Document Type : Research Paper
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
Department of Medical Biotechnology, Applied Biophotonics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran
Center of Excellence in Electrochemistry, University of Tehran, Tehran, Iran
Biosensor Research Center, Endocrinology & Metabolism Molecular, Cellular Sciences Institute
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
Background: Superoxide dismutases (SODs) are categorized as antioxidant enzymes that are involved in many processes such as stress signalling responses and cell protection against free radical species. The primary function of SOD is the removal of produced radical species like superoxide ions in different physiological processes. There are various isozymes of SODs which are classified according to the metal cofactor in their active sites into four general types of Fe-SOD, Mn-SOD, Cu/Zn-SOD and Ni-SOD. Among metal nanoparticles, gold nanoparticles (AuNPs) are useful for biological purposes as sensing probe for determining critical analysis based on surface plasmon resonance and colorimetric method. In this study, the human Cu-Zn SOD expressed, purified, and its interaction with AuNPs based on a new colorimetric method was investigated.
Objectives: In this approach, a colorimetric detection method for SOD activity was developed based on the carboxylic stabilized AuNPs.
Material and Methods: The Ni-NTA Sepharose affinity column was performed for the purification process of enzyme. Following SOD purification, the enzyme activity in presence of AuNPs due to the possible etching in the presence of free radicals which are produced by riboflavin, methionine, Na2CO3 and potassium phosphate buffer, have been performed. In addition, Fluorescence spectroscopy analysis toward SOD and gold nanoparticle were performed.
Results: Superoxide radicals generated from the enzymatic reaction would preferentially etch AuNPs and resulted in remarkable changes of localized surface plasmon resonance of AuNPs, which is reduced in the presence of SOD. Under the optimized experimental conditions assay (pH~7.8 and 25 ˚C), better selectivity and sensitivity toward SOD activity was shown.
Conclusions: In this context, an indirect new colorimetric method for determining of SOD activity based on gold nanoparticles (AuNPs) was evaluated. According to the presented result, it may be concluded that by scavenging of free superoxide radicals in the presence of SOD, the amount of AuNP absorbance can be replenished.