Superoxide dismutase activities in the midgut of Helicoverpa armigera larvae: identification and biochemical properties of a manganese superoxide dismutase

Figures & data

Figure 1 Visualization of SOD activity isoforms and determination of total SOD activity at various developmental stages of Helicoverpa armigera larvae.

Notes: (A) Proteins were separated on 10% native polyacrylamide gel, and SOD activity isoforms were stained with NBT. Five SOD activity isoforms were detected and numbered as a–e. (B) Total SOD activity was measured from third to sixth instar of the larvae. The experiment was conducted in triplicate using three biological replicates (30 larvae for each replicate). Data were subjected to one-way ANOVA to verify the significance of the observed differences in enzyme activities during different instars. The data were mean ± SD. *P<0.05; **P<0.01.
Abbreviations: SOD, superoxide dismutase; NBT, nitroblue tetrazolium; ANOVA, analysis of variance; SD, standard deviation.

Figure 2 Effect of metal ions on Helicoverpa armigera total SOD activity.

Notes: Proteins were extracted form actively feeding H. armigera larvae and used as source of SOD. The enzyme extract was incubated with each metal ion (5 mM), and the incubated reaction mixture was subjected to SOD activity assay. SOD activity assay was carried out with various metal ions. The experiment was conducted in triplicate using three biological replicates (30 larvae for each replicate). Data were analyzed by Student’s t-tests (control vs treatment) to verify statistical significance. The data were mean ± SD. *P<0.05; **P<0.01.
Abbreviations: SOD, superoxide dismutase; SD, standard deviation.

Figure 3 Identification of Helicoverpa armigera MnSOD.

Notes: The midgut extract was pre-incubated with H₂O₂, and the mixture was processed to visualize SOD activity isoforms. The experiment was conducted in triplicate using three biological replicates (30 larvae for each replicate). Among all five SOD activity isoforms, only SOD b was found to be insensitive to H₂O₂ inhibition.
Abbreviations: MnSOD, manganese SOD; SOD, superoxide dismutase.

Table 1 Purification of Helicoverpa armigera MnSOD

Purification step	Total protein (mg)	Total activity (U)	Specific activity (U/mg/min)	Purification fold	% yield
Crude	52.27	4,1515.9	794.26	1.0	100
Acetone precipitation	17.39	17,390.0	1,000.0	1.25	33.26
Gel filtration	13.90	8,830.6	6,348.0	8.0	26.61

Abbreviation: MnSOD, manganese superoxide dismutase.

Figure 4 Molecular weight determination and visualization of purified MnSOD.

Notes: (A) Molecular weight determination of purified MnSOD. The identified MnSOD was partially purified by using gel filtration chromatography, and the purified protein was loaded onto 12% SDS polyacrylamide gel with protein molecular weight standards (14.3–97.4 kDa). Electrophoresis was carried out at constant current of 20 mA, and after electrophoresis, protein bands were visualized using CBBR-250 staining. Lane 1: standard molecular weight markers, lane 2: crude midgut extract, lane 3: acetone precipitate, and lane 4: gel filtration fraction containing partially purified MnSOD. (B) Activity visualization of purified MnSOD. The purified fraction was loaded onto 10% native polyacrylamide gel, and SOD activity was visualized. A single MnSOD activity band was detected on 10% native polyacrylamide gel.
Abbreviations: MnSOD, manganese superoxide dismutase; SDS, sodium dodecyl sulfate; CBBR-250, Coomassie Brilliant Blue R-250.

Figure 5 Optimum temperature and pH of Helicoverpa armigera MnSOD.

Notes: (A) Optimum temperature. Purified enzyme was incubated at various temperatures ranging from 10°C to 90°C with 10°C intervals. Reaction was initiated by the addition of xanthine oxidase (56 mU/mL) in reaction mixture and continued at 37°C for 20 minutes. Then, reaction was terminated by using 0.84 mM allopurinol, and the change in absorbance was measured at 560 nm. H. armigera MnSOD showed strong activity at broad range of temperature. The data were mean ± SD. (B) Optimum pH. The activity of MnSOD in various buffers was evaluated by pre-incubating the reaction mixture with 200 μL of each buffer ranging from pH 2.0 to 12.0. The assay conditions were similar as described for optimum temperature. H. armigera MnSOD showed strong activity at highly alkaline pH. The data were mean ± SD.
Abbreviations: MnSOD, manganese superoxide dismutase; SD, standard deviation.