Revisiting the fundamentals of p-nitrophenol analysis for its application in the quantification of lipases activity. A graphical update.
p-Nitrophenol (pNP) is a widely used compound for analytical determinations of several esterases (EC. 3.1.1.X), including lipases (E.C. 18.104.22.168). Most enzymatic measurements employ pNP derivatives such as esters, which are broken down by enzymatic hydrolysis, releasing pNP that is quantified by its absorbance at 410 nm. Although this type of methods was developed a few decades ago, the spectrophotometric analysis of pNP requires analytical measurements of pH and temperature to achieve reliable determinations. The aim of this paper is to offer a graphical update of how pH and temperature affect the p-nitrophenol absorbance at different wavelengths in lipase emulsified media, due to its relevance for the quantitative determination of lipase activity using spectrophotometric methods. To highlight the importance of each variable involved in this analysis, we dissolved pNP in emulsified media (for lipase activity quantification) at several pH values from 4.00 to 11.00, and measured its absorbance in a range of 270 nm – 500 nm and at several temperatures from 25°C to 50°C. The absorption patterns of pNP under the established conditions were graphed in 3D plots. The constructed 3D plots showed that, regardless of the temperature, below pH 6.00, pNP predominantly absorbs at 317 nm, due to the greater abundance of its protonated form, which is completely predominant at pH 3.50 and below. On the other hand, at pH 10.0 and above, the major absorption occurs at about 401 nm, confirming that the equilibrium is completely shifted to the pNP anionic form. These results also indicate that close to neutral pH value pNP, it displays a temperature dependence effect, increasing absorbance to 410 nm at higher temperatures. Due to many analytical determinations of enzymatic activities, the release of pNP is carried around pH 7.00. It is necessary to consider the determinant role of both pH and temperature over these measurements, how these variables must be strictly controlled, and how the calibration curves and blanks should take the reaction media pH and temperature into account.
Biggs, A. I. (1954). A spectrophotometric determination of the dissociation constants of p-nitrophenol and papaverine. Transactions of the Faraday Society, 50(0), 800. doi: https://doi.org/10.1039/tf9545000800
Daiha, K. de G.; Angeli, R.; de Oliveira, S. D. & Almeida, R. V. (2015). Are Lipases Still Important Biocatalysts? A Study of Scientific Publications and Patents for Technological Forecasting. PLOS ONE, 10(6), e0131624. doi: https://doi.org/10.1371/journal.pone.0131624
Farnet, A. M.; Qasemian, L.; Goujard, L.; Gil, G.; Guiral, D.; Ruaudel, F. & Ferre, E. (2010). A modified method based on p-nitrophenol assay to quantify hydrolysis activities of lipases in litters. Soil Biology and Biochemistry, 42(2), 386–389. doi: https://doi.org/10.1016/J.SOILBIO.2009.11.015
Fourage, L.; Helbert, M.; Nicolet, P. & Colas, B. (1999). Temperature Dependence of the Ultraviolet–Visible Spectra of Ionized and Un-ionized Forms of Nitrophenol: consequence for the Determination of Enzymatic Activities Using Nitrophenyl Derivatives—A Warning. Analytical Biochemistry, 270(1), 184–185. doi: https://doi.org/10.1006/ABIO.1999.4071
Gupta, N.; Rathi, P. & Gupta, R. (2002). Simplified para-nitrophenyl palmitate assay for lipases and esterases. Analytical Biochemistry, 311(1), 98–99. doi: https://doi.org/10.1016/S0003-2697(02)00379-2
Hernández-García, S.; García-García, M. I. & García-Carmona, F. (2017). An improved method to measure lipase activity in aqueous media. Analytical Biochemistry, 530, 104–106. doi: https://doi.org/10.1016/J.AB.2017.05.012
Kapoor, M. & Gupta, M. N. (2012). Lipase promiscuity and its biochemical applications. Process Biochemistry, 47(4), 555–569. doi: https://doi.org/10.1016/J.PROCBIO.2012.01.011
Kumar, A.; Dhar, K.; Kanwar, S. S. & Arora, P. K. (2016). Lipase catalysis in organic solvents: advantages and applications. Biological Procedures Online, 18(2),1-11. doi: https://doi.org/10.1186/s12575-016-0033-2
Lam, Q.; Cortez, A.; Nguyen, T. T.; Kato, M. & Cheruzel, L. (2016). Chromogenic nitrophenolate-based substrates for light-driven hybrid P450 BM3 enzyme assay. Journal of Inorganic Biochemistry, 158, 86–91. doi: https://doi.org/10.1016/J.JINORGBIO.2015.12.005
Liu, Z.-Q.; Zheng, X.-B.; Zhang, S.-P. & Zheng, Y.-G. (2012). Cloning, expression and characterization of a lipase gene from the Candida antarctica ZJB09193 and its application in biosynthesis of vitamin A esters. Microbiological Research, 167(8), 452–460. doi: https://doi.org/10.1016/J.MICRES.2011.12.004
Lopes, D. B.; Fraga, L. P.; Fleuri, L. F. & Macedo, G. A. (2011). Lipase and esterase: to what extent can this classification be applied accurately? Ciência e Tecnologia de Alimentos, 31(3), 603–613. doi: https://doi.org/10.1590/S0101-20612011000300009
Mayordomo, I.; Randez-Gil, F. & Prieto, J. A. (2000). Isolation, Purification, and Characterization of a Cold-Active Lipase from Aspergillus nidulans. Journal of Agricultural and Food Chemistry, 48(1), 105–109. doi: https://doi.org/10.1021/jf9903354
Orlando Beys Silva, W.; Mitidieri, S.; Schrank, A. & Vainstein, M. H. (2005). Production and extraction of an extracellular lipase from the entomopathogenic fungus Metarhizium anisopliae. Process Biochemistry, 40(1), 321–326. doi: https://doi.org/10.1016/J.PROCBIO.2004.01.005
Palacios, D.; Busto, M. D. & Ortega, N. (2014). Study of a new spectrophotometric end-point assay for lipase activity determination in aqueous media. LWT - Food Science and Technology, 55(2), 536–542. doi: https://doi.org/10.1016/j.lwt.2013.10.027
Peng, Y.; Fu, S.; Liu, H. & Lucia, L. A. (2016). Accurately Determining Esterase Activity via the Isosbestic Point of p-Nitrophenol. BioResources, 11(4), 10099-10111. doi: https://doi.org/10.15376/biores.11.4.10099-10111
Pratama, L.; Helianti, I.; Suryani, A. & Wahyuntari, B. (2017). Isolation, Characterization, and Production of Lipase from Indigenous Fungal for Enzymatic Interesterification Process. Microbiology Indonesia, 11(2), 35-45. doi: https://doi.org/10.5454/MI.11.2.%P
Sandoval, M.; Hoyos, P.; Cortés, A.; Bavaro, T.; Terreni, M. & Hernáiz, M. J. (2014). Development of regioselective deacylation of peracetylated β- d -monosaccharides using lipase from Pseudomonas stutzeri under sustainable conditions. RSC Adv., 4(98), 55495–55502. doi: https://doi.org/10.1039/C4RA10401C
Sandoval, M.; Ferreras, E.; Pérez-Sánchez, M.; Berenguer, J.; Sinisterra, J. V. & Hernaiz, M. J. (2012). Screening of strains and recombinant enzymes from Thermus thermophilus for their use in disaccharide synthesis. Journal of Molecular Catalysis B: Enzymatic, 74(3–4), 162–169. doi: https://doi.org/10.1016/J.MOLCATB.2011.09.012
Serjeant, EP.; Dempsey, B. (1979). Ionisation Constants of Organic Acids in Aqueous Solution. In: IUPAC Chemical Data Series No 23 (p. 989). New York, USA: Pergamon Press.
Shen, Y.-Y.; Sun, Y.; Zhou, L.-N.; Li, Y.-J. & Yeung, E. S. (2014). Synthesis of ultrathin PtPdBi nanowire and its enhanced catalytic activity towards p-nitrophenol reduction. Journal of Materials Chemistry A, 2(9), 2977. doi: https://doi.org/10.1039/c3ta14502f
Stoytcheva, M.; Montero, G.; Zlatev, R.; A. Leon, J. & Gochev, V. (2012). Analytical Methods for Lipases Activity Determination: a Review. Current Analytical Chemistry, 8(3), 400–407. doi: https://doi.org/10.2174/157341112801264879
Tang, J.; Tang, D.; Su, B.; Huang, J.; Qiu, B. & Chen, G. (2011). Enzyme-free electrochemical immunoassay with catalytic reduction of p-nitrophenol and recycling of p-aminophenol using gold nanoparticles-coated carbon nanotubes as nanocatalysts. Biosensors and Bioelectronics, 26(7), 3219–3226. doi: https://doi.org/10.1016/J.BIOS.2010.12.029
Winkler, U. K. & Stuckmann, M. (1979). Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. Journal of Bacteriology, 138(3), 663–670. doi: http://www.ncbi.nlm.nih.gov/pubmed/222724
Zhang, P.; Sui, Y.; Xiao, G.; Wang, Y.; Wang, C.; Liu, B. & Zou, B. (2013). Facile fabrication of faceted copper nanocrystals with high catalytic activity for p-nitrophenol reduction. J. Mater. Chem. A, 1(5), 1632–1638. doi: https://doi.org/10.1039/C2TA00350C
Authors who publish with this journal agree to the following terms:
1. Authors guarantee the journal the right to be the first publication of the work as licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgment of the work's authorship and initial publication in this journal.
2. Authors can set separate additional agreements for non-exclusive distribution of the version of the work published in the journal (eg, place it in an institutional repository or publish it in a book), with an acknowledgment of its initial publication in this journal.
3. The authors have declared to hold all permissions to use the resources they provided in the paper (images, tables, among others) and assume full responsibility for damages to third parties.
4. The opinions expressed in the paper are the exclusive responsibility of the authors and do not necessarily represent the opinion of the editors or the Universidad Nacional.
Uniciencia Journal and all its productions are under Creative Commons Atribución-NoComercial-SinDerivadas 4.0 Unported.
There is neither fee for access nor Article Processing Charge (APC)
Comentarios (ver términos de uso)
Most read articles by the same author(s)
- Luis Diego Mesén-Mora, Yendry Carvajal-Miranda, Victor Álvarez-Valverde, Gerardo Rodríguez-Rodríguez, Bioprospecting study, antibiotic and antioxidant activity of the santol’s fruit (Sandoricum koetjape) , Uniciencia: Vol 33 No 1 (2019): Uniciencia. January-june, 2019