FTIR-based L-asparaginase activity assay enables continuous measurements in optically dense media including blood plasma

• Published in: Analytical biochemistry Vol. 598; p. 113694
• Main Authors: Kudryashova, Elena V., Pokrovskaya, Marina V., Alexandrova, Svetlana S., Vinogradov, Alexander A., Sokolov, Nikolay N.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-06-2020
• Subjects: Asparaginase - analysis; Asparaginase - metabolism; Biocatalysis; Blood plasma; Catalytic activity; Chitosan - chemistry; FTIR spectroscopy; Humans; l-asparaginase; Oligomeric composition; Pectobacterium carotovorum - enzymology; PEG-chitosan; Polyethylene Glycols - chemistry; Reversed micelles; Spectroscopy, Fourier Transform Infrared; PEG-chitosan; Reversed micelles; Catalytic activity; Oligomeric composition; l-asparaginase; FTIR spectroscopy; Blood plasma
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2020.113694

Complex heterogeneous systems, such as micelles or blood plasma, represent a particularly challenging environment to measure the catalytic parameters of some enzymes, including l-asparaginase. Existing methods are strongly interfered by the presence of plasma proteins, amino acids, as well as other components of plasma. Here we show that FTIR spectroscopy enables continuous real-time measurement of catalytic activity of l-asparaginase, in native and in PEG-chitosan conjugated form, in aqueous solutions as well as in heterogeneous non-transparent multicomponent systems, including colloidal systems or blood plasma, with minimal or no sample preparation. The approach developed is potentially applicable to other enzymatic reactions where the spectroscopic properties of substrate and product do not allow direct measurement with absorption or fluorescence spectroscopy.
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•New method is developed to determine catalytic parameters of l-asparaginase using FTIR spectroscopy.•The new method works well in complex heterogeneous systems, including blood plasma and reversed micelles.•Using the new method we performed the first ever direct measurement of dimeric L-asparaginase activity.