Purification, characterization and ion binding properties of human brain S100b protein

Abstract

Human brain S100b (ββ) protein was purified using zinc-dependent affinity chromatography on phenyl-Sepharose. The calcium- and zinc-binding properties of the protein were studied by flow dialysis technique and the protein conformation both in the metal-free form and in the presence of Ca 2+ or Zn 2+ was investigated with ultraviolet spectroscopy, sulfhydryl reactivity and interaction with a hydrophobic fluorescence probe 6( p-toluidino)naphthalene-2-sulfonic acid (TNS). Flow dialysis measurements of Ca 2+ binding to human brain S100b (ββ) protein revealed six Ca 2+-binding sites which we assumed to represent three for each β monomer, characterized by the macroscopic association constants K 1 = 0.44·10 5 M −1; K 2 = 0.1·10 5 M −1 and K 3 = 0.08·10 5 M −1. In the presence of 120 mM Kcl, the affinity of the protein for calcium is drastically reduced. Zinc-binding studies on human S100b protein showed that the protein bound two zinc ions per β monomer, with macroscopic constants K 1 = 4.47·10 7 M −1 and K 2 = 0.1·10 7 M −1. Most of the Zn 2+-induced conformational changes occurred after the binding of two zinc ions per mole of S100b protein. These results differ significantly from those for bovine protein and cast doubt on the conservation of the S100 structure during evolution. When calcium binding was studied in the presence of zinc, we noted an increase in the affinity of the protein for calcium, K 1 = 4.4·10 5 M −1; K 2 = 0.57·10 5 M −1; K 3 = 0.023·10 5 M −1. These results indicated that the Ca 2+- and Zn 2--binding sites on S100b protein are different and suggest that Zn 2+ may regulate Ca 2+ binding by increasing the affinity of the protein for calcium.