[Proof of a disulfide bridge accessible to disulfide exchange between the heavy chains of IgG].
The paper deals with the direct experimental proof that human immunoglobulin G1 (IgG1) contains a reactive disulfide bond that can be opened by 3,3'-dithiobis(6-nitrobenzoate) (DTNB) within 24 h by a SH-catalysed disulfide exchange reaction. These results were obtained with the purified IgG1 myeloma protein and confirm earlier indirect evidence based on correlation analysis of DTNB reactivity and quantitative IgG1 determination. The reactive disulfide bond is most likely the one between Cys235 of the heavy chains in the "hinge"-region, activated for the disulfide exchange by the protonated amino groups of Lys231 as turned out by analysis of IgG1. As with the whole molecule, one mol of reactive disulfide was found per mol of the Fc-fragment. 0.8 mol of labile S-S bonds was detected per mol of F(ab)2. After separation of the excess of reagent, the sedimentation pattern still corresponded with the dimer. The unaltered antigenic properties as well as the crystallizability speak against any severe conformational changes. Therefrom it was concluded that in approximately 80% of the F(ab)2 molecules one of the two inter heavy chain-bridges was opened. With the isolated F(ab)-fragment a reaction with DTNB was ascertained to an extent of 20%, which is probably due to an altered stability of the heavy-light chain-SS-bridge. However, no influence on the sedimentation pattern was observed. The intrachainar disulfide bonds of neither the heavy nor the light chain reacted with DTNB to a measurable extent.
Schauenstein
E
E
Institut für Biochemie, Universität Graz.
Palm
W
W
Reiter
M
M
D004220:Disulfides / Q000032:analysis*; D004228:Dithionitrobenzoic Acid; D006868:Hydrolysis; D007120:Immunochemistry; D007074:Immunoglobulin G / Q000032:analysis*; D007202:Indicators and Reagents; D007474:Ion Exchange; D010206:Papain; D010434:Pepsin A; D014461:Ultracentrifugation
D004220:Disulfides; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D004228:Dithionitrobenzoic Acid; D010206:Papain; D010434:Pepsin A
Nachweis einer dem Disulfidaustausch zugänglichen S-S-Brücke zwischen den schweren Ketten im Immunglobulin G.