A Guide to Enzyme Assay Kits

Enzymes are diverse proteins and so are their biological substrates, products, and biochemical mechanisms. Given the diversity of enzymatic reactions occurring in cells and organisms, a variety of enzymatic assays have been developed to study the enzyme-driven processes underlying different biological pathways. These manufactured pre-packaged kits contain optimized, ready-to-use reagents and protocols for the biochemical assay of a wide range of enzymes such as kinases, phosphatases, proteases, deacetylases, peroxidases, lipases, and more. Assay kits are versatile and reliable tools that can serve many research areas, including studying protein characterization, biochemical pathways, disease states, toxicology, and pharmaceutical screening. The Biocompare catalog of enzyme assay kits lists a wide and growing selection of kits from reliable suppliers. Here we present a guide to help users decide in the selection of the most appropriate enzyme assay kit for unique research needs.

What’s in an enzyme assay kit?

Commercial assay kits for enzymatic activity generally contain a number of components.

• Substrate - This core reagent is specific to the target enzyme, which can come in a range of forms such as a labeled peptide, small molecule compound, or metabolite derivative.
• Detection reagent - If the reaction byproduct is not readily detectable, an a additional detection reagent will be supplied, usually in the form of a chromogenic dye or fluorescent probe.
• Enzyme - In enzyme-coupled reactions, an additional enzyme will also be included to produce a detectable byproduct.
• Reaction buffer - A buffer solution maintains the physiological pH and ionic strength required for the enzyme's optimal activity.
• Lysis reagent - Some kits will include a lysis buffer or reagent to release intracellular enzymes.
• Cofactors or activators - Some enzymes require cofactor molecules or metal ions to function optimally.
• Reaction stopper - A stop solution or enzyme inhibitor may be used to halt the reaction at a specific point.
• Standards and controls - Known quantities of the enzyme or reaction products are included for calibration or creating a standard curve for quantification. Positive and negative controls also ensure that the assay is working correctly.
• Reaction vessels - Microplates, test tubes, or vials may be provided for convenience.
• Detailed protocol - Instructions outlining the steps for preparing and conducting the assay, including incubation times, temperatures, detection methods, and sample recommendations.

Enzyme assay methods

Enzyme assays can be broadly categorized based on how substrates are used to measure enzyme activity. Substrate-based assays focus on the direct conversion of a specific substrate by the enzyme into a product, which is then measured to determine enzyme activity. These assays are straightforward and commonly used. Alternatively, coupled enzyme assays involve secondary reactions in which the product of the initial enzyme reaction serves as a substrate for another enzyme, ultimately leading to a detectable signal. This approach is often implemented when the primary reaction’s product is as easy to measure.

Assay kits also vary by colorimetric, fluorometric, or luminescent detection. Colorimetric assays, which use chromogenic probes or dyes, detect changes in color as the enzyme reaction progresses. These assays are simple and cost-effective and generally require only a UV-Vis spectrophotometer as a means of detection. Fluorometric assays rely on the emission of fluorescent dyes, offering higher sensitivity and specificity. These are ideal for detecting very low concentrations of enzyme products. Luminescent assays are perhaps the most sensitive, capable of detecting very low levels of enzyme activity and accommodating small sample volumes. Luminescent signals typically have low background interference compared to other detection methods like fluorescence or absorbance. Examples of some assay methods are highlighted below:

Alkaline Phosphatase (ALP) assay - ALP dephosphorylates the substrate p-nitrophenyl phosphate (pNPP), which turns the reaction yellow. ALP enzyme activity is determined using colorimetric measurement. A fluorometric version of the assay will utilize a non-fluorescent substrate (such as 4-methylumbelliferyl phosphate) to generate a fluorescent signal.

Beta Galactosidase assay - The beta-Gal enzyme hydrolyzes colorimetric substrates (such as ONPG) or fluorometric substrates (such as 4-MUG) to generate measurable signals proportional to the enzyme activity.

Adenosine Deaminase (ADA) assay - This assay utilizes several enzyme-coupled reactions that convert adenosine to hypoxanthine riboside, followed by the production of hydrogen peroxide, followed by a reaction with a colorimetric probe. The measurable end product is a red change in color. Alternatively, a fluorescent variant of this assay will utilize the conversion of a byproduct with a fluorometric probe.

Aldehyde Dehydrogenase (ALDH) assay - The reaction of ALDH with an aldehyde substrate is coupled to the reduction of NAD to NADH. NADH subsequently reacts with a colorless probe to form a colored product.

Trypsin assay - Active trypsin cleaves a specific labeled peptide substrate to release a chromophore such as p-nitroaniline (p-NA). The color change from the reaction can be detected colorimetrically to measure trypsin activity.

Nitric Oxide Synthase (NOS) assay - Active NOS enzymes catalyze L-arginine and oxygen to produce nitric oxide (NO) byproducts. NO-reactive fluorescent dyes and chromogens subsequently react with NO to generate fluorometric or colorimetric signals for detection.

Creatine Kinase (CK) assay - This assay involves the conversion of phosphocreatine and ADP to creatine and ATP, followed by an enzyme-coupled reaction to form NADPH. NADPH is measured colorimetrically to determine CK activity.

Methyltransferase assay - Histone methyltransferases (HMT) and DNA methyltransferases will catalyze the methylation of a target protein or nucleotide. One method of detection is through the use of labeled antibodies, which recognize specific phosphorylated histones or nucleotides. A fluorescent version of the assay uses a continuous enzyme-coupled assay that couples the removal of a methyl group to ultimately generate a hydrogen peroxide byproduct. H2O2 reacts with a fluorogenic probe to generate a signal.

Considerations in enzyme assay kit selection

Choose what is appropriate for your sample type - Most enzyme assay kits are generally designed to accommodate either cell-based or cell-free samples. This is important to consider because cytoplasmic enzymes will be less accessible to assay reagents when enclosed within an intact cell membrane. Some assay kits include a step for cell lysis to release the enzymes into solution; these are ideal for tissue samples or cell culture pellets. However, those who wish to analyze enzymatic activity in the context of living cells should choose a cell-based assay kit.

Review the protocol - The enzyme assay kit protocol is an excellent resource that can provide important bits of information. By reading the protocol a prospective user can learn the mechanism of the assay, how the enzymatic activity is defined (or units of activity), the complexity of the experimental procedure, what sample types are compatible and how they should be prepared, what materials are provided, and what additional materials or instruments will be required. Such information can be useful when deciding on which assay method is the most appropriate for a particular sample type or application. Protocols, if not displayed on the product page, are often listed as downloadable documents or booklets.

Check for citations - Some enzyme assay kits may have been cited in a published article. Reviewing how the assay has been used by other researchers can provide useful insight in areas such as sample types, host tissues or organisms, research areas, detection range, and more. Biocompare provides citation and figures data for assay kits when available. Citation information may also be found on the supplier's product pages.