Enzyme therapy: II. Effect of covalent attachment of polyethylene glycol on biochemical parameters and immunological determinants of beta-glucosidase and alpha-galactosidase.
The covalent attachment of polyethylene glycol (PEG) to beta-glucosidase from sweet almonds and alpha-galactosidase from green coffee beans results in alterations of their catalytic properties and masking of specific determinant sites on the enzymes. Both enzymes have increased Km and decreased Vmax values against their respective p-nitrophenyl substrate analogs after PEG attachment. When PEG is attached to 30% of alpha-galactosidase epsilon-amino groups, 12% activity remains against ceramide trihexoside, while its ability to convert type B erythrocytes to type H specificity is lost. However, it still is able to cleave terminal galactose residues from human saliva blood group substance B. PEG-beta-glucosidase (38%) did not elicit the production of complement-fixing antibodies, nor did it react with antibodies produced against the native enzyme. Antibody and lectin-specific binding were lost from both modified enzymes (PEG-beta-glucosidase and PEG-alpha-galactosidase). After conjugation with PEG, beta-glucosidase lost its ability to bind to concanavalin A-Sepharose. Antibodies directed against native alpha-galactosidase blocked its enzyme activity, but lost their ability to inhibit activity in progressively higher modified preparations of the enzyme. Antisera against PEG-alpha-galactosidase (53%) did not inhibit enzyme activity in any alpha-galactosidase or PEG-alpha-galactosidase preparation. These results indicate that PEG tends to cover lectin-specific carbohydrate moieties and antigenic determinants and that these sites probably remain cryptic during in vivo processing of PEG-enzymes.
D000936:Antigen-Antibody Complex; D003069:Coffee; D003168:Complement Fixation Tests; D057487:Enzyme Therapy; D004800:Enzymes, Immobilized / Q000378:metabolism*; D000939:Epitopes / Q000032:analysis*; D005696:Galactosidases / Q000378:metabolism*; D005915:Globosides; D005959:Glucosidases / Q000378:metabolism*; D006801:Humans; D007106:Immune Sera; D007700:Kinetics; D010944:Plants / Q000201:enzymology; D011092:Polyethylene Glycols; D000519:alpha-Galactosidase / Q000276:immunology / Q000378:metabolism*; D001617:beta-Glucosidase / Q000276:immunology / Q000378:metabolism*
D000936:Antigen-Antibody Complex; D003069:Coffee; D004800:Enzymes, Immobilized; D000939:Epitopes; D005915:Globosides; D007106:Immune Sera; D011092:Polyethylene Glycols; D005696:Galactosidases; D005959:Glucosidases; D001617:beta-Glucosidase; D000519:alpha-Galactosidase