| Description |
A glycopeptide has been isolated in a highly purified form from a papain hydrolysate of bovine immune lactoglobulin. This glycopeptide (glycopeptide 2) behaved as a homogeneous substance under the conditions of gel filtration on G-25 Sephadex and when submitted to paper electrophoresis-chromatography; its purity is further demonstrated by its composition. Other glycopeptides (glycopeptide fraction 3, 5 and 6) were isolated from the hydrolysate in a less pure form; each of these was orcinol-positive and was found to contain glucosamine. Because of the lack of purity no further carbohydrate analyses have been attempted on these fractions. The approximate amino acid compositions of glycopeptide fractions 3, 5 and 6 have been estimated by quantitative analysis; the results show that the amino acid compositions of these fractions are very similar to the amino acid composition of glycopeptide 2. The order of electronegativity of these substances at pH 6.4 is 6>5>2. The carbohydrate components of bovine immune lactoglobulin were identified as glucosamine, galactose, mannose, fucose and sialic acid. Quantitative analyses indicate that there are about 9 residues of hexose, 2 of fucose and 1 to 2 of sialic acid per molecule of protein, based on a, molecular weight of 160,000 for the glycoprotein. Galactose and mannose are in the approximate molar ratio of 6 to 3, as estimated from paper chromatograras. The same preparation of immune lactoglobulin has been reported by Smith et al.to contain 1.5% hexosamine (as glucosamine), which is equivalent to about 15 residues of glucosamine. The carbohydrate composition of glycopeptide 2 clearly does not account for all of the carbohydrate in the intact glycoproteins the glycopeptide contains fewer residues of hexose, glucosamine and fucose and no sialic acid. The estimated ratio of galactose to mannose (2 to 3) in glycopeptide 2 indicates that it contains all of the mannose of the intact immune lactoglobulin. A comparison of the gel filtration properties of glycopeptide 2 with those of a mixture of all the glycopeptides isolated from the papain hydrolysate of immune lactoglobulin indicates that most of the glycopeptides were considerably larger than glycopeptide 2, and the similarities in the amino acid compositions of the various glycopeptide fractions suggest that they may have come from the same amino acid sequence in the glycoprotein. The greater acidity of glycopeptides 5 and 6 is consonant with the absence from these glycopeptides of lysine, proline and arginine, which occupy the amino-terminal position in glycopeptide 2. These considerations suggest that the carbohydrate moiety of glycopeptide 2 may have arisen from a larger structure, part of which was lost during isolation. The evidence shows that the carbohydrate moiety of the glycopeptide is attached to the peptide moiety by way of the aspartyl residue and establishes the amino acid sequence. Inasmuch as both glycopeptide 2 and a glycopeptide isolated from a papain hydrolysate of glycopeptide 2 were neutral at pH 6.4 and their electrophoretic properties can be accounted for by their amino acid compositions alone, the amino groups of the glucosamine residues in these glycopeptides must have been in a bound form. As suggested in the case of rabbit gamma-globulin, it is likely that the glucosamine occurs as the N-acetyl derivative. As mentioned previously, only covalent bonds would be expected to survive the conditions to which glycopeptide 2 was subjected. Thus, although further work will be required to determine the exact nature and number of carbohydrate moieties in bovine immune lactoglobulin, it can be concluded that at least part of the carbohydrate components of this glycoprotein are bound to the protein by a covalent bond involving an aspartyl residue. |
