PURIFICATION, CHARACTERIZATION AND ION BINDING-PROPERTIES OF HUMAN-BRAIN S100B PROTEIN

Human brain S100b (.beta..beta.) protein was purified using Zn-dependent affinity chromatography on phenyl-Sepharose. The Ca- and Zn-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 or Zn was investigated with UV spectroscopy, SH reactivity and interaction with a hydrophobic fluorescence probe 6-(p-toluidino)naphthalene-2-sulfonic acid (TNS). Flow dialysis measurements of Ca2+ binding to human brain S100b (.beta..beta.) protein revealed 6 Ca-binding sites which are assumed to represent 3 for each .beta. monomer, characterized by macroscopic association constants K1 = 0.44 .cntdot. 105 M-1; K2 = 0.1 .cntdot. 105 M-1 and K3 = 0.08 .cntdot. 105 M-1. In presence of 120 mM KCl, the affinity of the protein for Ca is drastically reduced. Zn-binding studies on human S100b protein showed that the protein bound 2 Zn ions per .beta. monomer, with macroscopic constants K1 = 4.47 .cntdot. 107 M-1 and K2 = 0.1 .cntdot. 107 M-1. Most of the Zn-induced conformational changes occurred after the binding of 2 Zn ions/mol 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 Ca binding was studied in the presence of Zn, an increase in the affinity of the protein for Ca was noted, K1 = 4.4 .cntdot. 105 M-1; K2 = 0.57 .cntdot. 105 M-1; K3 = 0.023 .cntdot. 105 M-1. The Ca- and Zn-binding sites of on S100b protein are probably different. Zn may regulate Ca-binding by increasing the affinity of the protein for Ca.