MODIFIED HIGH-AFFINITY BINDING OF NI2-ALBUMIN AND PROALBUMIN(, CA2+ AND ZN2+ TO NATURAL MUTANTS OF HUMAN SERUM)

High-affinity binding of radioactive Ni2+, Ca2+ and Zn2+ to six geneti c albumin variants and to normal albumin isolated from the same hetero zygote carriers was studied by equilibrium dialysis at pH 7.4. The thr ee cations bind differently to albumin. Ni2+ binds to a site in the N- terminal region of the protein which is partially blocked by the prese nce of a propeptide as in proalbumin (proAlb) Varese (Arg(-2) --> His) , proAlb Christchurch (Arg(-1) --> Gln) and proAlb Blenheim (Asp(1) -- > Val) and by the presence of only an extra Arg residue (Arg(-1)) as i n Arg-Alb and albumin (Alb) Redhill. The association constants are dec reased by more than one order of magnitude in these cases, suggesting biological consequences for the ligand. The additional structural chan ges in Alb Redhill have no effect on Ni2+ binding. Finally, the modifi cation of Alb Blenheim (Asp(1) --> Val) reduces the binding constant t o 50%. Ca2+ binding is decreased to about 60-80% by the presence of a propeptide and the mutation Asp(1) --> Val. Arg(-1) alone does not aff ect binding, whereas Alb Redhill binds Ca2+ more strongly than the nor mal protein (125%). In contrast with binding of Ni2+ and Ca2+, albumin shows heterogeneity with regard to binding of Zn2+, i.e. the number o f high-affinity sites was calculated to be, on average, 0.43. The bind ing constant for Zn2+ is increased to 125% in the case of proAlb Vares e, decreased to 50-60% for proAlb Christchurch and Alb Redhill but is normal for proAlb Blenheim, Alb Blenheim and Arg-Alb. The effects of t he mutations on binding of Ca2+ and Zn2+ indicate that primary binding , when operative, is to as yet unidentified sites in domain I of the a lbumin molecule.