CBS (Cystathionine-β-synthase)
This enzyme is useful for enzymatic determination of L-homocysteine when coupled with CBS and LDH in clinical analysis.
Preparation and specification
|
Appearance |
Yellow amorphous powder, lyophilized |
|
Protein purity |
≥90% (from SDS-PAGE) |
|
Activity |
≥8 U/mg solid |
|
Glucose-6-phosphate dehydrogenase |
≤0.01% |
|
Lactate dehydrogenase |
≤0.01% |
Properties
|
EC number |
4.2.1.22 (Recombinant from microorganism) |
|
Molecular weight |
51 kDa (SDS-PAGE) |
|
Isoelectric point |
5.4 |
|
Michaelis constants |
8.0×10-4 M (L-Serine),1.6×10-4M(L-Homocysteine) |
|
Inhibitors |
Not inhibited by NaN3 |
|
Optimum pH |
8.0 Fig. 1 |
|
Optimum temperature |
40℃ Fig. 2 |
|
pH stability |
pH 6.0-10.0 (25℃, 16 h) Fig. 3 |
|
Thermal stability |
Below 45 ℃ (pH 8.0, 30min) Fig. 4 |
|
Storage stability |
At least one year at -25～-15℃ Fig. 5 |
|
Stabilizers |
Triton X-100 |
Storage conditions
Store at -25~-15°C, sealed, dry and protected from light. Valid for 2 year.
Figures
Assay principle
Unit definition
One unit (U) is defined as the amount of enzyme which consumes 1 μmol of NADH per min under the conditions described below.
Reagents preparation
Reagent I: 50 mM pH 8.0 PBS, contains 10 mM L-Serine, 2 mM DL-Homocysteine, 0.38 mM S-adenosyl-methionine, 0.25 mM PLP, 1 mM DTT, 5 U/mL CBL, 0.2 mM NADH, 5 U/mL LDH. Enzyme diluent: 50 mM pH 8.0 PBS buffer.
Sample: The enzyme was diluted to 0.8-2.4U/mL with the enzyme diluent.
Procedure
1. Add 1 mL Reagent I to the 1 mL cuvette and preheat it at 37℃ for 5 min.
2. Add 0.02 mL enzyme solution to the cuvette and mix.
3. Record the ΔAs at 340 nm in 1 minute in a spectrophotometer thermostated at 37℃.
At the same time, measure the blank rate ΔAb by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution
∆A = ∆As -∆Ab
Calculation
1.02: Total volume (mL)
0.02: enzyme volume (mL)
1.0: Light path length (cm);
df: dilution factor 6.22: Millimolar extinction coefficient of NADH under 340nm (cm2/μmol)
