A GENETIC VARIANT OF ALBUMIN (ALBUMIN ASOLA, TYR140-]CYS) WITH NO FREE -SH GROUP BUT WITH AN ADDITIONAL DISULFIDE BRIDGE

A slow migrating variant of human serum albumin, present in lower amou nt than the normal protein, has been detected by routine clinical elec trophoresis at pH 8.6 in two members of a family living in Asola (Lomb ardia, Italy). Ion-exchange chromatography of serum samples failed to separate the normal protein from the variant. Analysis of the albumin peak by SDS/PAGE revealed that the variant had a lower apparent molecu lar mass than its normal counterpart. However, the abnormal band was n ot detectable when the separation was performed under reducing conditi ons or when both albumins were carboxymethylated. Isoelectric-focusing analysis of CNBr fragments localized the mutation to fragment CNBr 3 (residues 124-298). This fragment was isolated on a preparative scale and subjected to tryptic digestion. Sequence determination of the abno rmal tryptic peptide revealed that the variant arises from a Tyr140--> Cys substitution. This result was confirmed by DNA sequence analysis, which showed a single transition of TAT-->TGT at nucleotide position 5 074. Despite the presence of an additional cysteine residue, several l ines of evidence indicated that albumin Asola has no free -SH group; t herefore, we propose the formation of a new S-S bond between Cys140 an d Cys34, the only free sulphydryl group present in the normal protein. The relatively low level of the variant in serum and its abnormal mob ility on cellulose acetate electrophoresis and SDS/PAGE are probably c aused by a gross conformational change of the molecule induced by the new S-S bridge.