RESUMO
In order to examine whether oxygen radicals could be responsible for aggregation of recombinant hepatitis B surface antigen (HBsAg) during its assembly in yeast, purified HBsAg was oxidized with ammonium peroxodisulphate (AP) and analyzed by non-denaturing and denaturing size exclusion chromatography, immunoassay and immunoelectron microscopy. As a result, peroxodisulphate radicals induced a reproducible aggregation of HBsAg. At 44 mM AP, the aggregation process took a few hours and the resulting structures were large, branched and non-antigenic. During more gentle oxidation with 9 mM AP and 20-80 microM Cu2+, a continuous structural modification to HBsAg delaying for tens of hours preceded the aggregation event. During this pre-aggregation period, peroxidation of HBsAg lipids and covalent cross-linking of S protein chains occurred that led a complete loss of antigenicity of oxidized particles. In contrast, yeast-derived HBsAg aggregate is decomposed to S monomers under reducing conditions and recognized by anti-HBsAg polyclonal and monoclonal antibodies, suggesting that is has been assembled in vivo from antigenic and reversibly cross-linked particles. Based on these observations, we conclude that oxidation, at least with respect to the specific molecular sites oxidized by AP, is not a primary event in HBsAg aggregate formation in vivo. Since oxidized HBsAg was shown to be irreversibly cross-linked and non-antigenic, there are no suitable techniques for detection HBsAg oxidation in biological samples. Hence, at present, the magnitude of the in-vivo oxidative damage to HBsAg cannot be evaluated and thus, whether the plasma-derived HBsAg undergoes radical-induced oxidation in the course of viral hepatitis remains to be established. If this occurs, this process is expected to contribute to low HBsAg levels in chronic hepatitis B carriers, failure of the currently available immunoassays to identify HBsAg-positive blood donors and inconsistency in the results provided by HBsAg- and anti-HBsAg-based tests in several recent reports.