重组麦芽糖磷酸化酶 (MPL-EP)

The enzyme is useful for the determination of α-amylase and inorganic phosphate in clinical analysis. PROPERTIES Molecular weight: ca. 220 kDa (gel filtration) Structure: 2 subunits of 90 kDa (SDS-PAGE) Michaelis constants: 1.9×10^−3 M (maltose); 3.4×10 ^ −3 M (phosphate); 8.3×10 ^ −3 M (arsenate) pH Optimum: 6.5–7.5 (Fig. 1) pH Stability: 5.5–8.0 (Fig. 2) Optimum temperature: 45–50°C (Fig. 3) Thermal stability: below 55°C (Fig. 4) Stability (liquid form): stable at 37°C for at least one week (Fig. 5) Stability (powder form): stable at 30°C for at least four weeks (Fig. 6) Inhibitors: Hg ²⁺ , Ag ⁺ , Zn ²⁺ , Cu ²⁺ STABILIZER: lactose, EDTA ASSAY PROCEDURE Principle The appearance of d-glucose is measured spectrophotometrically at 505 nm. Reagents A. HEPES–NaOH buffer, 50 mM; pH 7.0: dissolve 2.38 g of HEPES in 160 ml of distilled water, adjust to pH 7.0 with 1 N NaOH and dilute with distilled water to 200 ml. B. Maltose solution, 0.2 M: 3.60 g of maltose monohydrate/50 ml of HEPES–NaOH buffer (Reagent A). C. Phosphate solution, 0.2 M: dissolve 1.36 g of KH ₂ PO₄ in 40 ml of HEPES–NaOH buffer (Reagent A), adjust to pH 7.0 with 4 N NaOH and dilute with Reagent A to 50 ml. D. HCl solution, 5 N: 43 ml of concentrated HCl/100 ml of distilled water. E. pH Adjusting solution (NaOH solution, 1 n): 4.0 g of NaOH/100 ml of distilled water. F. Glucose assay kit: “GLUCOSE C II-TESTWAKO” (Wako Pure Chemical), or a similar glucose assay kit. Sample: dissolve the lyophilized enzyme to a volume activity of 0.15–0.55 U/ml with ice-cold HEPES–NaOH buffer (Reagent A) immediately before measurement. Procedure 1. Pipette the following reagents into a test tube. 0.2 ml HEPES–NaOH buffer (Reagent A) 0.1 ml Maltose solution (Reagent B) 0.1 ml Phosphate solution (Reagent C) 2. Equilibrate at 30°C for about 5 min. 3. Add 0.1 ml of sample and incubate for 10 min at 30°C [test].The blank solution is prepared by adding HEPES–NaOH buffer (Reagent A) instead of sample [blank]. 4. Add 0.1 ml of HCl solution (Reagent D) to stop the reaction. 5. Add 0.5 ml of pH adjusting solution (Reagent E). 6. Pipette 0.3 ml of the test and blank mixture into respective test tubes. 7. Add 3.0 ml of glucose assay kit (Reagent D) and incubate for about 5 min at 37°C. 8. Read the absorbance at 505 nm in a cuvette (light path: 1 cm) [test: AS, blank: A0]. Calculation Activity can be calculated by using the following formula: 6.24: Millimolar extinction coefficient of quinoneimine dye under the assay conditions (cm ² /μmol) 1/10: A factor for correction of the reaction volume df: Dilution factor C: Content of maltose phosphorylase preparation in sample (mg/ml) APPLICATIONS The enzyme is useful for the determination of α-amylase and inorganic phosphate in clinical analysis. EXPERIMENTAL DATA

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