HIGH-AFFINITY BINDING OF LAURATE TO NATURALLY-OCCURRING MUTANTS OF HUMAN SERUM-ALBUMIN AND PROALBUMIN

Binding of laurate (n-dodecanoate) to genetic variants of albumin or i ts proprotein and to normal albumin isolated from the same heterozygou s carriers was studied by a kinetic dialysis technique at physiologica l pH. The first stoichiometric association constant for binding to pro albumin Lille (Arg(-2) --> His) and albumin (Alb) Roma (Glu(321) --> L ys) was increased to 126% and 136% respectively compared with that for binding to normal albumin, whereas the constant for Alb Maku (Lys(541 ) --> Glu) was decreased to 80%. In contrast, normal laurate-binding p roperties were found for as many as nine other albumin variants with s ingle amino acid substitutions. Because the net charges of all these m utants were different from that of normal albumin, the results suggest that the examples of modified laurate binding are not caused by long- range electrostatic effects. Rather, the three positions mentioned are located close to different binding sites for the fatty acid anion. Th e most pronounced effect was observed for the glycosylated Alb Casebro ok, the binding constant of which was decreased to 20%. Binding to the glycosylated Alb Redhill was also decreased, but to a smaller extent (68%). These decreases in binding are caused by partial or total block ing of the high-affinity site by the oligosaccharides, by the negative charges of the oligosaccharides, and/or by conformational changes ind uced by these bulky moieties. Laurate binding to two chain-termination mutants (Alb Catania and Alb Venezia) was normal, indicating that the C-terminus of albumin is not important for binding. By using differen t preparations of normal albumin as controls in the binding experiment s, it was also possible to compare the effect of various methods for i solation and defatting on laurate binding.