GLUCOSE–6–PHOSPHATE　DEHYDROGENASE [G6PDHⅡ] (T-51)

T-51 PDF download
(Diagnostic Reagent Grade) Nagase Diagnostics ENZYMES T-51REACH適合品

GLUCOSE–6–PHOSPHATE　DEHYDROGENASE
[G6PDHⅡ]

from Bacillus sp.
(D–Glucose–6–phosphate: NADP+ 1–oxidoreductase, EC 1.1.1.49)

D–Glucose–6–phosphate + NADP+ →
D–Glucono–δ–lactone–6–phosphate + NADPH + H+

Preparation and Specification

Appearance
: White amorphous powder, lyophilized
Specific activity
: More than 100 U/mg solid

Properties

Substrate specificity
: See Table 1
Molecular weight
: 342 kDa (gel filtration)
Isoelectric point
: pH 6.13
Michaelis constants
: NADP+ 8.3 × 10-6M
G–6–P 1.2 × 10-4M
Optimum pH
: pH 8.4 (Tris–HCl) Figure 1
pH stability
: pH 6.0–8.0 (75℃, 15 min) Figure 2
Optimum temperature
: 75℃Figure3
Thermal stability
: Stable at 65℃ and below (pH 7.5, 15 min) Figure4
Effect of various
chemicals
: See Table 2
Inhibitors
: Mn2+, Cu2+, Al3+
Stabilizer
: BSA

Applications for Diagnostic Test

This enzyme is useful for enzymatic determination of glucose or ATP when coupled with hexokinase (T–50) .

 HK Ⅱ
D－Glucose + ATP→D－Glucose－6－phosphate + ADP
 G6PDH Ⅱ
D－Glucose－6－phosphate + NADP＋→D－Glucono－δ－lactone－6－phosphate+ NADPH + H＋

 

Table 1. Substrate specificity

SubstrateRelative activity
(%)
Glucose-6-phosphate100
Galactose-6-phosphate16
Mannose-6-phosphate33
Fructose-6-phosphate0
Glucose-1-phosphate0

 

Table 2. Effect of various chemicals on G6PDH II activity

AdditivesConsentrationRelative activity
(%)
None–100
NaCI10mM100
KCI10mM100
LiCI1mM100
MgCI210mM100
CaCl210mM100
BaCl210mM97
MnCl21mM42
EDTA1mM100
CuCl21mM22
Triton X-1001%155
Adekatol PC-81%161
Nikkol OP-101%155
Tetronic 7041%117

Fig.1 Optimum


40 mM buffer, 37℃
〇: Phosphate buffer
□: Tris-HCI buffer

Fig.2 pH Stability


40 mM buffer, 75℃, 15 min.
〇: Phosphate buffer
●: 3,3-Dimethylglutarate-NaOH buffer
□: Tris-HCI buffer

Fig.3 Optimum Temperature


pH 7.5
40mM Phosphate buffer

Fig.4 Thermal Stability


pH 7.5, 15 min.
40 mM Tris-HCI 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 reaction:

 G6PDH Ⅱ
D–Glucose–6–phosphate+NADP+→D–Glucono–δ–lactone–6–phosphate+ NADPH+ H+

NADP: Nicotineamide adenine dinucleotide phosphate
Unit definition

  1. One unit is defined as the amount of enzyme which oxidizes 1 μmole of D–glucose–6–phosphate to D–glucono–δ–lactone–6–phosphate per minute at 37℃ under the conditions specified in the assay procedure.

Reagents
  1. Reaction mixture
    0.2 M KH2PO4–K2HPO4 buffer pH 7.51.5 ml
    2.0% (W/V) BSA solution0.3 ml
    10 mM NADP solution0.3 ml
    0.1 M D–Glucose–6–phosphate solution0.3 ml
    Distilled water0.6 ml
  2. Enzyme dilution buffer
    10 mM KH2PO4–K2HPO4 buffer pH 7.5
  3. Reagents
    NADP (oxidized form) :
    FUJIFILM Wako Pure Chemical Corporation #308–50463
    D–Glucose–6–phosphate: Sigma Chemical Co. #G–7250
    BSA: Millipore Fraction V pH5.2　#81–053
Enzyme solution

  1. 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
  1. Pipette accurately 3.0 ml of reaction mixture into a small test tube and preincubate at 37℃.
  2. After 5 min, add exactly 50 μl of enzyme solution and mix to start the reaction at 37℃.
    ※In the case of a test blank, add 50 μl of enzyme dilution buffer in place of enzyme solution.
  3. After starting the reaction, measure the rate of increase per minute 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
    △A/min = As/min－Ab/min
    0.030 Abs/min ≦ △A/min ≦ 0.050 Abs/min
Calculation
  1. Activity (U/mg of powder) = {(△A/min)/6.22} × 3.05/0.05 × 1/x
    6.22 :millimolar extinction coefficient of NADPH at 340 nm
    (cm2/ μmole)
  1. 3.05 :final volume (ml)
    0.05 :volume of enzyme solution (ml)
    X :concentration of the sample in enzyme solution
    (mg/ml)
Storage

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

References
  1. Haberstich, H. V. and Zuber, H. (1971) Arch. Biochem. Biophys., 144, 245–252.
  2. Muramatsu, N. (1974) Arch. Microbiol., 98, 275–289.
  3. Ishaque, A., Milhausen, M., Levy, H. R. (1974) Biochem.
    Biophys. Res. Commun., 59, 894–901.
  4. Milhausen, M. and Levy, H. R. (1975) Eur. J. Biochem., 50, 453–461.
  5. Olive, C., Geroch, M. E. and Leuy, H. R. (1971) J. Biol. Chem., 246, 2043–2057.
  6. Coe, E. C. and Hsu, L. H. (1973) Biochem. Biophys. Res. Commun., 53, 66–69.
  7. Olive, C. and Levy, H. R. (1967) Biochemistry., 6, 730–736.
  8. Metzger, R. P., Metzger, S. A. and Parsons, R. L. ( 1972) Arch. Biochem. Biophys., 149, 102–109.

G6PDH Ⅱ活性測定法 (Japanese)

試薬液
  1. 反応試薬混合液
    0.2M KH2PO4–K2HPO4 緩衝液 pH7.51.5 ml
    2.0% (W/V) BSA 溶液0.3 ml
    10mM NADP 溶液0.3 ml
    0.1M G–6–P 溶液0.3 ml
    精製水0.6 ml
  2. 酵素溶解希釈用液
    10mM KH2PO4–K2HPO4 緩衝液 pH7.5
  3. 試薬
    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
    G–6–P (D–Glucose–6–phosphate) ：
    シグマ製　#G–7250
    BSA: Millipore 製　Fraction V pH5.2 #81–053
酵素試料液
  1. 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
  1. 小試験管に反応試薬混合液3.0ml を正確に分注し、37℃で予備加温する。
  2. 5 分経過後、酵素試料液50 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
    50 μl を加える。
  3. 反応開始後、340nm 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の試料液はAs/min、盲検液は
    Ab/min とする。
    0.030 Abs/min ≦ΔA/min = (As/min−Ab/min)
    ≦ 0.050 Abs/min
計算
活性 (U/mg) = {(△ A/min)/6.22} × 3.05/0.05 × 1/x
6.22 :NADPH の340nm におけるミリモル分子吸光係数
( cm2/ μmole)
3.05 :反応総液量 (ml)
0.05 :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)
T-51 PDF download