Detection of time-dependent and oxidatively induced antigens of bovine liver rhodanese with monoclonal antibodies.

G A Merrill,P M Horowitz,S Bowman,K Bentley,R Klebe

doi:10.1016/s0021-9258(19)77637-6

Abstract

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.

Highlights

Rhodanese has been extensively utilized as a model of nearly equal size which are joined by a short tether
Specificity of monoclonal antibodies (MABs) to Rhodanese-Anti-rhodanese MABs were selected for ascites tumor production based on a direct ELISA where bovine liver rhodanese was the immobilized antigen
Twenty eight ascites fluids were produced from 15 hybridomas secreting anti-rhodanese immunoglobins

Summary

THEJOURNAOFLBIOLOGICAL CHEMISTRY

Val. 263,No.36,Issue of December 25,pp. 19324-19330,1988 Printed in U.S.A. Detection of Time-dependent and Oxidatively Induced Antigens of Bovine Liver Rhodanese with Monoclonal Antibodies*. MABs recognizedmutually exclusive epitopes was provided by analy- to a second microtiter plate to which rhodanese had been immobilized sis of the fragments to which each of the MABs bound following and excess protein adsorption sites had been blocked with bovine partial digestion of rhodanese with proteolytic enzymes. Indirect ELISA assays to measure the time course of rhodanese inactivation were performed essentially as described in the indirect assay above except a t selected times (240 to 0 min) rhodanese (100 r l a t 50 pg/ml) in either the E or ES form, diluted in 20 mM trisbuffered saline,pH 7.5, with 0.05% (v/v)Tween 20 (TTBS),was removed from ice and added to wells of BSA/casein-blocked microtiter plates kept ina 37 “C incubator such that rhodanese thermally treated for 240 min was added to the microtiter plate 240 min prior to rhodanese thermally treated for 0 min. Fluorescence Spectra-Fluorescence emission spectra from 300 to 400 nm were obtained on rhodanese sampluetsilizing an SLM Aminco SPF500C spectrofluorometer (SLM Instruments, Urbana, IL) with excitation a t 295 nm and a path length of 1 cm

RESULTS

TABLEI the criteria used in the original screening assayfor detection

Soluble rhodanese form

DISCUSSION