Separation and characterization of two different species of rat angiotensinogen

Two distinct species of rat angiotensinogen (A-1 and A-2) were purified from plasma of nephrectomized rats by combining ammonium sulfate fractionation, chromatography on Blue Sepharose and SP-Sephadex, gel filtration and preparative polyacrylamide gel electrophoresis. Separation of the two species was accomplished in the SP-Sephadex chromatography step, A-1 eluting before A-2. The two angiotensinogen species had identical electrophoretic mobilities on analytical polyacrylamide gel electrophoresis, but differed in their apparent molecular weights as obtained by SDS-gel electrophoresis (A-1, Mr 60 000; A-2, Mr 56 400). In analytical isoelectric focusing each species displayed a characteristic double band with isoelectric points of 4.54 and 4.60 for A-1, and 4.69 and 4.76 for A-2. These physicochemical differences can be accounted for by the difference in carbohydrate content: A-1, when compared to A-2, had a higher content of sialic acid (5.0 and 2.1 mol/mol), neutral hexoses (10.2 and 5.9 mol/mol) and aminohexoses (10.5 and 7.0 mol/mol, respectively). Antiserum raised against rat angiotensinogen crossreacted completely with both angiotensinogens. Both species could also be isolated from plasma of non-nephrectomized rats, which indicates that they may be present under physiological conditions. The physiological significance of the occurrence of these species of angiotensinogen is still unknown.