BETA-2-MICROGLOBULIN CAN BE REFOLDED INTO A NATIVE-STATE FROM EX-VIVOAMYLOID FIBRILS

beta 2-microglobulin fibrils have been extracted from the femoral head of a patient who has been under chronic haemodialysis for 11. years. The primary structure of the N-terminal portion of the protein and mas s determination by electrospray mass spectrometry demonstrate that bet a 2-microglobulin, extracted as fibrils by the water extraction proced ure, was not glycated and that Asn17 was not deamidated. Limited prote olysis was observed in more than 20% of beta 2-microglobulin molecules and the main cleavage sites were at the C-terminus of Lys6 and Tyr10. beta 2-microglobulin from fibrils has been purified by gel filtration in 6 M Gdn/HCl and submitted to a refolding procedure. The refolding conditions have been determined through the study of the unfolding pat hway of the native protein. beta 2-microglobulin is stable at neutral pH where it displays a lower tendency to self-aggregate than in acidic conditions. Pulse dilution and extensive dialysis in refolding buffer at pH 7.5 yields beta 2-microglobulin with a tertiary structure ident ical to that of the native form. The CD spectrum in the near-ultraviol et region and the spectrum of the intrinsic fluorescence of Trp overla p those of the native protein, but the CD spectrum in the far-ultravio let region is affected by the contribution of oligomers created by bet a 2-microglobulin fragments that reduce the positive light polarisatio n at 205 nm typical of native beta 2-microglobulin.