The influence of Cu2+ on the unfolding and refolding of intact and proteolytically processed β2‐microglobulin

Human β 2 -microglobulin (β 2 m) is an amyloidogenic protein in patients suffering from chronic kidney disease and especially in those patients that need intermittent hemodialysis for longer periods, e.g., when awaiting transplantation. While many in vitro conditions induce β 2 m-amyloid formation from wild-type (wt) β 2 m and while a number of structurally altered β 2 m molecules are known to be conformationally unstable and amyloidogenic on their own, it is not known why β 2 m-amyloid is generated in some dialysis patients. For many amyloid proteins it is known that divalent metal ions, especially Cu 2+ , display strong binding and distinct destabilizing effects on protein conformation. The present study uses CE to assess conformational states of wt and cleaved β 2 m (dK58-β 2 m, β 2 m cleaved at lysine-58, a modification found in the circulation of hemodialysis patients) in the presence of divalent metal ions. The experiments provide both qualitative and quantitative data showing the specific destabilizing effects of Cu 2+ -ions on the folding of wt β 2 m. Both refolding after acid denaturation and solution structure of β 2 m under otherwise native conditions are severely influenced by Cu 2+ . An increased unfolding, aggregation, and induction of Congo red-reactive molecular species in Cu 2+ -incubated wt-β 2 m could be demonstrated while the refolding kinetics of dK58-β 2 m, already slower than the wt molecule, appeared not to be further decreased by Cu 2+ . Given the interest in the actions of metal ions in other types of amyloidosis, including, e.g., Alzheimer's disease and the prion encephalopathies, the use of microelectrophoretic methods to monitor unfolding and refolding of biomolecules available in scarce amounts as shown in this study is an attractive option.