title: Superoxide dismutase isozyme detection using two-dimensional gel electrophoresis zymograms.
abstract: Superoxide dismutases (SODs) are ubiquitous antioxidant enzymes involved in cell protection from reactive oxygen species. Their antioxidant activities make them of interest to applied biotechnology industries and are usually sourced from plants. SODs are also involved in stress signaling responses in plants, and can be used as indicators of these responses. In this article, a suitable method for the separation of different SOD isoforms using two-dimensional-gel electrophoresis (2D-GE) zymograms is reported. The method was developed with a SOD standard from bovine erythrocytes and later applied to extracts from Stemona tuberosa. The first (non-denaturing isoelectric focusing) and second (denaturing sodium dodecylsulphate-polyacrylamide gel electrophoresis) dimensions of duplicate 2D-GE gels were stained with either Coomassie brilliant blue G-250 for total protein visualization, or SOD activity (zymogram) using riboflavin/nitroblue tetrazolium. For confirmation, putative SOD activity positive spots were subject to trypsin digestion and nano-liquid chromatography tandem mass spectrometry, followed by searching the MASCOT database for potential identification. The method could separate different SOD isoforms from a plant extract and at least partially maintain or allow renaturation to the native forms of the enzyme. Peptide sequencing of the 2D-GE suggested that the SODs were resolved correctly, identifying the control CuZn-SOD from bovine erythrocytes. The two SODs from S. tuberosa tubers were found to be likely homologous of a CuZn-SOD. SOD detection and isoform separation by 2D-GE zymograms was efficient and reliable. The method is likely applicable to SOD detection from plants or other organisms. Moreover, a similar approach could be developed for detection of other important enzymes in the future.
authors: Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
authors: Ploypat
authors: P
authors: Niyomploy
authors: Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok 10210, Thailand.
authors: Chantragan
authors: C
authors: Srisomsap
authors: Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok 10210, Thailand.
authors: Daranee
authors: D
authors: Chokchaichamnankit
authors: Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
authors: Nawaporn
authors: N
authors: Vinayavekhin
authors: Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok 10330, Thailand.
authors: Aphichart
authors: A
authors: Karnchanatat
authors: Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. Electronic address: polkit@gmail.com.
authors: Polkit
authors: P
authors: Sangvanich
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000975:Antioxidants / Q000032:analysis / Q000378:metabolism; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004912:Erythrocytes / Q000201:enzymology*; D007525:Isoelectric Focusing / Q000379:methods; D007527:Isoenzymes; D015203:Reproducibility of Results; D020539:Sequence Analysis, Protein / Q000379:methods; D041901:Stemonaceae / Q000201:enzymology*; D013482:Superoxide Dismutase / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D000975:Antioxidants; D007527:Isoenzymes; D013482:Superoxide Dismutase
keywords: Liquid chromatography–mass spectrometry (LC–MS); Stemona tuberosa; Superoxide dismutase; Two-dimensional gel electrophoresis