Hexokinase Colorimetric Assay Kit

Catalog #: K789 | abID: ab136957

Product Details

abID ab136957
Cat # +Size K789-100
Size 100 assays
Detection Method Absorbance (450 nm)
Species Reactivity Mammalian
Applications Measurement of Hexokinase activity in various tissues/cells. Analysis of glucose metabolism and cell signaling in various cell types. Screening anti-diabetic drugs.
Features & Benefits • Can detect hexokinase activity even less than 0.1 mU/well
Kit Components • HK Assay Buffer
• HK Substrate
• HK Coenzyme (lyophilized)
• HK Enzyme Mix (lyophilized)
• HK Developer (lyophilized)
• NADH Standard (lyophilized)
• HK Positive Control (lyophilized)
Storage Conditions -20°C
Shipping Conditions Gel Pack
USAGE For Research Use Only! Not For Use in Humans.


Hexokinases (HK) are found in many organisms including bacteria, plants and mammals and play an important role in glucose metabolism. Hexokinases phosphorylate glucose and generate glucose-6-phosphate for glycolysis. Hexokinases have four isoforms (HK-I, II, III and IV). HK-I, HK-II and HK-III have low Km, while HK-IV has 100 fold high Km. Hexokinase deficiency leads to severe human diseases such as X-linked muscular dystrophy and a rare autosomal recessive hemolytic anemia. On the other hand, increased hexokinase activity is detected in various human tumors and is associated with metastasis. Early detection of abnormal hexokinase activity is crucial for diagnosis, prediction and treatment of the disease. In BioVision’s Hexokinase Assay kit, glucose is converted to glucose-6-phosphate by hexokinase; the glucose-6-phosphate is oxidized by glucose-6-phosphate dehydrogenase to form NADH, which reduces a colorless probe to a colored product with strong absorbance at 450 nm. The assay is simple, sensitive and rapid and can detect hexokinase activity even less than 0.1 mU/well.

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In measuring D-Glucose-rich sample (such as muscle), is there a possibility that the background control shows slightly higher values than the sample itself?
If there is Higher NADH in the muscle sample than D-glucose, then background can be equivalent or higher. Since OD is measured in a kinetic mode, time points can be chosen within the linear regime such that background values are lower than D-glucose reactions. Sample volume in the well can also be optimized such that very high (~50ul) volumes are not used. Low volumes will prevent saturation and will allow better distinction between sample and background.
What time should the standard data be read at?
Standards should be read at end-point (at the highest timepoint for the sample readings).
Do hemolysed pieces affect the assay?
Any particulate matter in suspension/turbidity affects absorbance measurements. Hemoglobin color or hemolysis in the sample can influence the OD measurements but typically OD 450nm is the brown/yellow range and hence red color does not influence much. I would suggest diluting the sample, removing the hemolyzed chunks and then using the sample.
Does this kit measure activity of HK-1 or HK-2 or other isoforms also? What about muscle tissue? What is the source of the positive control enzyme?
This kit is based on function of the Hexokinase enzyme (conversion of glucose to glucose-6-phosphate is measured). This kit does not distinguish between the isoforms. HKI, HKII and HKIII have low Km, while HKIV has 100 fold high Km. Hence it is very likely that you will measure hexokinase activity from I, II and III and small contribution if any from HK IV depending on the relative amounts of these isoforms in your sample. HK-II is the predominant form in adipose and muscle cells. HKII is insulin-sensitive. So if the samples are from adipose tissue or muscle you will measure HK-II activity. Our positive control is HK-II from Bacillus sp.
The substrate of G6PDH should be NADP, not NAD, right ?
We use NAD and not NADP for G6PDH. G6PDH enzyme can work with both NAD and NADP depending on the species origin (sometimes there is absolute specificity like the G6PDH from Aspergillus does not work with NADP at all, but our enzyme works with both NAD and NADP). We believe the Km is better with NAD and hence we use NAD in our assay.
Can I use other hexose-sugars as substrates, eg Fructose?
This kit assays hexokinase activity in a variety of samples. Theoretically hexokinases in general can phosphorylate hexose sugars including fructose which is then phosphorylated to fructose-6-P. Nevertheless, the primary substrate is glucose for these enzymes in mammalian cells. Hexokinase IV (fastest hexokinase) is actually a glucokinase meaning it acts on glucose to form G6P. It might be possible to use fructose as a substrate to test since NADH formed after phosphorylation reacts with the probe to generate color. But this depends on whether the sample has fructokinase activity or is mainly a glucokinase. The substrate provided in the kit is D-glucose.
What is the activity in the positive control?
The positive control is a purified enzyme and the specific activity changes from lot to lot. The positive control is provided to be used as a benchmark sample to make sure all components of the kit are working. The positive control is not to be used to compare activity in the sample.
Can frozen samples be used with this assay?
Fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
Is it possible to use a different wavelength than recommended for the final analysis?
It is always recommended to use the exact recommended wavelength for the most efficient results. However, most plate readers have flexibility in their band width of detection in increments of +/- 10 nm. Depending on this flexibility range, you can deviate from the recommended wavelengths within limits.
Are trial sizes of this kit available?
Unfortunately, we do not have trial sizes of this kit available. However,for customers based in the US or Canada, a 10% off list price introductory discount can be offered on the list price. For all other customers based out of this area our regional BioVision distributor should be contacted
Can individual components of this kit be purchased separately?
Yes, any of the kits components can be purchased separately without having to buy the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Can alternate buffers be used for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.
In your data sheet it says glucose-6-phosphate is oxidized by glucose-6-phosphate dehydrogenase to form NADH, which reduces a colorless probe to a colored product with strong absorbance at 450 nm. Is NADPH not the by-product of this reaction?
Yes, in our data sheet it says glucose is converted to glucose-6-phosphate by hexokinase. Glucose-6-phosphate is oxidized by glucose-6-phosphate dehydrogenase to form NADH and we detect NADH formed and not NADPH. Oxidation of glucose-6-phosphate can be carried out in the presence of either NADP+ and/or NAD+. Both NAD and NADP can serve as coenzyme with the intrinsic reaction velocity of NAD being approximately 1.8 times greater than that of NADP (Olive and Levy 1967). We have used NAD as a coenzyme in this kit and NADH is what is detected specifically in this assay. That’s why the standard provided is also NADH.
Which kit is better for measurement of hexokinase activity? Is measuring NADPH a direct method for detecting hexokinase activity as in K769?
Both K789 and K769 are different. In K789 NADH produced is detected and in K769 NADPH produced is detected. Both NAD and NADP can function as co-enzymes to promote the oxidation of Glucose-6-Phosphate. Both K789 and K769 detect the activity of hexokinase, with just a difference in the detection method. Both are equally relevant. In both kits, activity of hexokinase is measured through a series of reactions resulting either in the generation of a fluorescent product or a colored product. Additionally, fluorometric assays are more sensitive than colorimetric assays.
Saet-Byeol Kim, Alleviation of catabolite repression in Kluyveromyces marxianus: the thermotolerant SBK1 mutant simultaneously coferments glucose and xylose. Biotechnol Biofuels, Apr 19; 31044003.
Hyun Jik Lee, O-cyclic phytosphingosine-1-phosphate stimulates HIF1α-dependent glycolytic reprogramming to enhance the therapeutic potential of mesenchymal stem cells. Cell Death Dis, Aug 2019;  31383843.
Wang et al., A Missense Mutation in HK1 Leads to Autosomal Dominant Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci., Nov 2014; 55: 7159 - 7164.
Ayllón et al., Anaplasma phagocytophilum Inhibits Apoptosis and Promotes Cytoskeleton Rearrangement for Infection of Tick Cells. Infect. Immun., Jul 2013; 81: 2415 - 2425.
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