Fructosyl-peptide oxidase (FPOX), CAS No.rp216180

CAS: rp216180 Cat. No.: rp216180 Molecular Weight: 60 kDa EC Number: EC1.5.3

GRADE & PURITY BioReagent, EnzymoPure™, ≥90%(SDS-PAGE), ≥ 4 U/mg

Overview

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).

Specifications

Product Name

Fructosyl-peptide oxidase (FPOX), CAS No.rp216180

Grade

BioReagent, EnzymoPure™

Specifications & Purity

EnzymoPure™, BioReagent, ≥90%(SDS-PAGE), ≥ 4 U/mg

CAS

rp216180

Molecule Type

Enzyme

Storage and Shipping

Concentration

≥ 4 U/mg

Reconstitution

50 - 100 mM Tris-HCl buffer solution, pH 7.5 - 8.5

Storage

Store at -20°C,Avoid repeated freezing and thawing

Shipped In

Ice chest + Ice pads

Stability And Storage

Store at -20℃ long term (12 months). Upon receipt, it is recommended to aliquot. Avoid freeze/thaw cycle.