficient of NTB at 550nm
    ( cm2 /μmol)
    3.05 :final volume (ml)
    0.05 :volume of enzyme solution (ml)
    10 :reaction time (min)
    X :concentration of the sample in enzyme solution
    (mg/ml)
Storage

  1. Storage at −80 ℃ in the presence of a desiccant is recommended.

AG6PDH Ⅱ活性測定法 (Japanese)

試薬液
  1. 反応試薬混合液−Ⅰ
    0.2M トリス−HCl 緩衝液pH7.5
    		0.10 ml
    0.4M ADP 溶液 (pH7.5)0.05 ml
    0.4M 塩化マグネシウム溶液0.05 ml
    200U/ml ADP–HKT Ⅱ溶液0.10 ml
    精製水0.15 ml
  2. 基質溶液 (400mM 1,5–AG 溶液)
    1,5–AG 65.6mg を精製水1.0ml で溶解。
  3. 反応試薬混合液−Ⅱ
    0.2M EDTA・2Na (pH10.0) 溶液
    		0.05 ml
    1.0M Glycine–NaOH 緩衝液pH10.00.10 ml
    2.0% トリトンX–100 溶液0.05 ml
    0.5% NTB 溶液0.05 ml
    100U/ml DIP 溶液0.05 ml
    20mM NADP 溶液0.10 ml
    精製水0.10 ml
  4. 酵素溶解希釈用液
    10mM トリス−HCl 緩衝液pH9.0 (25℃)
  5. 反応停止液
    0.1N HCl
  6. 試薬
    トリス (ヒドロキシメチル) アミノメタン：
    シグマ製　#T–1503
    ADP (アデノシンニリン酸・2Na) ：
    オリエンタル酵母製
  1. ADP–HKT Ⅱ：ナガセダイアグノスティックス製　#T–93
    1,5–Anhydro–D–Sorbitol
    (1,5–Anhydro–D–Glucitol と同) ：
    富士フイルム和光純薬製　#012–13533
    EDTA・2Na (エチレンジアミン四酢酸ニナトリウム)
    ：キシダ化学製 #060–29133
    Glycine (グリシン) ：
    富士フイルム和光純薬製　特級　#077–00735
    トリトンX–100：Dow Chemical 製
    NTB (ニトロテトラゾリウムブルー) ：
    同仁化学工業製　#344–02033
    DIP (ジアフォラーゼ) ：ナガセダイアグノスティックス製　#T–10
    NADP (ニコチンアミドアデニンジヌクレオチド・
    リン酸酸化型) ：
    富士フイルム和光純薬製　#308–50463
酵素試料液
  1. 検品約10mg を精密に量り、酵素溶解希釈用液で溶解して全容10.0ml 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
  1. 小試験管に反応試薬混合液–Ⅰを0.45ml を正確に分注する。
  2. 1,5–AG 基質液を0.05ml を正確に加えて混和する。
    ※盲検は1.5–AG 基質液の代わりに精製水0.05ml を加える。
    ※上記混合液は使用するまで氷中に保存する。
  1. 次に37℃で第一反応を開始し、15 分後反応試薬混合液−Ⅱを0.5ml ずつ正確に加えて混和し、更に37℃で5 分間放置する。
  2. 5 分経過後、酵素試料液0.05ml を正確に加えて混和し、37℃で反応を開始する。
  3. 10 分間酵素反応を行った後、反応停止液 (0.1N HCl)
    2.0ml を加えて混和し、反応を停止する。
    反応を停止後、550nm における吸光度を測定する。
    求められた吸光度変化を
    試料液についてはAs
    盲検液についてはAb とする。
    ※吸光度範囲：△ A = (As−Ab) ; 0.2～0.5Absの範囲とする。
計算
以下の計算式に従い、活性 (U/mg) を計算する。
活性 (U/mg) = △ A/17.0 × 3.05/0.05 × 1/10 × 1/x
17 :NTB の550nm におけるミリモル分子吸光係数
(cm2 / μmol)
3.05 :反応総液量 (ml)
0.05 :反応に供した酵素試料液量 (ml)
10 :酵素反応時間 (min)
X :酵素試料液中の検品濃度 (mg/ml)
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