FRUCTOSYLAMINE OXIDASE[FOD III] (T-191)

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(Diagnostic Reagent Grade) Nagase Diagnostics ENZYMES T-191REACH適合品

FRUCTOSYLAMINE OXIDASE[FOD III]

from Microorganism
(Ketoamine oxidase, EC 1.5.3)

D‒Glucose + acceptor → D‒glucono‒1,5-lactone + reduced acceptor

Preparation and Specification

Appearance
: Yellowish lyophilized powder
Specific activity
: More than 10 U/mg solid

Properties

Molecular weight
: 49 kDa (SDS-PAGE)
Michaelis constant
: 1.61 × 10-3M (Fructosyl valylhistidine)
Optimum pH
: See Figure 1
pH stability
: See Figure 2
See Figure 2
: See Figure 3
Thermal stability
: See Figure 4
Substrate specificity
: See Table 1
Effect of various chemicals on FOD Ⅲ activity
: See Table 2 and Table 3

Applications for Diagnostic Test

This enzyme is useful for the measurement of the glycated hemoglobin (HbA1c) in human whole blood.

Fig.1 Optimum pH


Fig.2 pH Stability


Fig.3 Optimum Temperature


Fig.4 Thermal Stability


 

Table 1. Substrate specificity

SubstrateRelative activity
(%)
Fructosyl Valine436.0
Fructosyl Valine-Histidine100.0
Fructosyl Valine-Leucine0.9
Fructosyl Valine-Histidine-Leucine0.0
Fructosyl Valine-Histidine-Leucine-Threonine0.0
Fructosyl Valine-Histidine-Leucine-Threonine-Proline0.0
Fructosyl Valine-Leucine-Threonine-Proline-Leucine0.0

 

Table 2. Effect of various chemicals on FODIII activity

AdditivesConcentrationRelative activity (%)
None-100
MgCl20.5mM101
MnCl20.5mM103
CaCl20.5mM103
LiCl0.5mM103
NaCl0.5mM110
CoCl20.5mM12
CoCl20.5mM44
KCl0.5mM107
EDTA1mM109
TritonX-1000.1%100
Sodium cholate0.1%98
Tween 800.1%103
Tween 600.1%103
Briji 350.1%105

 

Table 3. Effect of various chemicals on FODIII activity

AdditivesConcentrationRelative activity (%)
None-100
KCl1mM101
20mM95
100mM84
NaCl1mM97
20mM93
100mM86
250mM68
Sodium lauryl sulfate40.01%94
0.03%77
0.05%3
0.10%0
Ethylene glycol1%89
2%76
5%57
10%39
20%17
Dimethyl Sulfoxide1%87
2%77
5%61
10%42
20%22
2-Hydroxypropyl-β-cyclodextrin1%97
3%92
5%111

 

Table 4. Effect of various chemicals on FODIII stability

AdditiveResidual activity
(%)
None (40mM Tris-HCl pH7.5)71
+ 5mM EDTA85
+ 250mM KCl85
+ 250mM NaCl89
+ 20mM Sodium glutamate85
+ 20% Sucrose94
+ 20% Ethylene glycol86
+ 20% Glycerol91
+ 0.1% Triton X-10064
+ 4% Sorbitol99
+ 0.1% Briji 3565
+ 0.1% Tween 6074
+ 0.002mM Flavin adenine dinucleotide76
+ 0.02mM Flavin adenine dinucleotide76
+ 0.002mM Flavin mononucleotide75
+ 0.02mM Flavin mononucleotide74
+ 10mM NH4Cl77
Residual activity after heating for 50 °C, 10 min.
(3U/ml Enzyme solution)

Assay

Principle

  1. The assay is based on the increase in absorbance at 555 nm as the formation of quinoneimine dye proceeds in the following reactions:

FOD Ⅲ
1-deoxyfructosyl-valinyl-histidine+O2+H2O→Glucosone+Valinyl-histidine+H2O
POD
2H2O2+4–AA+TOOS→quinoneimine dye+4H2O
Unit definition

  1. One unit is defined as the amount of enzyme which converts 1 μ mole of deoxyfructosyl-valinyl-histidine to H2O2 per minute at 37 ℃ under the conditions specified in the assay procedure.

Reagents
  1. Reaction mixture
    50mM Tris-HCl buffer pH 7.5 containing 1.0mM 1-deoxyfructosyl-valinyl-histidine and 0.03% 4-AA and 0.02% TOOS and 5.0U/mL POD
  2. Reaction stopper
    0.5% SDS solution
  3. Enzyme dilution buffer
    10mM Tris-HCl buffer pH7.5
  4. Reagents
    Tris (hydroxymethyl) aminomethane: Sigma #T-1503
    1-deoxyfructosyl-valinyl-histidine：Peptide Institute. Inc.
    4-AA (4-Aminoantipyrine) : nacalai tesque #01907-52
    TOOS (N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, sodium salt, dihydrate) : DOJINDO LABORATORIES #OC13
    SDS (Sodium lauryl sulfate) : nacalai tesque #31606
    POD (Peroxidase) : Sigma Type Ⅱ #P-8250
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 0.5 ml of reaction mixture into a small test tube and preincubate at 37 ℃.
  2. After 5 min, add 10 μl of enzyme solution and mix to start the reaction at 37 ℃.
  3. At 5min after starting the reaction, add 1.0 ml of reaction stopper and mix to stop the reaction.
    ※IIn the case of a test blank, add 10 μl of enzyme dilution buffer in place of enzyme solution after stopping the reaction.
  4. Measure the absorbance at 555 nm.
    Absorbance sample : As
    blank : Ab
    △ A = (As−Ab) ≦ 0.050 ～ 0.800 Abs
Calculation
  1. Activity (U/mg of powder) = {(△A/5min)/(39.2×1/2)} × 1.51/0.01 × 1/X
    = (△A/min×1.541)/X
  2. 39.2 :millimolar extinction coefficient of quinoneimine dye at 555 nm (cm2/ μmole)
    1/2 :a multiplier derived from the fact that 2 mole of H2O2 produces 1 mole of quinoneimine dye
    1.51 :final volume (ml)
    0.01 :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.

FOD Ⅲ活性測定法(Japanese)

試薬液
  1. 反応試薬混合液
    1.0mM 1-deoxyfructosyl-valinyl-histidine、0.03% 4-AA、0.02% TOOS、5.0U/ml POD を含む50mM トリス-HCl 緩衝液 pH7.5
  2. 反応停止液
    0.5% SDS 溶液
  3. 酵素溶解希釈溶液
    10mM トリス-HCl 緩衝液 pH7.5
  4. 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
  1. 1-deoxyfructosyl-valinyl-histidine：ペプチド研究所製
    4-AA (4- アミノアンチピリン) ：ナカライテスク製 特級 #01907-52
    TOOS：同仁化学製 #OC13
    SDS (ドデシル硫酸ナトリウム) ：ナカライテスク製 #31606
    POD (パーオキシダーゼ) ：シグマ製　Type Ⅱ #P-8250
酵素試料液
  1. 検品約20mg を精密に量り、酵素溶解希釈用液に溶解して全容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0.5ml ずつを正確に分注し、37℃で予備加温する。
  2. 5 分経過後、酵素試料液10 μl を正確に加えて混和し、37℃で反応を開始する。
  3. 5 分経過後、反応停止液1.0ml を加えて混和し、反応を停止する。
    ※盲検は反応停止後に酵素試料液10 μl を加える。
  4. 555nm における吸光度を測定する。
    求められた吸光度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 ΔA ＝ ( As － Ab ) ＝ 0.050 ～ 0.800Abs
計算
活性 (U/mg) = {(△A/5min)/(39.2×1/2)} × 1.51/0.01 × 1/X
={(△A/min)×1.541}/X
39.2 :キノンイミン色素の555nm におけるミリモル分子吸光係数 (cm2/μmol)
1/2 :H2O2 2 モルからキノンイミン色素1 モルが生成することによる係数
1.51 :反応総液量 (ml)
1.51 :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)
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