Hexokinase (HK) Activity Assay Kit (UV Colorimetric Method) - BioReagent, high purity

Cat. No.: H1501281

GRADE & PURITY BioReagent

Overview

Introduction
Hexokinase (HK) is widely present in animals, plants, microorganisms, and cultured cells. It is the first key enzyme in the glucose degradation pathway, catalyzing the conversion of glucose to glucose-6-phosphate, which is the intersection point of glycolysis and the pentose phosphate pathway.

Assay Principle
HK catalyzes the synthesis of Glucose-6-Phosphate (G6P) from Glucose. Glucose-6-Phosphate Dehydrogenase (G6PDH) then further catalyzes the dehydrogenation of G6P, generating NADPH. NADPH has a characteristic absorption peak at 340 nm.

Component	50T	Storage
Extraction Buffer	60 mL	2-8℃
Reagent 1	30 mL	2-8℃
Reagent 2	1EA	2-8℃
Reagent 3	5 mL	2-8℃
Reagent 4	1EA	-20℃
Reagent 5	1EA	-20℃
Reagent 6	1EA	-20℃

Reagent 2: Powder × 1 bottle. Dissolve in 30 mL distilled water before use. Aliquot and store unused portions at -20°C. Avoid repeated freeze-thaw cycles.

Reagent 4: Powder × 1 tube. Dissolve in 4 mL distilled water before use. Aliquot and store unused portions at -20°C. Avoid repeated freeze-thaw cycles.

Reagent 5: Powder × 1 tube. Dissolve in 2 mL distilled water before use. Aliquot and store unused portions at -20°C. Avoid repeated freeze-thaw cycles.

Reagent 6: Powder × 1 tube. Dissolve in 250 μL Reagent 1 and 250 μL distilled water before use. Aliquot and store unused portions at -20°C. Avoid repeated freeze-thaw cycles.

Required Materials and Equipment (Not Provided)
UV spectrophotometer, constant temperature water bath, benchtop centrifuge, adjustable pipettes, 1 ml quartz cuvette, mortar and pestle, ice, and distilled water.

Sample Preparation:

1. Bacteria or Cultured Cells:

   • Collect cells by centrifugation and discard the supernatant.

   • Add Extraction Buffer at a ratio of 1 ml per 5-10 million cells (e.g., 1 ml for 5 million cells).

   • Sonicate on ice (20% power or 200W, pulse 3s on/10s off, repeat 30 times).

   • Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.

2. Tissues:

   • Homogenize tissue on ice in Extraction Buffer at a ratio of 1:5-10 (w/v) (e.g., 0.1 g tissue in 1 ml buffer).

   • Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.

3. Serum (or Plasma) Samples:

   • Assay directly.

Assay Procedure:

1. Preheat the spectrophotometer for at least 30 min. Set wavelength to 340 nm. Zero with distilled water.

2. Pre-warm Reagents 1, 2, 3, 4, and 5 to 37°C (for mammalian samples) or 25°C (for other species) for 10 min.

3. Pipette into a 1 ml quartz cuvette in the following order:

Reagent	Volume (μL)
Reagent 1	400
Reagent 2	400
Reagent 3	80
Reagent 4	80
Reagent 5	40
Reagent 6	8
Sample	30

• Mix immediately upon sample addition and start the timer.

• Record the initial absorbance (A₁) at 20 seconds and the final absorbance (A₂) at 5 minutes and 20 seconds (320 sec total) at 340 nm.

• Calculate ΔA = A₂ - A₁.

Notes:

1. To minimize operational error, it is recommended to pre-mix Reagents 1, 2, 3, 4, and 5 in the stated proportions. Pre-warm this Master Mix for 10 min. Then add 30 μl sample + 8 μl Reagent 6 + 1 ml Master Mix to the cuvette. Mix and proceed with the assay.

2. HK activity varies across different tissues. Perform a pilot test with 1-2 samples before formal assay. If ΔA > 0.5, the tissue activity is too high. Dilute the supernatant with Extraction Buffer (include dilution factor D in calculations) or shorten the reaction time to 2 min to ensure ΔA < 0.5 and improve detection sensitivity.

HK Activity Calculation:

General Parameters:

• Vₜₒₜₐₗ (Total reaction volume) = 1.038 × 10⁻³ L (1038 μL)

• ε (NADPH molar extinction coefficient) = 6.22 × 10³ L/mol/cm

• d (Cuvette light path) = 1.0 cm

• Vₛₐₘₚₗₑ (Sample volume in reaction) = 0.03 mL (30 μL)

• T (Reaction time) = 5 min

• Cpr (Sample protein concentration, mg/mL)

• W (Sample mass, g)

• Vₛₐₘₚₗₑₜₒₜₐₗ (Total extract volume) = 1.0 mL (for tissue/cell calculations)

• 500 (Cell/Bacteria count in millions for example calculation: 5 million)

1. For Serum (Plasma):

• Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per ml of serum.

• Calculation:
  HK Activity (nmol/min/ml) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ Vₛₐₘₚₗₑ ÷ T
  Simplified Formula: HK (nmol/min/ml) = 1113 × ΔA

2. For Tissues, Bacteria, or Cells:
a. Based on Sample Protein Concentration:
* Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per mg of protein.
* Calculation:
HK Activity (nmol/min/mg prot) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ × Cpr) ÷ T
Simplified Formula: HK (nmol/min/mg prot) = 1113 × ΔA ÷ Cpr

b. Based on Sample Fresh Weight:
* Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per gram of fresh tissue.
* Calculation:
HK Activity (nmol/min/g fresh weight) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (W × Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ T
Simplified Formula: HK (nmol/min/g fresh weight) = 1113 × ΔA ÷ W

c. Based on Bacterial or Cell Density:
* Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per 10⁴ cells.
* Calculation (example for 5 million cells in 1 ml extract):
HK Activity (nmol/min/10⁴ cell) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (500 × Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ T
Simplified Formula: HK (nmol/min/10⁴ cell) = 2.226 × ΔA

Precautions
Perform a pilot assay with 2-3 samples expected to have significant activity differences before the formal determination.

Storage and Shipping

Storage

Protected from light,Store at -20°C

Shipped In

Ice chest + Ice pads

Stability And Storage

Each component has a shelf life of 6 months under corresponding storage conditions.