Description
ApplicationOxidized glycosylated amino acids, used in the development and mass preparation of enzymatic glycosylated hemoglobin reagents.Enzymatic propertiesSource: MicroorganismEnzymology Committee Number: EC1.5.3Molecular weight: 60 kDa (SDS-PAGE)Isoelectric point: 6.4Km value: 4.0×10-3M (fructosyl-Val-His)Inhibitors: Hg²⁺, Pb²⁺ Optimum pH: 6.5-7.5 Figure 1Optimum temperature: 37℃ Figure 2pH stability: pH 6.5-9.5 (25℃,16h) Figure 3Thermal stability: Stable below 40℃ (pH8.0, 30min) Figure 4Stability: -25 ~ -15℃ standing store for 12 monthsMore than 90% activity Figure 5Protective agent: glycerin, trehalose Assay method for activity1. Principle The resulting Quinoneiminedye can be detected at 555nm.2. Definition of enzyme activityUnit enzyme activity is defined as the amount of enzyme required to catalyze the production of 1µmol H2O2 per minute under the following conditions3. Reagent preparationReagent I: 0.1M potassium phosphate buffer, pH8.0Reagent 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: 10mLReagent II: 0.1mLReagent III: 1mLReagent IV: 1mLReagent V: 10mLDouble steam water set volume to 100mLSample: Diluted with enzyme diluent 20mMTris-HCl, pH8.0.4. Operation procedure4.1 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-∆Ab5. 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)