Hp is an intestinal pPD oxidase and ferroxidase. (A) In-gel pPD oxidase activity was measured after separating 30 μg mouse enterocyte protein extract (C57/BL/6J), isolated under nondenaturing conditions, by native gel electrophoresis. Gels were immediately immersed in pPD solution and photographed as color developed. (B) Immunoblot of a duplicate sample of the enterocyte extract (C57/BL/6J) using Hp1a antiserum to the C-terminus of Hp. (C) In-gel pPD oxidase activity of 30 μg HT29 cell protein extract (HT29). (D) Immunoblot of a duplicate sample of HT29 cell extract (HT29) using Hp1a antiserum. Human ceruloplasmin (Cp) (10 μg) was used as a positive control. The upper and lower arrows indicate the mobilities of Hp and Cp, respectively. (E) In-gel ferroxidase activity was measured by separating serial dilutions of mouse enterocyte protein extracts (C57/BL/6J) under nondenaturing conditions by nondenaturing, nonreducing electrophoresis. Gels were immediately immersed in ferrous ammonium sulfate solution, developed with ferrozine, and scanned as color developed. Ferroxidase signals were analyzed by densitometric measurement expressed as relative densitometric units (RDUs). (F) Immunoblot and densitometric analysis expressed as RDU of replicate samples of mouse intestinal extracts (C57/BL/6J) using Hp1a antiserum. Purified human Cp was used as control.