Abstract
Metal binding strategies employing low molecular weight chelators and equilibrium dialysis were used to investigate several unresolved aspects of zinc and copper binding to serum albumin. Direct measurement of histidine binding to bovine serum albumin when the histidine is presented either as a metal-chelate or alone provides no evidence for an albumin-metal-histidine ternary complex. Using previously determined intrinsic constants for Zn(II) and Cu(II), we have measured zinc binding to bovine serum albumin in the presence of saturating amounts of copper. The results of these experiments unambiguously show that zinc and copper bind at separate noninteracting sites on this protein. The intrinsic constants for zinc and copper binding to dog serum albumin have been determined. Contrary to previous reports, we find that dog serum albumin has a specific high affinity site for copper, log10K 10.17 for Cu(II) compared to 6.85 for Zn(II) at the separate site.
Highlights
James Masuokal and Paul Saltman From the Department of Biology, University of California at Sun Diego, La Jolla, California 92093-0322
Further,althoughother periments unambiguously show that zinc and copper groups have reported the existence of a ternary complex, it was bind at separate noninteracting sites on this protein. a negligible component at the metacloncentrations used in the
Ternary Complex Formation-Previous studies of zinc and small additions of 0.1 N NaOH, and the solution was brought to final copper binding t o BSA, HSA,and DSA suggested that thehigh volume with water
Summary
The formation of a n albumin-metal-chelator ternary comweight chelators and equilibriumdialysis were used to plexhas been proposed by previous investigators [2,3,4]. Afundamental parameter of metal binding activity is the intrinsic binding constant, which describes the affinity with which metal ions are bound to specific protein investigated zinc and copper binding to HSA and DSA by equilibrium dialysis. We used this strategy to determine the has been reportedto havevery different copper binding activity intrinsic binding constants for Zn(I1) and Cu(I1) binding to due to theabsence of His-3 [13, 14].Appleton and Sarkar[13] bovine serum albumin(BSA1.l Wehave applied this experimen- compared the copper binding activity of HSA and DSA and tal system to other aspeocftZs n(I1) and Cu(I1)binding to BSA. Inparticular, we tested whether an albumin-metal-chelator Giroux and Schoun [5]reported an apparent constant ternary complex formed and whether thehigh affinity sites for for copper binding to DSA which was 3 orders of magnitude. Cu(I1) and Zn(I1) do not compete for sites on albumin, and the nature of Cu(I1) and Zn(I1) binding sites on DSA closely resembles those for BSA and HSA
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