FIGURE 7.

Binding of PvTRAg38 fragments and synthetic peptides to Band 3. A, binding of PvTRAg38 fragments with purified Band 3 by solid phase ELISA. Each well of the plate was coated with 50 nm of Band 3 and then allowed to react with increasing concentrations (0–2 μm) of histidine-tagged PvTRAg38 or its fragments. The plates were developed with mouse anti-His₆ monoclonal antibody as described in the text. Recombinant bacterial thioredoxin was taken as negative control. B, specificity of M-PvTRAg38 binding to Band 3 was determined by competition assay. Increasing concentrations of untagged PvTRAg38 was mixed with fixed concentration (0. 5 μm) of histidine-tagged M-PvTRAg38 and allowed to react with 50 nm immobilized Band 3 for 4 h at 37 °C. Bound recombinant histidine-tagged M-PvTRAg38 was detected with mouse anti-His₆ monoclonal antibody as described in the text. Binding in the absence of untagged PvTRAg38 was taken as percentage control for rest of the concentrations. The data shown are the means ± S.D. of three independent experiments. C, SPR analysis of Band 3 interaction with M-PvTRAg38. Three different concentrations of M-PvTRAg38 (0.2, 0.8, and 1.4 μm) were injected at flow rate of 30 μl/min over the immobilized Band 3 on the cell of CM5 chip. Sensogram curves show dose-dependent response of M-PvTRAg38 binding with Band 3. D, binding of PvTRAg38 peptides to Band 3 by solid phase ELISA. The plate was coated with purified Band 3 (50 nm) protein and reacted with different concentrations (0–2 μm) of histidine-tagged PvTRAg38, P-2, or P-4. Plate was developed with mouse anti-His₆ monoclonal antibody as described in the text. Histidine-tagged bacterial thioredoxin from D. desulfuricans was used as negative control. The data shown are the means ± S.D. of three independent experiments. E, specificity of binding of Band 3 to peptide P-4 by competition assay. Recombinant GST-PvTRAg38 (0.5 μm) was mixed with increasing concentrations (0–4 μm) of peptide P-4. Then this mixture was added to Band 3 (50 nm) coated ELISA plate and incubated for 4 h at 37 °C. The plate was then incubated with mouse anti-GST monoclonal antibody and HRP-conjugated secondary antibody as described in the text. Binding in the absence of respective untagged peptides was taken as percentage control for rest of the concentrations. F, SPR analysis of Band 3 interaction with P-4. Three different concentrations of P-4 (0.2, 0.6, and 1.2 μm) were injected at flow rate of 30 μl/min over the immobilized Band 3 on the cell of CM5 chip. Sensogram curves show dose-dependent response of P-4 binding with Band 3.