Electrically neutral microheterogeneity of human plasma transthyretin (prealbumin) detected by isoelectric focusing in urea gradients

Mutants of the human plasma transthyretin (TTR, prealbumin) have attracted interest due to their rather frequent association with the autosomal domina nt disease familial amyloidotic polyneuropathy (FAP). Some three quarters o f known TTR mutations produce electrically neutral amino acid substitutions undetectable via separation by charge. We have developed an electrophoreti c procedure sensitive to differences in the stability of tetramers and mono mers under partially denaturing conditions. The differential folding states were found to be fully reversible. Applying the procedure we found 14 elec trically silent mutants of TTR among 2 000 plasma samples from German donor s. We demonstrate that the normal TTR monomer exists in different forms of variable stability and/or charge due to binding of sulfhydryls from plasma to the unique cysteine at position 10 of the primary structure as well as d ue to modification by treatment with an oxidant. We found that reduction of Cys10 increases the stability of the folded monomeric and tetrameric confo rmations. The conformational changes of TTR induced by isoelectric focusing in a urea gradient were found to be associated by a gain of three positive charge units. Using published crystallographic data we present structural sites in the TTR molecule which could explain the observed effects.