α-GLU (α-Glucosidase)
This enzyme is useful for diagnostic tests for the determination of α-amylase.
Specification
|
Appearance |
White amorphous powder, lyophilized |
|
Protein purity |
≥90% (from SDS-PAGE) |
|
Activity |
≥40 U/mg solid |
|
α-amylase |
≤0.00001% |
Properties
|
EC number |
3.2.1.20 (Recombinant from microorganism) |
|
Molecular weight |
65 kDa (SDS-PAGE) |
|
Isoelectric point |
5.2 |
|
Michaelis constants |
5.8×10-4 M (p-Nitrophenyl-α-D-glucopyranoside) |
|
Inhibitors |
SDS, Ag+, Hg2+, Cu2+ |
|
Optimum pH |
7.1 Fig. 1 |
|
Optimum temperature |
50 ℃ Fig. 2 |
|
pH stability |
pH 6.0-8.0 (25 ℃, 16 h) Fig. 3 |
|
Thermal stability |
Below 50 ℃ (pH 7.0, 10 min) Fig. 4 |
|
Storage stability |
At least one year at -25 ～-15℃ Fig. 5 |
|
Stabilizers |
BSA |
Figures
Assay Principle
Unit definition
One unit (U) is defined as the amount of enzyme which produces 1 μmol of PNP per min under the conditions described below.
Reagents preparation
Reagent I: 100 mM PBS, pH 7.0.
Reagent II: 20 mM p-Nitrophenyl-α-D-glucopyranoside (PNPG).
Reagent III: 200 mM Na2CO3.
Reagent IV: 200 mM, pH 7.0 potassium phosphate buffer, contain 0.05% Tween 20 and 1 mM EDTA.
Sample: Dilute the sample to 0.006-0.022 U/mL by Reagent IV.
Procedure
1. Add 1mL Reagent I, 0.5 mL Reagent II to a test tube, and preincubate for 5 min at 37 ℃ .
2. Add 0.5 mL sample and mix, react for 15 min in 37 ℃, then add 2 mL reagent III to stop the reaction.
3. Record the ΔAs at 400 nm in a spectrophotometer.
4. Exchange the adding order of reagent III and sample, and measure the blank absorban ΔAb.
∆A = ∆As - ∆Ab
Calculation
Vt: Total volume (4.0 mL)
Vs: enzyme volume (0.5 mL)
1.0: Light path length (cm)
df: Dilution factor
C: Enzyme concentration (mg/mL)
t: Reaction time (15 min)
18.1: Millimolar extinction coefficient of p-Nitrophenol under 400 nm (cm2 /μmol)
References
1. Ranscher, E. et al (1986). Analyt. Chem.324: 304.
2. Kruse – Jarres, J.D. et al (1989). J. Clin. Chem. 27: 103.
