Detection of time-dependent and oxidatively induced antigens of bovine liver rhodanese with monoclonal antibodies.
Rhodanese has been extensively utilized as a model protein for the study of enzyme structure-function relationships. An immunological study of conformational changes occurring in rhodanese as a result of oxidation or thermal inactivation was conducted. Three monoclonal antibodies (MABs) to rhodanese were produced. Each MAB recognized a unique epitope as demonstrated by binding of the MABs to different proteolytic fragments of rhodanese. While none of the MABs significantly bound native, soluble, sulfur-substituted bovine rhodanese, as indicated in indirect enzyme-linked immunosorbent assay experiments, each MAB was immunoadsorbed from solution by soluble rhodanese as a function of the time rhodanese was incubated at 37 degrees C. Thus, as rhodanese was thermally inactivated, conformational changes resulted in the expression of three new epitopes. Catalytic conformers demonstrated different rates of thermally induced antigen expression. Each MAB also recognized epitopes expressed when rhodanese was immobilized on microtiter plates at 37 degrees C. Two conformers resulting from oxidation of rhodanese by hydrogen peroxide were identified immunologically. All MABs recognized rhodanese that was oxidized with peroxide and allowed to undergo a secondary cyanide-dependent reaction which also resulted in a fluorescence shift and increased proteolytic susceptibility. Only one MAB was capable of recognizing an epitope expressed when rhodanese was oxidized with peroxide and then separated from the reactants to prevent induction of the secondary conformational changes.
Merrill
G A
GA
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284.
Horowitz
P M
PM
Bowman
S
S
Bentley
K
K
Klebe
R
R
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013466:Sulfurtransferases / Q000096:biosynthesis*; D013884:Thiosulfate Sulfurtransferase / Q000096:biosynthesis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase