Sucrose Phosphorylase (SPL-E) 60122 蔗糖磷酸化酶

  • 临床检测用酶
Sucrose Phosphorylase 蔗糖磷酸化酶

The enzyme is useful for the determination of inorganic phosphate in clinical analysis.

由来 recombinant E. coli
系统名称

Sucrose : orthophosphate α-D-glucosyltransferase

EC编号 2.4.1.7
反应式

Sucrose + Orthophosphate →→→ D-Fructose + α-D-Glucose 1-phosphate

规格

Appearance white lyophilizate
Activity ≧50 U/mg
Stabilizer sucrose
Storage condition below -20℃

特性

Molecular weight ca. 56 kDa (gel filtration)
Structure monomer of 56 kDa (SDS-PAGE)
Isoelectric point 4.6
Michaelis constant 3.9×10-2M (sucrose)
6.2×10-3M (phosphate)
pH Optimum 7.5
pH Stability 5.0–8.0
Optimum temperature 40℃
Thermal stability below 45℃
Stability (liquid form) stable at 37℃ for at least two weeks
Stability (powder form) stable at 30℃ for at least two weeks
Inhibitors glucose, glucono-1,5-lactone
Specificity sucrose (100), maltose (0), starch (0)

适用类型

The enzyme is useful for the determination of inorganic phosphate in clinical analysis.

ASSAY PROCEDURE

Principle

The appearance of NADPH is measured spectrophotometrically at 340 nm.

Definition of unit

One unit (U) is defined as the amount of enzyme which produces 1 μmol of NADPH per min at 25℃ and pH 6.8 under the conditions described below.

Reagents

  1. Triethanolamine (TEA) buffer, 0.1 m; pH 7.6: dissolve 1.86 g of triethanolamine hydrochloride in 90 ml of distilled water, adjust to pH 7.6 with 5 n NaOH and dilute with distilled water to 100 ml.
  2. Potassium phosphate buffer, (a) 0.1 M; pH 6.8, (b) 0.05 M; pH 6.8: (a) mix 0.1 M KH2PO4 and 0.1 M K2HPO4 to make a pH 6.8 solution. (b) dilute 0.1 m potassium phosphate buffer (Reagent B (a)) with same volume of distilled water.
  3. Sucrose solution, 0.32 M: 11.0 g of sucrose/100 ml of distilled water.
  4. EDTA solution, 9.9 mM: 37 mg of EDTA·Na2·2H2O/10 ml of potassium phosphate buffer (Reagent B (b)).
  5. NADP+ solution, 12 mM: 9.2 mg of NADP+·Na/1.0 ml of distilled water.
  6. D-Glucose-1,6-diphosphate (G-1,6-P2) solution, 0.1 mM: 1.0 mg of G-1,6-P2 cyclohexylammonium salt/10 ml of distilled water.
  7. MgCl2 solution, 1.0 M: 4.06 g of MgCl2·6H2O/20 ml of distilled water.
  8. α-Phosphoglucomutase (α-PGM) suspension, 2000 U/ml: crystalline suspension, 10 mg/ml (200 U/mg).
  9. Glucose-6-phosphate dehydrogenase (G6PDH) solution: 2000 U/ml of potassium phosphate buffer (Reagent B(b)).

Sample: dissolve the lyophilized enzyme to a volume activity of 0.8–1.5 U/ml in ice-cold TEA buffer (Reagent A) immediately before measurement.

Procedure

  1. Pipette the following reagents into a cuvette (light path: 1 cm).
    1.5 ml Potassium phosphate buffer (Reagent B (a))
    1.5 ml Sucrose solution (Reagent C)
    0.03 ml EDTA solution (Reagent D)
    0.1 ml NADP+ solution (Reagent E)
    0.1 ml G-1,6-P2 solution (Reagent F)
    0.05 ml MgCl2 solution (Reagent G)
    0.01 ml α-PGM suspension (Reagent H)
    0.01 ml G6PDH solution (Reagent I)
  2. Equilibrate at 25℃ for about 5 min.
  3. Add 0.02 ml of sample and mix.
  4. Add 0.1 mL of sample and mix.
  5. Record the increase of absorbance at 340 nm in a spectrophotometer thermostated at 25℃, and calculate the ΔA per min using the linear portion of the curve (ΔAS).
    The blank solution is prepared by adding TEA buffer (Reagent A) instead of sample (ΔA0).

Calculation

Activity can be calculated by using the following formula:

6.2 : Millimolar extinction coefficient of NADPH at 340 nm (cm2/μmol)
df : Dilution factor
C : Content of sucrose phosphorylase preparation in sample (mg/ml)

EXPERIMENTAL DATA

FAQ

What precautions should be taken when handling the product after opening?
How should SPL-E be stored?
What stabilizers are used in SPL-E?
How thermostable is SPL-E?
What are the optimal pH and temperature for SPL-E?
What is the molecular weight of SPL-E?
What are the main applications of SPL-E?

参考文献



TOP