Probing the interactions between carboxylated multi-walled carbon nanotubes and copper–zinc superoxide dismutase at a molecular level Jin Guan, a Guiliang Liu, b Kai Cai, c Canzhu Gao a * and Rutao Liu a * ABSTRACT: In order to evaluate the toxicity of multi-walled carbon nanotubes (MWCNTs-COOH) at a molecular level, the effect of MWCNTs-COOH on antioxidant enzyme copper–zinc superoxide dismutase (Cu/ZnSOD) was investigated using fluorescence spectroscopy, UV/vis absorption spectroscopy, circular dichroism (CD) spectroscopy and isothermal titration calorimetry (ITC). By deducting the inner filter effect (IFE), the fluorescence emission spectra and synchronous fluorescence spectra indicated that there were interactions between MWCNTs-COOH and Cu/ZnSOD. Moreover, the microenvironment of the amino acid residues in the enzyme was changed slightly. The UV/vis absorption and CD spectroscopic results showed appreciable conformational changes in Cu/ZnSOD. However, the results of a Cu/ZnSOD activity determination did not show any significant difference. In other words, MWCNTs-COOH has no significant effect on enzyme activity. The ITC results showed that the binding of MWCNTs-COOH to Cu/ZnSOD was a weak endothermic process, indicating that the predominant force of the binding was hydrophobic interaction. Moreover, it was essential to consider the IFE in fluorescence assays, which might affect the accuracy and precision of the results. The above results are helpful in evaluating the oxidative stress induced by MWCNTs-COOH in vivo. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: MWCNTs-COOH; Cu/ZnSOD; spectroscopy; ITC; activity inhibition Introduction Since their discovery by Iijima (1), carbon nanotubes (CNTs) have attracted much attention owing to their fascinating electronic, optical, thermal and mechanical assets. It cannot be denied that the extensive application of CNTs, as well as professional and public exposure to nanomaterials, is on the increase (2). CNTs are divided into single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes(MWCNTs). MWCNTs are more accostable than SWCNTs and there is widespread concern re- garding their ecological safety (3,4). A number of studies have shown that MWCNTs have toxic effects on animals (5,6) and humans (7). However, the results of CNT toxicity experiments are inconsistent and not easily com- pared (8–11). In addition, functionalized CNTs have better dispersibility in a medium and better biocompatibility with living organisms than nonfunctionalized CNTs (12). Therefore, carbox- ylated MWCNTs (MWCNTs-COOH) may better illustrate the prominent properties of MWCNTs. It has been found that CNTs can enter a cell wall, inducing oxidative stress and damage through the primary mechanism of a reactive oxygen species (ROS) burst (13,14). However, the effect of CNTs on antioxidant enzymes at the molecular level has received less attention. The superoxide anion radical (O 2 •– ), a type of ROS, is generated by the unavoidable reaction of aerobic metabolism (15–17). To defend against the production of this ROS, aerobic organisms have efficient defensive systems made up of the chemical and enzymatic antioxidants known as superoxide dismutases (SODs); these include copper–zinc superoxide dismutase (Cu/ZnSOD), manganese superoxide dismutase (MnSOD) and extracellular superoxide dismutase (ECSOD). The SODs play a key role in catalyzing the disproportionation of O 2 •– to H 2 O 2 and O 2 (18–20). The most common SOD in eukaryotes is Cu/ZnSOD, which is found within all eukaryotic cells (21). This study aimed to inves- tigate interactions between MWCNTs-COOH and Cu/ZnSOD at the molecular level using multiple spectroscopy and isothermal titration calorimetry (ITC) methods. The effects of MWCNTs- COOH on the activity and conformation of Cu/ZnSOD were investigated. In addition, the binding force of the interaction was explored. Because MWCNTs-COOH show strong absorption at the excitation and emission wavelengths of proteins, the inner filter effect can affect the accuracy of fluorescence experiments. The Lakowicz method was, therefore, used to correct for the inner filter effect (22). This study indicates a threat from MWCNTs- COOH to human health in vivo. * Correspondence to: C. Gao and R. Liu, School of Environmental Science and Engineering, Shandong University, Jinan 250100, People’s Republic of China. E-mail: gaocz@sdu.edu.cn; rutaoliu@sdu.edu.cn a Shandong Key Laboratory of Water Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Shandong University, Jinan 250100, People’s Republic of China b Shandong Institute for Food and Drug Control, Jinan 250101, People’s Re- public of China c Shandong Environmental Monitoring Centre, Shandong 250101, People’s Republic of China Luminescence 2014 Copyright © 2014 John Wiley & Sons, Ltd. Research article Received: 16 June 2014, Revised: 12 September 2014, Accepted: 24 September 2014 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/bio.2807 1