Hexokinase can catalyze the phosphorylation of the 6-hydroxyl group of hexose. When coupled with glucose-6-phosphate dehydrogenase, this enzyme can be used for the enzymatic determination of glucose, adenosine 5'-triphosphate (ATP) and creatine kinase.
Application：This product can be used for the rapid clinical detection of α-amylase content in human serum and urine samples, as well as the research and development and production of α-amylase detection kits.
Assay Procedure
PRINCIPLE
The appearance of NADH is measured at 340nm by spectrophotometry.
UNIT DEFINITION
One unit causes the formation of one micromole of NADH per minute at pH8.0 at 30°C.
Reagents:
A. Tris-HCl buffer, pH8.0: 50mM, containing 13.3mM MgCl₂
B. Glucose solution: 0.67M in Tris-HCl buffer solution(A)（The solution should be kept at room temperature at least for 1 hour before use）
C. ATP solution: 16.5mM in Tris-HCl buffer solution(A)（Should be prepared fresh）
D. NAD⁺ solution: 6.8mM in Tris-HCl buffer solution(A)（Should be prepared fresh）
E. G-6-PDH solution: 300U/ml（Dilute with Tris-HCl buffer solution(A)and store on ice）
F. Enzyme diluent: Tris-HCl buffer solution(A) contg.0.1% of bovine serum albumin
Procedure:
1. Prepare the following reaction mixture in a cuvette（d=1.0cm）and equilibrate at 30°C for about 5 minutes.
| 2.30ml | Tris-HCl buffer solution | （A） | | 0.50ml | Glucose solution | （B） | | 0.10ml | ATP solution | （C） | | 0.10ml | NAD⁺ solution | （D） | | 0.01ml | G-6-PDH solution | （E） |
|
Concentration in assay mixture | Tris-HCl buffer | 50mM | | Glucose | 0.11M | | ATP | 0.53mM | | NAD⁺ | 0.22mM | | MgCl₂ | 13mM | | BSA | 3.2μg/ml | | G-6-PDH | ca. 1.0U/ml |
|
2. Add 0.1ml of the enzyme solution* and mix by gentle inversion.
3. Record the increase of optical density at 340nm against water for 4~5minutes in a spectrophotometer thermostated at 30°C, and calculate the △OD per minute from the initial linear portion of the curve (△OD test).
At the same time, measure the blank rate（△OD Blank）by using the same method as the test expect that the enzyme diluent is added instead of the enzyme solution.
*:Dissolve the enzyme preparation in ice-cold enzyme diluent（F）and dilute to 0.1~0.3U/ml with the same buffer, immediately before assay.
Calculation:
Activity can be calculated by using the following formula:
Vt: Total volume（3.11ml）
Vs: Sample volume（0.1ml）
6.22: Millimolar extinction coefficient of NADH under the assay condition (cm²/μM)
1.0: Light path length（cm）
df: Dilution factor
C: Enzyme concentration in dissolution（c mg/ml）
ZJ26F0333378