Hexokinase (HK) is the first enzyme in glycolysis, where it phosphorylates glucose to glucose-6-phosphate. There are four distinct tissue-specific isoforms that manage this pathway: HK I-IV. HK I, II, and III possess a high affinity for glucose and are strongly inhibited by their product, while HK IV (glucokinase) features a low affinity for glucose and no inhibition by its product. Genetic deficiency in HK ultimately leads to hemolytic anemia, while altered expression of HK is linked to neurological disorders, rheumatoid arthritis, and type 2 diabetes. Overexpression of hexokinase increases glucose uptake during exercise in skeletal muscles but also fuels the Warburg effect, accelerating cancer cell growth and proliferation. Monitoring HK activity is an important indicator in cancer research and presents a potential therapeutic target.

Hexokinase Activity Assay Kit provides a simple, reproducible, and sensitive tool for measuring HK activity in plasma, serum, cell lysates, and other biological liquid samples. This assay is based on a coupled enzyme reaction. Hexokinase phosphorylates glucose to a colored product that can be quantified using a spectrophotometer or a microtiter plate reader at 450 nm. The change in absorbance is directly proportional to HK activity in the sample.

• Microplate reader capable of measuring absorbance at 450 nm
• Precision pipettes to deliver 2 µL to 1 mL volumes
• Tubes to prepare sample dilutions
• 5 mL tubes
• Multi-channel pipettes to deliver 20 µL to 200 µL volumes
• MilliQ water or another type of double distilled water (ddH₂O)

1. Add 50 µL of the diluted NADPH standards, samples, or positive control to the 96-well microtiter plate.
2. Add 50 µL Reaction Mix to each standard and one half of the paired sample wells, mix thoroughly.
3. Add 50 µL of Control Mix to other half of the paired sample wells. Carefully shake the plate for a few seconds to mix.
4. Kinetic reading: Immediately start measuring absorbance at 450 nm continuously at room temperature and record data every 2 minutes for 30 minutes.