enzyme assays


Summary: Methods used to measure the relative activity of a specific enzyme or its concentration in solution. Typically an enzyme substrate is added to a buffer solution containing enzyme and the rate of conversion of substrate to product is measured under controlled conditions. Many classical enzymatic assay methods involve the use of synthetic colorimetric substrates and measuring the reaction rates using a spectrophotometer.

Top Publications

  1. Lippi G, Simundic A, Musile G, Danese E, Salvagno G, Tagliaro F. The alcohol used for cleansing the venipuncture site does not jeopardize blood and plasma alcohol measurement with head-space gas chromatography and an enzymatic assay. Biochem Med (Zagreb). 2017;27:398-403 pubmed publisher
    ..It seems reasonable to conclude that using ethanol-containing antiseptics before venipuncture may not be causes of spurious or false positive results of alcohol measurement at least when ideal venipunctures can be performed. ..
  2. Etzerodt T, Wetterhorn K, Dionisio G, Rayment I. Functional characterization of a soluble NADPH-cytochrome P450 reductase from Fusarium graminearum. Protein Expr Purif. 2017;138:69-75 pubmed publisher
    ..graminearum which most likely is involved in mycotoxin biosynthesis or some primary metabolic pathway such as sterol biosynthesis in F. graminearum. ..
  3. Mair P, Gielen F, Hollfelder F. Exploring sequence space in search of functional enzymes using microfluidic droplets. Curr Opin Chem Biol. 2017;37:137-144 pubmed publisher
  4. Sato T, Irie T, Yoshino F. Heterologous expression of the Pleurotus ostreatus MnP3 gene by the laccase gene promoter in Lentinula edodes. Biosci Biotechnol Biochem. 2017;81:1553-1556 pubmed publisher
    ..edodes laccase 1 gene promoter into L. edodes for expression. The resulting transformant expressed the recombinant gene and showed a higher level of MnP activity than that of the wild-type strain. ..
  5. Yu Z, Zhao J, Hua Z, Wang X, Wang X, Wang H, et al. Novel 19 F-MRS ?-galactosidase reporter molecules incorporated nitrogen mustard analogues. Chem Biol Drug Des. 2017;90:719-729 pubmed publisher
  6. Muller J, Becher T, Braunstein J, Berdel P, Gravius S, Rohrbach F, et al. Profiling of active thrombin in human blood by supramolecular complexes. Angew Chem Int Ed Engl. 2011;50:6075-8 pubmed publisher
  7. Dick M, Erlenkamp G, Nguyen G, Förster K, Groth G, Gohlke H. Pyrazolidine-3,5-dione-based inhibitors of phosphoenolpyruvate carboxylase as a new class of potential C4 plant herbicides. FEBS Lett. 2017;591:3369-3377 pubmed publisher
    ..These findings make this compound class highly interesting for further investigations toward generating potent, C4 plant-selective herbicides with a low potential for unwanted effects. ..
  8. Zotter A, Bäuerle F, Dey D, Kiss V, Schreiber G. Quantifying enzyme activity in living cells. J Biol Chem. 2017;292:15838-15848 pubmed publisher
    ..This suggests substrate-limited reaction rates to be common. These findings indicate that in vitro data cannot be simply extrapolated to the crowded in vivo environment. ..
  9. Palir N, Ruiter J, Wanders R, Houtkooper R. Identification of enzymes involved in oxidation of phenylbutyrate. J Lipid Res. 2017;58:955-961 pubmed publisher
    ..e., long-chain 3-ketoacyl-CoA thiolase. Taken together, the enzymatic characteristics of the PB degradative pathway may lead to better dose finding and limiting the toxicity of this drug. ..

More Information


  1. El Alaoui M, Soulère L, Noiriel A, Queneau Y, Abousalham A. ?-Eleostearic acid-containing triglycerides for a continuous assay to determine lipase sn-1 and sn-3 regio-preference. Chem Phys Lipids. 2017;206:43-52 pubmed publisher
    ..This continuous high-throughput screening method could determine directly without any processes after lipolysis the regio-selectivity of various lipases. ..
  2. Küchler A, Messmer D, Schlüter A, Walde P. Preparation and Applications of Dendronized Polymer-Enzyme Conjugates. Methods Enzymol. 2017;590:445-474 pubmed publisher
    ..The nanoarmored enzyme-polymer conjugate indicated high stability and retention of enzymatic activity. ..
  3. Yamaguchi T, Yokoyama K, Nakajima C, Suzuki Y. Quinolone resistance-associated amino acid substitutions affect enzymatic activity of Mycobacterium leprae DNA gyrase. Biosci Biotechnol Biochem. 2017;81:1343-1347 pubmed publisher
    ..In addition, patterns of time-dependent conversion from relaxed circular DNA into supercoiled DNA by DNA gyrases with clinically unreported Asp95Gly and Asp95Asn were observed to be distinct from those by the other DNA gyrases. ..
  4. Canbolat M, Savas H, Gultekin F. Enzymatic behavior of laccase following interaction with ?-CD and immobilization into PCL nanofibers. Anal Biochem. 2017;528:13-18 pubmed publisher
    ..Among all test groups, the best activity result was recorded by laccase-?-CD complex encapsulated PCL nanofibers with 96.48 U/mg. ..
  5. Fratebianchi D, Cavello I, Cavalitto S. Purification and Biochemical and Kinetic Properties of an Endo-Polygalacturonase from the Industrial Fungus Aspergillus sojae. J Mol Microbiol Biotechnol. 2017;27:102-109 pubmed publisher
    ..The enzyme exhibits Michaelis-Menten kinetics, with KM and VMAX values of 0.134 mg/mL and 9.6 µmol/mg/min, respectively, and remained stable and active in the presence of SO2, ethanol, and various cations assayed except Hg2+. ..
  6. Koyama K, Miyazaki K, Abe K, Egawa Y, Fukazawa T, Kitta T, et al. Alternative Internal Standard Calibration of an Indirect Enzymatic Analytical Method for 2-MCPD Fatty Acid Esters. J Oleo Sci. 2017;66:585-590 pubmed publisher
    ..The relative standard deviation (RSDR) among the testing laboratories was ? 15% and the Horwitz ratio was ? 1.0, a satisfactory value. ..
  7. Leurs M, Tiller J. Nanoarmored Enzymes for Organic Enzymology: Synthesis and Characterization of Poly(2-Alkyloxazoline)-Enzyme Conjugates. Methods Enzymol. 2017;590:413-444 pubmed publisher
    ..The POx-enzyme conjugates generated by this modification strategy show modulated catalytic activity in both, aqueous and organic, systems. ..
  8. Bertozo L, Zeraik M, Ximenes V. Dansylglycine, a fluorescent probe for specific determination of halogenating activity of myeloperoxidase and eosinophil peroxidase. Anal Biochem. 2017;532:29-37 pubmed publisher
    ..In conclusion, this assay can differentiate the peroxidase and halogenating activity of MPO and EPO. Moreover, the method is adequate for real-time measurement of the production of HOCl and HOBr. ..
  9. Driscoll M, Rentergent J, Hay S. A quantitative fluorescence-based steady-state assay of DNA polymerase. FEBS J. 2014;281:2042-50 pubmed
    ..This approach is used in a steady-state assay of DNA polymerase Klenow fragment exo(?), where we determine kcat and Km values for the DNA polymerase that are in excellent agreement with literature values. ..
  10. Mao W, Qian X, Zhang J, Xia L, Xie H. Specific Detection of Extended-Spectrum ?-Lactamase Activities with a Ratiometric Fluorescent Probe. Chembiochem. 2017;18:1990-1994 pubmed publisher
    ..Further experiments have demonstrated that this ratiometric fluorescent probe can distinguish bacteria with extended-spectrum antibiotic resistance from a group of clinically important pathogens within a short period of time. ..
  11. Chen N, Rinner O, Czernik D, Nytko K, Zheng D, Stiehl D, et al. The oxygen sensor PHD3 limits glycolysis under hypoxia via direct binding to pyruvate kinase. Cell Res. 2011;21:983-6 pubmed publisher
  12. Qiao H, Zhang W, Guan W, Chen F, Zhang S, Deng Z. Enhanced expression of lipase I from Galactomyces geotrichum by codon optimisation in Pichia pastoris. Protein Expr Purif. 2017;138:34-45 pubmed publisher
    ..This lipase may prove useful to the detergent industry and in organic synthesis reactions. ..
  13. Tsurumaki H, Katano H, Sato K, Imai R, Niino S, Hirabayashi Y, et al. WP1066, a small molecule inhibitor of the JAK/STAT3 pathway, inhibits ceramide glucosyltransferase activity. Biochem Biophys Res Commun. 2017;491:265-270 pubmed publisher
    ..2 ?M, and that its action was independent of JAK/STAT3 pathway blockade. Moreover, the modes of inhibition of ceramide glucosyltransferase were uncompetitive with respect to both C6-NBD-cermide and UDP-glucose. ..
  14. Muhammad M, Falak S, Rashid N, Gardner Q, Ahmad N, Imanaka T, et al. Escherichia coli Signal Peptidase Recognizes and Cleaves Archaeal Signal Sequence. Biochemistry (Mosc). 2017;82:821-825 pubmed publisher
    ..coli signal peptidase. To the best of our knowledge, this is the first report describing an archaeal signal sequence recognized and cleaved by E. coli signal peptidase. ..
  15. Filoni D, Pesi R, Allegrini S, Camici M, Tozzi M. A native electrophoretic technique to study oligomerization and activity of cytosolic 5?-nucleotidase II. Anal Bioanal Chem. 2013;405:8951-4 pubmed publisher
    ..Furthermore, cN-II mutants K311A and G319Q in subunit interface assume a quaternary dimeric active form, while the only active quaternary structure of wild-type cN-II is the tetramer. ..
  16. Chen W, Hu H, Zhang C, Huang F, Zhang D, Zhang H. Adaptation response of Pseudomonas fragi on refrigerated solid matrix to a moderate electric field. BMC Microbiol. 2017;17:32 pubmed publisher
    ..Our study provides direct evidence that psychrotrophic P. fragi MC16 cultured on nutrient agar plates at 7 °C are capable of adapting to MEF treatment. ..
  17. McKee L. Measuring Enzyme Kinetics of Glycoside Hydrolases Using the 3,5-Dinitrosalicylic Acid Assay. Methods Mol Biol. 2017;1588:27-36 pubmed publisher
    ..Presented here is an application of the method in measuring the kinetics of a glycoside hydrolase reaction, including the optimization of the DNSA reagent, and the production of a standard curve of absorbance and sugar concentration. ..
  18. Ott C, Baljinnyam B, Zakharov A, Jadhav A, Simeonov A, Zhuang Z. Cell Lysate-Based AlphaLISA Deubiquitinase Assay Platform for Identification of Small Molecule Inhibitors. ACS Chem Biol. 2017;12:2399-2407 pubmed publisher
    ..We expect that the new platform can be readily adapted to other DUBs to allow the identification of more potent and selective small molecule inhibitors and chemical probes...
  19. Rossano R, Larocca M, Riviello L, Coniglio M, Vandooren J, Liuzzi G, et al. Heterogeneity of serum gelatinases MMP-2 and MMP-9 isoforms and charge variants. J Cell Mol Med. 2014;18:242-52 pubmed
    ..These observations open perspectives for better diagnosis and prognosis of many diseases and need to be critically interpreted when applying other methods for MS and other diseases. ..
  20. Mai Gisondi G, Master E. Colorimetric Detection of Acetyl Xylan Esterase Activities. Methods Mol Biol. 2017;1588:45-57 pubmed publisher
    ..g., acetylated p-nitrophenyl ?-D-xylopyranosides) and various acetylated xylans are also described. Strengths and limitations of the colorimetric assays are highlighted. ..
  21. Pensa A, Cinelli M, Li H, Chreifi G, Mukherjee P, Roman L, et al. Hydrophilic, Potent, and Selective 7-Substituted 2-Aminoquinolines as Improved Human Neuronal Nitric Oxide Synthase Inhibitors. J Med Chem. 2017;60:7146-7165 pubmed publisher
    ..Most importantly, these modifications preserved Caco-2 permeability and reduced off-target CNS binding. ..
  22. Nicolai E, Sinibaldi F, Sannino G, Lagana G, Basoli F, Licoccia S, et al. Omega-3 and Omega-6 Fatty Acids Act as Inhibitors of the Matrix Metalloproteinase-2 and Matrix Metalloproteinase-9 Activity. Protein J. 2017;36:278-285 pubmed publisher
    ..This observation is of interest since it assigns to these compounds a key role as MMPs inhibitors, and stimulates further study to better define their therapeutic potentialities in carious decay. ..
  23. Arnal G, Attia M, Asohan J, Brumer H. A Low-Volume, Parallel Copper-Bicinchoninic Acid (BCA) Assay for Glycoside Hydrolases. Methods Mol Biol. 2017;1588:3-14 pubmed publisher
  24. Ehlert F, Linde K, Diederich W. What Are We Missing? The Detergent Triton X-100 Added to Avoid Compound Aggregation Can Affect Assay Results in an Unpredictable Manner. ChemMedChem. 2017;12:1419-1423 pubmed publisher
  25. Smirnova J, Fernie A, Spahn C, Steup M. Photometric assay of maltose and maltose-forming enzyme activity by using 4-alpha-glucanotransferase (DPE2) from higher plants. Anal Biochem. 2017;532:72-82 pubmed publisher
    ..These data are presented together with assimilatory starch levels. ..
  26. Nadtochiy S, Wang Y, Zhang J, Nehrke K, Schafer X, Welle K, et al. Potential mechanisms linking SIRT activity and hypoxic 2-hydroxyglutarate generation: no role for direct enzyme (de)acetylation. Biochem J. 2017;474:2829-2839 pubmed publisher
    ..Overall, these data suggest that a causal relationship should not be assumed between acetylation of metabolic enzymes and their activities, canonical or otherwise. ..
  27. Noda T, Iimure K, Okamoto S, Saito A. Expression analysis of polyphenol oxidase isozymes by active staining method and tissue browning of head lettuce (Lactuca sativa L.). Biosci Biotechnol Biochem. 2017;81:1484-1488 pubmed publisher
    ..By expression analysis of the PPO isozymes after wounding, PPO isozymes that correlated with time-course of tissue browning were detected. The wound-induced PPO may play a key role in enzymatic browning...
  28. Grigoletto A, Mero A, Maso K, Pasut G. Transgultaminase-Mediated Nanoarmoring of Enzymes by PEGylation. Methods Enzymol. 2017;590:317-346 pubmed publisher
  29. Lamonte G, Almaliti J, Bibo Verdugo B, Keller L, Zou B, Yang J, et al. Development of a Potent Inhibitor of the Plasmodium Proteasome with Reduced Mammalian Toxicity. J Med Chem. 2017;60:6721-6732 pubmed publisher
    ..This study describes a promising new class of antimalarial compound based on the carmaphycin B scaffold, as well as several chemical structural features that serve to enhance antimalarial specificity. ..
  30. Matsumoto M, Fujimura Y, Wada H, Kokame K, Miyakawa Y, Ueda Y, et al. Diagnostic and treatment guidelines for thrombotic thrombocytopenic purpura (TTP) 2017 in Japan. Int J Hematol. 2017;106:3-15 pubmed publisher
    ..Recent reports show that the monoclonal anti-CD-20 antibody rituximab is effective in patients with refractory or relapsed TTP. ..
  31. Trani A, Gambacorta G, Loizzo P, Cassone A, Fasciano C, Zambrini A, et al. Comparison of HPLC-RI, LC/MS-MS and enzymatic assays for the analysis of residual lactose in lactose-free milk. Food Chem. 2017;233:385-390 pubmed publisher
    ..An LC-MS/MS method using the formate adduct was developed, and it allowed quantitation of lactose and lactulose in all samples at a high level of precision and repeatability. ..
  32. Hester K, Liu J, Flynn N, Sultatos L, Geng L, Brimijoin S, et al. Polyionic complexes of butyrylcholinesterase and poly-l-lysine-g-poly(ethylene glycol): Comparative kinetics of catalysis and inhibition and in vitro inactivation by proteases and heat. Chem Biol Interact. 2017;275:86-94 pubmed publisher
    ..Understanding the unique properties of PLL-g-PEG-BChE complexes may lead to enhanced approaches for use of BChE and other protein bioscavengers. ..
  33. Jones D, McLean R, Abbott D. An Improved Kinetic Assay for the Characterization of Metal-Dependent Pectate Lyases. Methods Mol Biol. 2017;1588:37-44 pubmed publisher
    ..We use an endolytic PL2 from Yersinia enterocolitica (YePL2A) as example to demonstrate how altering the catalytic metal within the reaction can modulate PL kinetics. ..
  34. Dimova D, Bajorath J. Assessing Scaffold Diversity of Kinase Inhibitors Using Alternative Scaffold Concepts and Estimating the Scaffold Hopping Potential for Different Kinases. Molecules. 2017;22: pubmed publisher
    ..Our analysis revealed that the consideration of conventional compound-based scaffolds most likely leads to an overestimation of scaffold hopping frequency, at least for compounds forming analog series. ..
  35. Conrad C, Miller M, Bartsch J, Schlomann U, Lauffenburger D. Simultaneous Detection of Metalloprotease Activities in Complex Biological Samples Using the PrAMA (Proteolytic Activity Matrix Assay) Method. Methods Mol Biol. 2017;1574:243-253 pubmed publisher
  36. Sthijns M, den Hartog G, Scasso C, Haenen J, Bast A, Haenen G. Activation versus inhibition of microsomal glutathione S-transferase activity by acrolein. Dependence on the concentration and time of acrolein exposure. Chem Biol Interact. 2017;275:116-120 pubmed publisher
    ..These opposite forms of direct adaptation on the level of enzyme activity further narrow the thin line between survival and promotion of cell death, governed by the level of exposure. ..
  37. Irague R, Massou S, Moulis C, Saurel O, Milon A, Monsan P, et al. NMR-based structural glycomics for high-throughput screening of carbohydrate-active enzyme specificity. Anal Chem. 2011;83:1202-6 pubmed publisher
    ..The results demonstrate the potential of high-throughput NMR technology in glycomics, to mine artificial and natural enzyme diversity for novel biocatalysts. ..
  38. Contreras Aguilar M, Escribano D, Martinez Subiela S, Martínez Miró S, Rubio M, Tvarijonaviciute A, et al. Influence of the way of reporting alpha-Amylase values in saliva in different naturalistic situations: A pilot study. PLoS ONE. 2017;12:e0180100 pubmed publisher
    ..Therefore, how sAA is reported and the factors involved in the different ways of expressing sAA, should be taken into consideration for an objective interpretation of sAA values. ..
  39. Danese E, Salvagno G, Negrini D, Brocco G, Montagnana M, Lippi G. Analytical evaluation of three enzymatic assays for measuring total bile acids in plasma using a fully-automated clinical chemistry platform. PLoS ONE. 2017;12:e0179200 pubmed publisher
  40. Raghupathi K, THAYUMANAVAN S. Nano-Armoring of Enzymes: Rational Design of Polymer-Wrapped Enzymes. Methods Enzymol. 2017;590:381-411 pubmed publisher
    ..Nano-armoring of the enzymes were nearly complete and provided high yields of the encased enzyme. ..
  41. Liu W, Wang Y, Zhang Z, Wang M, Lv Q, Liu H, et al. Generation and characterization of caprine chymosin in corn seed. Protein Expr Purif. 2017;135:78-82 pubmed publisher
    ..5 U/mg. These results indicate that we have successfully established a technology for generating corn seed-derived caprine chymosin for potential use in the dairy industry. ..
  42. Ramjee M, Patel S. Continuous-flow injection microfluidic thrombin assays: The effect of binding kinetics on observed enzyme inhibition. Anal Biochem. 2017;528:38-46 pubmed publisher
  43. Ngo M, Suits M. Methods for Determining Glycosyltransferase Kinetics. Methods Mol Biol. 2017;1588:59-70 pubmed publisher
  44. El Naggar N, Deraz S, Soliman H, El Deeb N, El Shweihy N. Purification, characterization and amino acid content of cholesterol oxidase produced by Streptomyces aegyptia NEAE 102. BMC Microbiol. 2017;17:76 pubmed publisher
    ..49 ?g/mL. Taking into account the extracellular production, wide pH tolerance, thermal stability and shelf life, cholesterol oxidase produced by Streptomyces aegyptia NEAE 102 suggested that the enzyme could be industrially useful. ..
  45. Park C, Lee K, Jun S, Song S, Song J. Ultra-performance liquid chromatography-tandem mass spectrometry-based multiplex enzyme assay for six enzymes associated with hereditary hemolytic anemia. J Chromatogr B Analyt Technol Biomed Life Sci. 2017;1060:76-83 pubmed publisher
    ..To overcome the drawbacks of traditional spectrophotometric enzyme assays, the present assay was based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)...
  46. Qian H, Xia B, He Y, Lu Z, Bie X, Zhao H, et al. Expression, purification, and characterization of a novel acidic Lipoxygenase from Myxococcus xanthus. Protein Expr Purif. 2017;138:13-17 pubmed publisher
    ..The synthetic dye aniline blue was decolorized 69.7 ± 3.5%, following incubation with rMxLOX for 35 min. These results reveal the potential for the use of rMxLOX in the pulp, textile, and wastewater treatment industries. ..
  47. Anzuay M, Ciancio M, Ludueña L, Angelini J, Barros G, Pastor N, et al. Growth promotion of peanut (Arachis hypogaea L.) and maize (Zea mays L.) plants by single and mixed cultures of efficient phosphate solubilizing bacteria that are tolerant to abiotic stress and pesticides. Microbiol Res. 2017;199:98-109 pubmed publisher
    ..Phosphate solubilizing bacteria used in this study showed efficient phosphate mineralizing and solubilization ability and would be potential P-biofertilizers for peanut and maize. ..
  48. Pettengill M, Matute J, Tresenriter M, Hibbert J, Burgner D, Richmond P, et al. Human alkaline phosphatase dephosphorylates microbial products and is elevated in preterm neonates with a history of late-onset sepsis. PLoS ONE. 2017;12:e0175936 pubmed publisher
    ..TNAP, the main circulating isozyme of ALP, de-phosphorylates TLR agonists, demonstrates a post-natal age dependent increase in preterm and term plasma across the first 4 weeks of life, and is elevated in association with preterm LOS. ..
  49. Matzel P, Krautschick L, Höhne M. Photometric Characterization of the Reductive Amination Scope of the Imine Reductases from Streptomyces tsukubaensis and Streptomyces ipomoeae. Chembiochem. 2017;18:2022-2027 pubmed publisher
    ..The nucleophile acceptor spectrum depended on the carbonyl substrate employed. The conversion of well-accepted substrates could also be detected if crude lysates were employed as the enzyme source. ..
  50. Elnegaard R, Møllegaard N, Zhang Q, Kjeldsen F, Jørgensen T. Uranyl Photocleavage of Phosphopeptides Yields Truncated C-Terminally Amidated Peptide Products. Chembiochem. 2017;18:1117-1122 pubmed publisher
    ..This could be of great interest to the pharmaceutical industry, as efficient peptide amidation reactions are one of the top challenges in green pharmaceutical chemistry. ..
  51. Chen X, Pradhan T, Wang F, Kim J, Yoon J. Fluorescent chemosensors based on spiroring-opening of xanthenes and related derivatives. Chem Rev. 2012;112:1910-56 pubmed publisher
  52. Danyluk H, Shum L, Zandberg W. A Rapid Procedure for the Purification of 8-Aminopyrene Trisulfonate (APTS)-Labeled Glycans for Capillary Electrophoresis (CE)-Based Enzyme Assays. Methods Mol Biol. 2017;1588:223-236 pubmed publisher
    ..This chapter describes a FACE/SPE procedure ideal for preparing glycans for capillary electrophoresis (CE)-based enzyme assays, as well as for the purification of rare, commercially unavailable glycans from tissue culture samples.