Immunoprecipitated Hp has a pPD oxidase and ferroxidase activity. (A) Enterocyte (lanes 2-4) and HT29 (lanes 5-7) extracts were precleared with protein G Sepharose, and supernatants were collected. Supernatants were incubated with preimmune serum or Hp1a antiserum with protein G Sepharose for 16 hours. Sepharose beads were separated by centrifugation and washed with reaction buffer, and bound Hp was eluted with peptide 1. Eluates were tested with the in-gel pPD oxidase assay. Lane 1 is purified human ceruloplasmin control, lanes 2 and 5 are precleared supernatant controls. Lanes 3 and 6 are eluates from preimmune immunoprecipitates, whereas 4 and 7 are eluates from Hp1a antiserum immunoprecipitates. (B) Enterocyte extracts incubated with protein G Sepharose in the presence of Hp1a antiserum and peptide 1 (lane 1) or Hp1a antiserum only (lane 2). Sepharose beads were separated by centrifugation and washed with reaction buffer, and bound Hp was eluted with peptide 1 and tested with the ferroxidase assay. Purified human ceruloplasmin was used as control (lane 3). (C) Supernatants of enterocyte extracts preincubated with protein G Sepharose in the presence of buffer (lane 2), preimmune serum (lane 3), or Hp1a antiserum (lane 4) were tested with the in-gel pPD oxidase assay. Supernatants of HT29 cell extracts incubated with protein G Sepharose in the presence of buffer (lane 5), preimmune serum (lane 6), or Hp1a antiserum (lane 7) were tested with the in-gel pPD oxidase assay. Purified human ceruloplasmin (lane 1) was used as control. (D) Supernatants of mouse enterocyte extracts preincubated with protein G Sepharose in the presence of Hp1a antiserum (lanes 2-3) or buffer only (lanes 4-5) were tested with the ferroxidase assay. Purified human ceruloplasmin (10 μg) (l ane 1) was used as a control.