Modified high-affinity binding of Ni2+, Ca2+ and Zn2+ to natural mutants of human serum albumin and proalbumin.
High-affinity binding of radioactive Ni2+, Ca2+ and Zn2+ to six genetic albumin variants and to normal albumin isolated from the same heterozygote carriers was studied by equilibrium dialysis at pH 7.4. The three cations bind differently to albumin. Ni2+ binds to a site in the N-terminal region of the protein which is partially blocked by the presence of a propeptide as in proalbumin (proAlb) Varese (Arg-2-->His), proAlb Christchurch (Arg-1-->Gln) and proAlb Blenheim (Asp1-->Val) and by the presence of only an extra Arg residue (Arg-1) as in Arg-Alb and albumin (Alb) Redhill. The association constants are decreased by more than one order of magnitude in these cases, suggesting biological consequences for the ligand. The additional structural changes in Alb Redhill have no effect on Ni2+ binding. Finally, the modification of Alb Blenheim (Asp1-->Val) reduces the binding constant to 50%. Ca2+ binding is decreased to about 60-80% by the presence of a propeptide and the mutation Asp1-->Val. Arg-1 alone does not affect binding, whereas Alb Redhill binds Ca2+ more strongly than the normal protein (125%). In contrast with binding of Ni2+ and Ca2+, albumin shows heterogeneity with regard to binding of Zn2+, i.e. the number of high-affinity sites was calculated to be, on average, 0.43. The binding constant for Zn2+ is increased to 125% in the case of proAlb Varese, decreased to 50-60% for proAlb Christchurch and Alb Redhill but is normal for proAlb Blenheim, Alb Blenheim and Arg-Alb. The effects of the mutations on binding of Ca2+ and Zn2+ indicate that primary binding, when operative, is to as yet unidentified sites in domain I of the albumin molecule.
Kragh-Hansen
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U
Institute of Medical Biochemistry, University of Aarhus, Denmark.
Brennan
S O
SO
Minchiotti
L
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Galliano
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M
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D014644:Genetic Variation; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009154:Mutation; D009532:Nickel / Q000378:metabolism*; D011228:Prealbumin / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D012709:Serum Albumin / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D011228:Prealbumin; D012709:Serum Albumin; D009532:Nickel; D015032:Zinc; D002118:Calcium