Figure 2

Redox titration of adiponectin oligomerization in glutathione-based redox buffers. (A) Native PAGE of adiponectin oligomers after collapse to trimers and subsequent incubation in various reduction potentials and time points as indicated. (B) Non-reducing denaturing SDS-PAGE analysis of disulfide-bonded dimers or reduced monomers at various time points. Purified bovine octadecameric adiponectin was collapsed to trimers as described in Methods. After collapse, different ratios of oxidized and reduced glutathione at reduction potentials ranging from -120 to -220 mV were added and allowed to incubate in an anaerobic chamber for up to 48 hrs. The total concentration of glutathione in each condition ranged from 1 to 3 mM. At each time point, aliquots were removed and further disulfide bond exchange was halted by treatment with NEM.