Inhibition of horseradish peroxidase activity by specific antibody: determinant specificity of anticatalytic antibodies.
Rabbit antisera specific for horseradish peroxidase inhibit the catalytic activity of the enzyme. All antibodies prepared against the holoenzyme react with the peroxidase apoenzyme. However, only a minority (30-45%) of the total antiperoxidase pool cross react with reduced and alkylated apoenzymes. The antibodies inhibiting peroxidase activity do not bind to S-carboxymethyl of S-carboxamidomethylated apoenzyme derivatives as measured by absorption and competition of inhibition experiments. Glycopeptides derived from horseradish peroxidase also failed to bind anticatalytic antibodies. Antibodies that inhibit enzyme activity have specificity for noncarbohydrate conformation dependent antigenic determinants of horseradish peroxidase. Additional experiments probed the mechanism by which inhibitory antibody decreases the catalytic activity of horseradish peroxidase. Absorption spectra of horseradish peroxidase that has bound Fab fragments sufficient to cause 90% inhibition of the enzyme activity determined that the enzyme retained the ability to bind hydrogen peroxide. Thus, anticatalytic antibodies do not prevent the formation of the first enzyme-substrate intermediate but mediate their inhibitory effects by disrupting a later step in the reaction mechanism.
D000918:Antibody Specificity; D001051:Apoenzymes / Q000276:immunology; D002384:Catalysis; D002850:Chromatography, Gel; D003429:Cross Reactions; D000939:Epitopes; D006020:Glycopeptides / Q000276:immunology; D006735:Horseradish Peroxidase / Q000037:antagonists & inhibitors / Q000276:immunology*; D006861:Hydrogen Peroxide; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D008745:Methylation; D010544:Peroxidases / Q000276:immunology*; D013053:Spectrophotometry
D001051:Apoenzymes; D000939:Epitopes; D006020:Glycopeptides; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D010544:Peroxidases