«Reagent-free» L-asparaginase enzyme activity assay based on CD spectroscopy and conductometryстатья

Статья опубликована в высокорейтинговом журнале

Информация о цитировании статьи получена из Scopus, Web of Science
Статья опубликована в журнале из списка Web of Science и/или Scopus
Дата последнего поиска статьи во внешних источниках: 30 марта 2016 г.

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1. Полный текст CD_aspaprginase_final.pdf 407,7 КБ 7 ноября 2016 [ElenaKudryashova]

[1] Кудряшова Е. В., Суховерков К. В. reagent-free l-asparaginase enzyme activity assay based on cd spectroscopy and conductometry // Analytical and Bioanalytical Chemistry. — 2016. — Vol. 408, no. 4. — P. 1183–1189. A new method to determine the catalytic parameters of L-asparaginase using CD spectroscopy has been developed. The assay is based on the difference in CD signal between the substrate (L-Asparagine) and the product (L-Aspartic acid) of enzymatic reaction. CD spectroscopy, being a direct method, enables continuous measurement, and thus differentiates from multistage and laborious approach based on the Nessler’s method, and overcomes limitations of conjugated enzymatic reaction methods. In this work we show robust measurements of L-Asparaginase activity in conjugates with PEG-chitosan copolymers, which otherwise would not have been possible. The main limitation associated with CD-method is that the analysis should be performed at substrate saturation conditions (Vmax regime). For KM measurement conductometry method is suggested, which can serve as a complimentary method to CD spectroscopy. The activity assay based on CD spectroscopy and conductometry was successfully implicated to examine the catalytic parameters of L-asparaginase conjugates with chitosan and its derivatives, and for optimization of molecular architecture and composition of such conjugates for improving biocatalytic properties of the enzyme in the physiological conditions. The approach developed is potentially applicable to other enzymatic reactions where the spectroscopic properties of substrate and product do not enable direct measurement with absorption or fluorescence spectroscopy. This may include a number of amino acid or glycoside transforming enzymes. [ DOI ]

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