Purification of multiple forms of plasma angiotensinogen: molecular weight and charge heterogeneity

A method of purifying angiotensinogen (C.A. 11002-13-14) from dog plasma using a monoclonal antibody is presented. More than 98% of applied angiotensinogen was sequestered by the 2-C: G7F7 anti-angiotensinogen monoclonal antibody in solid-phase adsorption assays. Employing immunoaffinity column chromatography, sufficient angiotensinogen was purified for subsequent investigation of angiotensinogen molecular heterogeneity. SDS-polyacrylamide gel electrophoretic analysis resolved two molecular weight forms, an alpha-form at Mr 59,000 and a beta-form at Mr 57,000. These alpha- and beta-plasma angiotensinogen forms were separated by anion-exchange HPLC; an additional gamma-form was also identified. Homogeneous dog angiotensinogen, recovered by reverse-phase HPLC, was partially sequenced. The dog angiotensinogen N-terminal sequence was shown to be more similar to rat than to human angiotensinogen. Since neuraminidase reduced the molecular weight of alpha-angiotensinogen by 4000 and beta-angiotensinogen by 2000, these two forms of the prohormone in dog plasma contain two and one carbohydrate side chain, respectively.