A fluorimetry study of N‐(1‐pyrenyl)iodoacetamide‐labelled bovine brain S‐100a protein

FEBS Letters

Kay, Cyril M.¹  Mani, Rajam S.¹ 

[1] Medical Research Council Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

关键词: S-100a protein;    Ca2+ effect;    Zn2+ effect;    Fluorescent probe;    PIA;    N-(1-pyrenyl)iodoacetamide;    CD;    circular dichroism;    DTNB;    5;    5'-dithiobis-(2-nitrobenzoic acid);    [14C]NEM;    N-ethyl-[2;    3-14C]maleimide;    UV;    ultraviolet;   

DOI  :  10.1016/0014-5793(85)80275-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

S-100 a protein has been modified with the thiol specific fluorescent label N-(l-pyrenyl) iodoacetamide. When the protein was reacted with the fluorophore in the presence of EDTA, only one sulfhydryl group was labelled. The labelled protein when excited at 344 nm had an emission maximum at 386 nm and a shoulder at 403 nm. Addition of calcium resulted in a quenching of the fluorescence intensity at 386 nm whereas Zn²⁺ addition caused an enhancement. Titration of labelled protein with CaCl₂ or ZnSO₄ yielded dissociation constants (K _D values) of 3 × 10⁻⁵ M and 8 × 10⁻⁶ M for Ca²⁺ and Zn²⁺, respectively. In the presence of 90 mM KCl the protein has a lower affinity towards calcium (K _D, 6 × 10⁻⁵ M). Acrylamide quenching studies revealed the probe to be more accessible to acrylamide in the plus calcium state. Reaction kinetics with DTNB in 0.1 M Tris buffer at pH 7.5 also suggested that the single reactive sulfhydryl group was more exposed to the solvent in the presence of Ca²⁺. Thus, the observed quenching of calcium addition may be the result of the probe moving to a relatively more polar environment. This would imply that the probe moves to a more nonpolar region in the presence of Zn²⁺. These results are in accord with our earlier near UV-CD studies where the metal ions, calcium and zinc were observed to induce different conformational changes in S-100a protein.

【 授权许可】

Unknown