Purification of human gastric proteases by immunoadsorbents: pepsinogen II-group.
The pepsinogen II group was prepared from gastric mucosal extract by a two-step procedure consisting of immunoadsorption to an anti-pepsinogen II column, followed by ion exchange chromatography on DEAE-Sephadex A-50. The final product was pure according to biochemical and immunochemical criteria. As determined by quantitative immunodiffusion, the enrichment factor of pepsinogen II was 34. A recovery of 55% was calculated. The effectiveness of this procedure was due to the use of purified anti-pepsinogen II antibodies for immunoadsorption. This was achieved by immunoadsorption to Sepharose bound crude pepsinogen and a further passage over an unrelated immunoadsorbent (human serum coupled to Sepharose). The immunoadsorbent prepared using purified anti-pepsinogen antibodies showed low non-biospecific binding of gastric extract proteins. Complete separation of pepsinogen II from pepsinogen I was observed in one single passage. Purified pepsinogen II showed two protein bands in polyacrylamide gel electrophoresis and four bands of proteolytic activity in agarose enzyme electrophoresis. In both methods, as well as in two-dimensional immunoelectrophoresis, components with the same electrophoretic mobility were detected in pure preparations of group and II pepsinogens. Consequently, only in pure preparations was it possible to define the exact number of bands belonging to each of the two groups and to assess the immunological specificity of every band. Upon hydroxyapatite chromatography purified pepsinogen II was further resolved into two fractions.
D001798:Blood Proteins; D002846:Chromatography, Affinity; D005753:Gastric Mucosa / Q000201:enzymology*; D006801:Humans; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D007164:Immunosorbents; D007527:Isoenzymes / Q000302:isolation & purification; D010435:Pepsinogens / Q000302:isolation & purification*
D001798:Blood Proteins; D007164:Immunosorbents; D007527:Isoenzymes; D010435:Pepsinogens