Because alterations in the carbohydrate portions of cell surface glycocnjugates are implicated in the antisocial nature of malignancy, it is important to identify the specific changes and to determine whether the changes are unique to transformation or represent inappropriate expression of a specific normal configuration. Alpha-fetoprotein (AFP) lectin-binding molecular variants synthesized by a series of transplantable rat hepatomas, each hepatoma line secreting a characteristic "mutant" pattern of variants, will serve as a model for glycosylation alterations associated with malignancy. Initial assessment of the degree and type of glycan heterogeneity in tumor-produced AFP will be made by lectin crossed immunoaffinoelectrophoresis. To identify specific changes or classes of changes in glycosylation, we will determine the primary structure of the oligosaccharide chain(s) of rat AFP, comparing the primary structures of the glycans of AFP secreted by normal hepatocytes (newborn liver) and by malignant hepatocytes (transplantable hepatomas). Achievement of these aims will allow us to determine whether or not the altered patterns of lectin-binding molecular variants secreted by malignant hepatocytes reflect abnormal proporations of AFP molecules with a normal oligosaccharide composition or characteristic proportions of AFP molecules with altered oligosaccharide composition. The general approach to the investigation will be physicochemical. The methods proposed are particularly applicable to determining the primary carbohydrate structures of small quantities of glycoprotein variants: isolation of AFP from new-born rat serum or hepatoma-bearing rat serum by immunoadsorbent chromatography, reduction and alkylation, lectin-affinity chromatography, quantitative hydrazinolysis and re-N-acetylation with 14C-acetic anhydride. Molar ratios of monosaccharides will be estimated by gas-liquid chromatography; glycan structure by thin layer chromatography and paper electrophoresis; linkages between monosaccharides by methylation analysis and mass spectrometry and nuclear magnetic resonance spectroscopy. Elucidation of the carbohydrate structures of the AFP lectin-binding variants also will provide information on the homogeneity or heterogeneity of glycosylation systems, which is relevant to the synthesi of carbohydrate units in normal cells.