Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

326 Kobayashi
that S-100A1 and S-100B also bind to phenothiazine- and W7-Sepharose conjugates
in a Ca 2+ -dependent manner and quantitatively compete with
calmodulin for binding to the immobilized drugs. They also reported the use of
calmodulin antagonist-Sepharose conjugates for the purification of S-100A1
and S-100B. Furthermore, studies concerning the binding of isolated domains
of several of these proteins have also been reported by Vogel et. al. (4). In this
chapter, attention is focused on the interaction of newly developed pharmacological
agents with nonneuronal S-100 family proteins.
1.1. Coupling of Ligands to Matrix for Drug-Affinity
Chromatography
Affinity matrices, coupling solvents, temperature and blocking methods
should be chosen based on coupling chemistry (5–9), solubility and stability
of the drug of interest, stability of the matrix in the organic solvent, and the
length of spacer arm of the matrix (10,11).
2. Materials
1. Drugs: W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide) are available
from Sigma Co. and Calbiochem Co. Fluphenazine·2HCl (4-[3-[2-(trifluoromethyl)-10H-phenothiazin-10-yl]propyl]-1-piperazine
ethanol) was obtained from
Research Biochemicals International, Inc. Amlexanox (2-amino-7-(1-methylethyl)-5-oxo-5H-[1]benzopyrano(2,3-b)pyridine-3-carboxylic
acid) was a generous
gift from Takeda Pharmaceutical Co., Japan. Cromolyn (cromoglycic acid)
sodium salt (5,5'-[(2-hydroxy-1,3-propanediyl)bis-(oxy)]bis[4-oxo-4H–1benzopyran-2-carboxylic
acid] disodium salt) can be purchased from Sigma Co.
Tranilast (rizaben, N-(3',4'-dimethoxycinnamoyl) anthranilic acid) was a generous
gift from Kissei Yakuhin Co. (Matsumoto, Japan). Structures of drugs mentioned
are in Fig. 1.
2. Affinity matrices: AF-amino Toyopearl 650M and Epoxy Toyopearl 650M,
hydrophilic vinyl polymer supports were obtained from Tosoh Co. (Tokyo,
Japan). These affinity matrices are relatively stable in organic solvents, such as
dimethylformamide and dioxane. Epoxy-activated Sepharose 6B was obtained
from Amersham Pharmacia Biotech (Uppsala, Sweden).
3. EDC (N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride).
4. DMF (N,N'-dimethylformamide).
5. Sodium acetate.
6. Acetic anhydride.
7. Ethanolamine.
8. Dioxane.
9. Rinsing Buffers A and B: 0.1 M Tris-HCl buffer, pH 7.6, and 100 mL 20 mM
Tris-HCl buffer, pH 7.6.
10. Buffer A: 20 mM Tris-HCl, 0.1 mM EGTA, pH 7.5.
11. Buffer B: 20 mM Tris-HCl, 0.5 mM CaCl2, pH 7.5.