Дискуссии - Наука

Еkaterina E. Vazhenkova, Ivan D. Shumov, Dmitry D. Zhdanov, Victoria V. Shumyantseva, Vadim S. Ziborov, Alexander N. Ableev, Andrey F. Kozlov, Oleg N. Afonin, Nikita V. Vaulin, Denis V. Lebedev, Anton S. Bukatin, Ivan S. Mukhin, Vadim Y. Tatur, Andrei A. Lukyanitsa, Irina N. Saraeva, Alexander I. Archakov, Yuri D. Ivanov

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

 

Abstract

L-asparaginase (L-ASNase) enzyme has found applications in medicine for treatment of various cancers. Herein, we report single-molecule study of thermal denaturation of L-ASNase 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-ASNase 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. The results of AFM experiments have been confirmed by fluorescence spectroscopy. Our results reported open up opportunities for further development of anti-cancer drugs.

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

1. Introduction

L-asparaginase (L-ASNase) 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 L-asparagine, in its turn, represents an essential amino acid (AA) for tumour cells [2], L-ASNase has found applications in cancer therapy: for instance, the use of L-ASNase for treatment of lymphoblastic leukemia [3,4], hepatocellular carcinoma, lymphosarcoma, pancreatic adenocarcinoma ant other cancers [5,6]. Of note, only Type II L-ASNase 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 [11,12,13].

The present research is aimed at single-molecule study of thermal denaturation of L-ASNase 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 thermal denaturation has also been studied by fluorescence spectroscopy. The results of the study can be of use in the development of novel anticancer drugs based on L-ASNase.

2. Materials and Methods

2.1. Enzyme and Chemicals

L-Asparaginase enzyme E. сarotovora was obtained and purified in IBMC (Laboratory of Medical Biotechnology) according to previously published protocols [17]. Lyophilized powder of the enzyme was dissolved in 2 mM Dulbecco’s modified phosphate buffered saline (PBSD) and diluted to the desired concentration. In all experiments, ultrapure (with a resistivity of 18.2 M Ω?cm) water obtained with a Simplicity UV purification system (Millipore, Molsheim, France) was used.

2.2. Solid State Nanopore-Based Electrical Detector

The SSNPED comprised a measuring cell fabricated from polydimethylsiloxane (PDMS). The cell was divided into two chambers with a holder, in which a nanopore chip was fixed. Namely, 40-nm-thick silicon nitride chip with a single ~6 nm nanopore was inserted into and fixed in the holder. The nanopore was formed in the chip by electron beam drilling (EBD) with a JEM 2000F transmission electron microscope (TEM; JEOL Ltd., Akishima, Tokyo, Japan). Figure 1 displays a TEM image of the nanopore.

Each of the two chambers was filled with 700 µL of 1 mM PBSD (pH 7.6). Ag/AgCl electrodes were immersed into the solution in each chamber in order to perform electrical measurements.

2.3. Atomic Force Microscopy

In AFM experiments, samples of enzyme solution were first warmed up to the desired temperature in a test tube; secondly, the enzyme was adsorbed from the warmed-up solution onto mica AFM substrates. This procedure was performed in the following way.

Firstly, 0.1 µM solution of L-ASNase in 2 mM PBSD (pH 7.4) was placed in an Eppendorf Thermomixer Comfort shaker (Eppendorf, Germany), and sequentially warmed up to the desired temperatures within the studied temperature range (25°С, 30°С, 45°С, 55°С, and 60°С). At each of these temperature points, two 1-mL samples of the solution were taken, and the warming-up was continued until the 60°С value was reached.

Secondly, the enzyme from the samples taken as described above was directly adsorbed [15] onto bare mica AFM substrates. Each 1-mL sample of the enzyme solution was pipetted into an 1.7-mL Eppendorf-type test tube, and a 7?15 mm rectangular piece of muscovite mica (SPI, USA) was immersed into the solution. The test tube was then placed into an Eppendorf Thermomixer Comfort shaker (Eppendorf, Germany) and continuously shaken at 60 rpm and room temperature (25°С) for ten minutes. Each of the AFM substrates was then evacuated from the enzyme solution, placed in another test tube with 1 mL of ultrapure water and, again, shaken at 60 rpm and room temperature (25°С) for ten minutes in order to wash off buffer salts from the substrate surface.

All AFM measurements were performed with a NTEGRA PRIMA atomic force microscope (NT-MDT, Zelenograd, Russia). Prior to the meas urements, the microscope was calibrated with a TGZ1 calibration grating (NT-MDT, Zelenograd, Russia). The AFM scanning was performed in taping mode in air at controlled laboratory conditions (25 °C temperature, 55% air humidity). For each AFM substrate, no less than twenty-five 4 µm?4 µm scans with a resolution of 256?256 were obtained.

The analysis of the AFM data was performed with a NOVA Px software (NT-MDT, Zelenograd, Russia) and odAFM specialized software developed in IBMC (Rospatent registration No. 2010613458, 05/26/2010), and the main criterion for assessing the size of enzyme molecules was the height of their AFM images. The processing of the AFM data included calculation of the distributions of AFM images of the enzyme molecules by height ρ(h) and the normalized number N400 of the enzyme molecules per 400 µm2 area of the AFM substrate; these calculations were performed as described elsewhere [16].

2.4. Spectrophotometry

Absorbance spectra of 1 µM L-ASNase solution in 2 mM PBSD were recorded at 25 °C within 190 to 100 nm range with an Agilent 8453 UV-Vis spectrophotometer employing a 10 µL quartz micro-cell with an optical pathlength of 2 mm.

2.5. Fluorescence Spectroscopy

In the fluorescence spectroscopy experiments, samples of L-ASNase solution were placed in standard Eppendorf-type test tubes and warmed up with a TS-100 thermoshaker (Vector-Best, Russia). The warming-up was performed stepwisely from 25 to 60 °C with a 5 °C step. In order to additionally perform more precise monitoring of the sample temperature, similar test tube with the same volume of blank enzyme-free solution was placed in the shaker, and the sensor of a FY-10 digital thermocouple-based thermometer was placed into this solution. Each to be studied in the fluorescence spectroscopy experiments was removed from the shaker two minutes after reaching the desired temperature indicated by the thermocouple-based thermometer.

Fluorescence spectra of 0.1 µM L-ASNase solution in 2 mM PBSD (pH 7.4) were acquired at an excitation wavelength of 280 nm within emission wavelength range from 290 to 400 nm with an LS55 luminescent spectrometer (Perkin Elmer, USA) using a 300 µL quartz fluorimetric cell with an optical pathlength of 1 cm. In each temperature point, fluorescence spectra were recorded in three technical replicates. At the most temperatures studied, the central maximum of fluorescence intensity was observed at 308 nm. Background signal was subtracted using a LabView (v. 6.1) software. Processing of fluorescence spectroscopy data is described in detail in °endix A.

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

DOI: https://doi.org/10.20944/preprints202601.0032.v2

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