Abstract
1. α1-Acid glycoprotein (α1-AGP) was digested with pronase and the digest was fractionated by chromatography on DEAE-cellulose, which gave six well separated glycopeptide fractions in good yield. Five out of the six glycopeptide fractions were also homogeneous on high-voltage paper electrophoresis. The glycopeptides were designated GP-I to GP-VI in the order in which they were eluted from the DEAE-cellulose column.
2. Amino acid sequences of individual glycopeptides were deter-mined using proteolytic enzymes, mainly carboxypeptidases A and B [EC 3. 4. 2. 1 and 3. 4. 2. 2] and chemical degradations including dinitrophenylation, periodate oxidation, alkali treatment and the Edman procedure. The peptide sequences were found to be: GP-I, Asp-Lys; GP-II, Thr-Pro-Asp-Lys; GP-III, Asp-Lys; GP-IV, Thr-Asp-Lys; GP-V, Asp-Thr-(Ser, Ala)-Gly; GP-VI, Glu-(Ser)-Asp-Thr-(Gly). The sequences of GP-V and GP-VI were only tentative.
3. The linkage between the amino acid and polysaccharide unit in GP-I, GP-II, GP-III and GP-IV were determined using β-aspartylglycosylamine amide hydrolase (“Amidase”). In GP-II, enzymatic breakdown of the linkage took place during incubation of the sialic-acid-free glycopeptide with the carboxypeptidase B preparation. The linkages in GP-V and GP-VI could not be determined because “Amidase” could not be used effectively due to the complex structures of these glycopeptides.
4. All the glycopeptides were found to contain sialic acid, glucosamine and neutral sugars. The ratios of sialic acid to either glucosamine or neutral sugars increased approximately in the order of elution i.e. GP-I to GP-VI. In GP-I the ratio of sialic acid/glucosamine was 0.47 whilst in GP-VI it was 0.66. The ratio of neutral sugars to glucosamine did not differ markedly. Analysis of neutral sugar components by gasliquid chromatography showed that the ratios of fucose or galactose to mannose increased from GP-I to GP-VI. GP-I differed considerably from the other glycopeptides in that it contained no fucose and had a considerably lower galactose/mannose ratio. On the basis of these analytical results, tentative assignment of molar sugar composition for each polysaccharide unit was presented and it was concluded that there were at least six different polysaccharide units present in α1-AGP, all of which appeared to have some degree of heterogeneity and which could not be fractionated further by the present procedures.
