果糖基肽氧化酶（FPOX）

用途 氧化糖化氨基酸，用于酶法糖化血红蛋白试剂的研发和大量配制。 酶学性质 来源： 微生物 酶学委员会编号： EC1.5.3 分子量： 60 kDa (SDS-PAGE) 等电点： 6.4 Km值： 4.0×10-3M (fructosyl-Val-His) 抑制剂： Hg²⁺, Pb²⁺ 最适pH： 6.5-7.5 图1 最适温度： 37℃ 图2 pH稳定性： pH 6.5-9.5 (25℃,16h) 图3 热稳定性： 40℃以下稳定(pH8.0，30min) 图4 稳定性： -25～-15℃静置保存12个月保持 90%以上活性 图5 保护剂： 甘油、海藻糖 活性测定方法 1、原理 反应生成的醌亚胺（Quinoneiminedye）可在555nm检测。 2、酶活定义 单位酶活定义为在下述条件下，每分钟催化生成1μmol H2O2所需的酶量 3、试剂准备 试剂I：0.1M磷酸钾缓冲液，pH8.0。 试剂II：1kU/mLPOD。 试剂III：50mMTOOS溶液（1.477gTOOS溶于100mLUP水）。 试剂IV：50mM4-AA溶液（1.016g4-AA溶于100mLUP水）。 试剂V：200mM糖化缬氨酸。 按如下配制反应混合物： 试剂I 10mL 试剂II 0.1mL 试剂III 1mL 试剂IV 1mL 试剂V 10mL 双蒸水 定容至100mL 样品：用酶稀释液20mMTris-HCl，pH8.0稀释。 4、操作步骤 1.向1mL比色皿中加入980μL反应混合物。 2.37°C孵育5min。 3.向反应混合物中加入20μL待测酶液。 4.37°C反应，用分光光度计在555nm检测样品1min内的吸光度变化（∆As）。 *用酶稀释液代替待测酶液，测定样品1min内的吸光度变化（∆Ab）。 ∆A=∆As-∆Ab 5、 活力计算 Vt：反应液总体积(1.0mL)； Vs：酶液体积(0.02mL)； t：反应时间(1min)； df：稀释倍数； C：酶浓度(mg/mL)； 1.0：光路长度(cm)； 1/2：1摩尔过氧化氢生成1/2摩尔醌亚胺染料； 39.2：标准反应条件下，生色基团在555nm处毫摩尔吸光系数(cm2/μmol)。 Application Oxidized glycosylated amino acids, used in the development and mass preparation of enzymatic glycosylated hemoglobin reagents. Enzymatic properties Source: Microorganism Enzymology Committee Number: EC1.5.3 Molecular weight: 60 kDa (SDS-PAGE) Isoelectric point: 6.4 Km value: 4.0×10-3M (fructosyl-Val-His) Inhibitors: Hg²⁺, Pb²⁺ Optimum pH: 6.5-7.5 Figure 1 Optimum temperature: 37℃ Figure 2 pH stability: pH 6.5-9.5 (25℃,16h) Figure 3 Thermal stability: Stable below 40℃ (pH8.0, 30min) Figure 4 Stability: -25 ~ -15℃ standing store for 12 months More than 90% activity Figure 5 Protective agent: glycerin, trehalose Assay method for activity 1. Principle The resulting Quinoneiminedye can be detected at 555nm. 2. Definition of enzyme activity Unit enzyme activity is defined as the amount of enzyme required to catalyze the production of 1μmol H2O2 per minute under the following conditions 3. Reagent preparation Reagent I: 0.1M potassium phosphate buffer, pH8.0 Reagent II: 1kU/mLPOD. Reagent III: 50mMTOOS solution (1.477gTOOS dissolved in 100mLUP water). Reagent IV: 50 mm4-AA solution (1.016g4-AA dissolved in 100mLUP water). Reagent V: 200mM glycosylated valine. Sample: Diluted with enzyme diluent 20mMTris-HCl, pH8.0. Prepare the reaction mixture as follows: Reagent I: 10mL Reagent II: 0.1mL Reagent III: 1mL Reagent IV: 1mL Reagent V: 10mL Double steam water set volume to 100mL Sample: Diluted with enzyme diluent 20mMTris-HCl, pH8.0. 4. Operation procedure 4.1 Add 980μL reaction mixture to 1mL colorimetric dish. 4.2 Incubate at 37°C for 5min. 4.3 Add 20μL of enzyme solution to the reaction mixture. 4.4 Reaction at 4.37°C, the absorbance change (∆As) within 1min of the sample is detected by spectrophotometer at 555nm. * Replace the enzyme solution to be tested with enzyme diluent and determine the absorbance change (∆Ab) of the sample within 1min. ∆A=∆As-∆Ab 5. Vitality computing Vt: total volume of reaction liquid (1.0mL); Vs: enzyme liquid volume (0.02mL); t: Reaction time (1min); df: dilution ratio; C: Enzyme concentration (mg/mL); 1.0: optical path length (cm); 1/2:1 mole of hydrogen peroxide to generate 1/2 mole of quinone imide dye; 39.2: Under standard reaction conditions, the millimolar absorption coefficient of the color group at 555nm (cm2/μmol).

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