title: "Reagent-free" L-asparaginase activity assay based on CD spectroscopy and conductometry.
abstract: A new method to determine the catalytic parameters of L-asparaginase using circular dichroism spectroscopy (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 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 the CD method is that the analysis should be performed at substrate saturation conditions (V max regime). For K M measurement, the 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 the 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.
authors.affiliation: Faculty of Chemistry, M.V. Lomonosov Moscow State University, 119991, Moscow, Russia. Helena_Koudriachova@hotmail.com.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Kudryashova
authors.affiliation: Faculty of Chemistry, M.V. Lomonosov Moscow State University, 119991, Moscow, Russia.
authors.forename: Kirill V
authors.initials: KV
authors.lastname: Sukhoverkov
mesh_terms: D001215:Asparaginase / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D001216:Asparagine / Q000737:chemistry / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D048271:Chitosan / Q000737:chemistry; D002942:Circular Dichroism / Q000379:methods*; D003217:Conductometry / Q000379:methods*; D011092:Polyethylene Glycols / Q000737:chemistry; D015203:Reproducibility of Results
chemical_list: D001224:Aspartic Acid; D011092:Polyethylene Glycols; D001216:Asparagine; D048271:Chitosan; D001215:Asparaginase
keywords: Branched copolymers; CD spectroscopy; Catalytic activity; Conductometry; L-Asparaginase; PEG–chitosan