Heterogeneity in the carbohydrate structure of rat angiotensinogen

Angiotensinogen (Ao), the precursor of the peptide hormone angiotensin, exists in two different forms in plasma, Ao-1 and Ao-2. The completely separated and purified molecules show distinct differences in sodium dodecyl sulfate (SDS)-disc electrophoresis and in analytical isoelectric focusing. Ao-1 is about 3500 Da heavier and contains more acid isoelectric points than Ao-2. In analytical isoelectric focusing Ao-1 displays five bands which overlap with two of the three bands of Ao-2. This difference in charge was eliminated by treatment with neuraminidase for 92 h at 37 degrees C. Thereafter both molecules display an identical-pattern two bands in isoelectric focusing. Treatment of these N-acetylneuraminic acid (NeuNAc)-free forms with renin leads to a shift of these double bands to a more acid pH, indicating heterogeneity within the protein structure. By affinity chromatography on concanavalin A (ConA)-Sepharose, both forms can be separated in three fractions. The analysis of these fractions and of their NeuNAc-free derivatives by SDS-disc electrophoresis, as well as by analytical isoelectric focusing, leads to the conclusion that Ao-1 contains two Asn-linked N-glycan residues with 4-8 mol of NeuNAc, while the smaller form, Ao-2, contains one carbohydrate residue with 2-4 mol of NeuNAc.