Kragh-Hansen U, Brennan S O, Minchiotti L, Galliano M
Institute of Medical Biochemistry, University of Aarhus, Denmark.
Biochem J. 1994 Jul 1;301 ( Pt 1)(Pt 1):217-23. doi: 10.1042/bj3010217.
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.
通过在pH 7.4条件下的平衡透析，研究了放射性Ni2+、Ca2+和Zn2+与六种遗传性白蛋白变体以及从相同杂合子携带者中分离出的正常白蛋白的高亲和力结合。这三种阳离子与白蛋白的结合方式不同。Ni2+与蛋白质N端区域的一个位点结合，在原白蛋白（proAlb）瓦雷泽（Arg-2→His）、proAlb克赖斯特彻奇（Arg-1→Gln）和proAlb布伦海姆（Asp1→Val）中，该位点部分被前肽的存在所阻断，在精氨酸-白蛋白（Arg-Alb）和白蛋白（Alb）雷丁希尔中，仅被一个额外的精氨酸残基（Arg-1）所阻断。在这些情况下，缔合常数降低了一个多数量级，表明对配体有生物学影响。Alb雷丁希尔中的额外结构变化对Ni2+结合没有影响。最后，Alb布伦海姆（Asp1→Val）的修饰使结合常数降低到50%。前肽的存在和Asp1→Val突变使Ca2+结合降低到约60-80%。单独的Arg-1不影响结合，而Alb雷丁希尔比正常蛋白质更强烈地结合Ca2+（125%）。与Ni2+和Ca2+的结合不同，白蛋白在Zn2+结合方面表现出异质性，即高亲和力位点的数量平均计算为0.43。在proAlb瓦雷泽的情况下，Zn2+的结合常数增加到125%，在proAlb克赖斯特彻奇和Alb雷丁希尔中降低到50-60%，但在proAlb布伦海姆、Alb布伦海姆和Arg-Alb中正常。突变对Ca2+和Zn2+结合的影响表明，当起作用时，主要结合是到白蛋白分子结构域I中尚未确定的位点。
