An improved method for simple and accurate colorimetric determination of l-asparaginase enzyme activity using Nessler's reagent

Author(s)

Simas, Rodrigo G. ^{[1, 2]} ; Krebs Kleingesinds, Eduardo ^{[1, 2]} ; Pessoa Junior, Adalberto ^{[1, 2]} ; Long, Paul F. ^{[1, 2]}

Affiliation

^[1] Univ Sao Paulo, Fac Ciencias Farmaceut, Sao Paulo - Brazil

^[2] Kings Coll London, Fac Life Sci & Med, 150 Stamford St, London SE1 9NH - England

• Document type: Journal article
• Source: JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY; v. 96, n. 5 JAN 2021.
• Web of Science Citations: 0

Abstract

BACKGROUND: Colorimetric determination of l-asparaginase enzyme activity most commonly uses Nessler's reagent, but often yields unreliable results due to precipitation of tetraiodomercurate salts. Interference caused by some commonly used inorganic and organic compounds of biochemical buffers, reagents and microbiology fermentation media was determined. The stabilising effects of tartrate and polyvinyl alcohol upon the impact of these salts was next evaluated using the following two-step assay in 96-well microtitre plates: 160 mu L of 50 mmol L-1 Tris-HCl pH 8.6, 32 mu L of 100 mmol L-1 l-asparagine, 16 mu L analyte and 128 mu L water were added to each well. After incubation at 37 degrees C for 1 h, 16 mu L of 1.5 mol L-1 trichloroacetic acid was added to stop the reaction. A 35 mu L volume was next transferred to a fresh well containing 35 mu L Nessler's reagent, 35 mu L stabiliser solution (4 mmol L-1 disodium tartrate and 10 mg L-1 polyvinyl alcohol) and 245 mu L water. After incubation at room temperature for 10 min, absorbance was recorded at 436 nm and the enzyme activity calculated by extrapolation of a (NH4)(2)SO4 calibrated standard curve. RESULTS: Addition of tartrate and polyvinyl alcohol proved to be effective in reducing interference caused by precipitation of salts, allowing determination of l-asparaginase activity in a range from 0.050 to 0.796 IU mL(-1), ideal for monitoring enzyme production in fermentations. CONCLUSION: A method is offered for rapid and reliable determination of l-asparaginase activity, particularly in complex samples such as microbial fermentation broths where existing methodologies do not account for interferences that frequently yield overvalues. (c) 2020 Society of Chemical Industry (AU)

• FAPESP's process: 19/06919-2 - In vivo test of humanized L-asparaginase free and nano-encapsulated in liposomes
• Grantee: Eduardo Krebs Kleingesinds
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• FAPESP's process: 19/12295-1 - Identification of stress factors and their influence on genotype to phenotype changes during recombinant L-Asparaginase production by Escherichia coli
• Grantee: Rodrigo Gabriel Simas
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate