CD81 or heparin binding in isolation does not affect the oxidation state of E1 or E2. HCVpp were incubated with heparin sodium salt, dimeric MBP-LEL(113–201), or mutant MBP-LEL(113–201)F186S prior to purification and labeling with maleimide- or NHS-biotin. Particles were lysed and reduced by addition of DTT then applied to streptavidin agarose for capture of biotinylated proteins. Samples were run on reducing SDS-PAGE and Western blotted with anti-E2 and anti-E1 mAbs (H52 and A4, respectively). The ratio of maleimide-biotin and NHS-biotin labeling was calculated for E1 and E2 ± heparin, MBP-LEL(113–201), and MBP-LEL(113–201)F186S by densitometry analyses of Western blots produced from three independent experiments (means ± S.E.).
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