PDI-mediated disulfide bond reduction in vitronectin

The free thiols of platelet-poor plasma proteins were alkylated by the addition of 50 mM NEM for 20 min at 23° in the dark. After dialysis, the NEM-pretreated plasma was recalcified to 1 mM CaCl₂. PDI–CCCC, PDI–AAAA or ERp57–CCCC as indicated was added to 100 μl of NEM-pretreated plasma in 0.3 mM DTT. The reaction was monitored and the reaction terminated by alkylation of new thiols using 20 mM of PEG₅₀₀₀-maleimide (Sigma-Aldrich) or PEG₂₀₀₀-maleimide (Nanocs). Aliquots were examined by SDS–PAGE and probed for vitronectin by Western blotting. The PDI-mediated reduction of vitronectin was monitored by changes in the electrophorectic mobility due to alkylation with PEG₂₀₀₀-maleimide or PEG₅₀₀₀-maleimide.