Eghtedari Masoumeh, Jafari Porzani Samaneh, Javanmardi Masoud, Ganjali Mohammad Reza, Hosseinkhani Saman
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.
Iran J Biotechnol. 2021 Jul 1;19(3):e2741. doi: 10.30498/ijb.2021.225724.2741. eCollection 2021 Jul.
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.
In this approach, a colorimetric detection method for SOD activity was developed based on the carboxylic stabilized AuNPs.
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, NaCO and potassium phosphate buffer, have been performed. In addition, Fluorescence spectroscopy analysis toward SOD and gold nanoparticle were performed.
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.
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.
超氧化物歧化酶（SODs）被归类为抗氧化酶，参与许多过程，如应激信号反应和细胞免受自由基的保护。SOD的主要功能是在不同生理过程中清除产生的自由基，如超氧离子。SOD有多种同工酶，根据其活性位点的金属辅因子分为四种常见类型：铁超氧化物歧化酶（Fe-SOD）、锰超氧化物歧化酶（Mn-SOD）、铜/锌超氧化物歧化酶（Cu/Zn-SOD）和镍超氧化物歧化酶（Ni-SOD）。在金属纳米颗粒中，金纳米颗粒（AuNPs）作为基于表面等离子体共振和比色法进行关键分析的传感探针，在生物学领域具有重要用途。在本研究中，对表达、纯化的人铜锌超氧化物歧化酶及其与金纳米颗粒基于一种新比色法的相互作用进行了研究。
在本方法中，基于羧基稳定的金纳米颗粒开发了一种用于检测SOD活性的比色检测方法。
使用镍-氮三乙酸琼脂糖亲和柱进行酶的纯化过程。在SOD纯化后，研究了在核黄素、蛋氨酸、碳酸钠和磷酸钾缓冲液产生的自由基存在下，金纳米颗粒存在时酶的活性，这可能会导致蚀刻。此外，还对SOD和金纳米颗粒进行了荧光光谱分析。
酶促反应产生的超氧自由基会优先蚀刻金纳米颗粒，导致金纳米颗粒的局域表面等离子体共振发生显著变化，在SOD存在时这种变化会减小。在优化的实验条件（pH约7.8和25℃）下，该方法对SOD活性表现出更好的选择性和灵敏度。
在此背景下，评估了一种基于金纳米颗粒（AuNPs）测定SOD活性的间接新比色法。根据给出的结果，可以得出结论，在SOD存在下清除游离超氧自由基时，可以补充金纳米颗粒吸光度的量。
