Impact of selected amino acids of HP0377 (Helicobacter pylori thiol oxidoreductase) on its functioning as a CcmG (cytochrome c maturation) protein and Dsb (disulfide bond) isomerase
Fig 8
Analysis of HP0377-apocytochrome c complex formation.
(A) Size exclusion profile of the HP0377, apocytochrome, HP0377-apocytochrome complex and HP0377-apocytochrome complex treated with DTT separated on an Enrich™ sec70 column (Biorad) and monitored by absorbance at 280 nm. HP0377 elutes as a peak at 10.7 min, with an estimated mass of 23.5 kDa, consistent with the size of the monomer. Apocytochrome elutes at 12.9 min, with estimated mass of 9.5 kDa. Complex eluted at 8.9 min, with estimated mass of 56 kDa. Complex treated with DTT elutes as two peaks at 12.9 min and 10.7 min, consistent with the sizes of the HP0377 and apocytochrome. The column was calibrated with Gel Filtration Standard (Bio-Rad): Thyroglobulin (670 kDa), γ-globulin (158 kDa), Ovalbumin (44 kDa), Myoglobin (17 kDa), Vitamin B12 (1.35 kDa). The relative positions of the chosen standards are marked with arrows. (B) SDS-PAGE analysis of HP0377-apocytochrome complex formation. Complexes formed between HP0377CSYA and apocytochrome were first purified using Ni-NTA resin, and then separated on a size exclusion column. The samples were next analyzed by SDS-PAGE with or without DTT.