¹ Institute of Biomedical Chemistry, Moscow, Russia

² Joint Institute for High Temperatures of the Russian Academy of Sciences, Moscow, Russia

³ Alferov Federal State Budgetary Institution of Higher Education and Science Saint Petersburg National Research Academic University of the Russian Academy of Sciences, St. Petersburg, Russia

⁴ Foundation of Perspective Technologies and Novations, Moscow, Russia

⁵ Moscow State University, Faculty of Computational Mathematics and Cybernetics, Moscow, Russia

⁶ Р.N. Lebedev Physical Institute of the Russian Academy of Sciences, Moscow, Russia

L-asparaginase (L-Aspase) enzyme has found applications in medicine for treatment of various cancers. Herein, we report single-molecule study of thermal denaturation of L-Aspase within 25°C to 60°C temperature range by atomic force microscopy (AFM) and by single-molecule sensing with a (solid state nanopore)-based electrical detector (SSNPED). AFM has allowed us to reveal a thermally induced changes in aggregation state of L-Aspase and in its adsorbability on mica. At the same time, the configuration of the enzyme’s globule spatial conformation has been found to alter according to data obtained with the SSNPED. Our results reported open up opportunities for further development of anti-cancer drugs.

Keywords: L-asparaginase; solid-state nanopore; thermal denaturation; atomic force microscopy

L-asparaginase (L-Aspase) enzyme pertains to hydrolases; the molecular weight of its monomer is about 36 kDa [1]. This enzyme catalyzes conversion of L-asparagine into aspartate [2]. Since Lasparagine, in its turn, represents an essential amino acid (AA) for tumour cells [2], L-Aspase has found applications in cancer therapy: for instance, the use of L-Aspase for treatment of lymphoblastic leukemia [3,4], hepatocellular carcinoma, lymphosarcoma, pancreatic adenocarcinoma ant other cancers [5,6]. Of note, only Type II L-Aspase is suitable for medical applications, while Type I enzyme lacks therapeutic activity [7].

Molecular detectors, which include atomic force microscope and nanopore-based detectors, allow one to perform single-molecule studies of biological macromolecules, such as proteins (including enzymes) [8,9] and DNA [10]. While atomic force microscopy (AFM) allows for visualization of enzyme macromolecules [8,9], (solid state nanopore)-based electrical detectors (SSNPEDs) enable registration of functional activity of single enzyme molecules [11–13].

The present research is aimed at single-molecule study of thermal denaturation of L-Aspase E. сarotovora. Functionality of the enzyme has been checked at 25°C with an SSNPED, whose nanopore was formed in a silicon nitride (SiN) membrane. Thermal denaturation and dependence of aggregation state of the enzyme on temperature have subsequently been studied by AFM within 25 to 60 °C temperature range. The results of the study can be of use in the development of novel anticancer drugs based on L-Aspase.

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Preprints ID: 10.20944/preprints202601.0032.v1

https://www.preprints.org/manuscript/202601.0032

https://doi.org/10.20944/preprints202601.0032.v1