Spectral [corrected] studies on the cadmium-ion-binding properties of bovine brain S-100b protein.
The effect of Cd2+ binding on bovine brain S-100b protein was studied using c.d. u.v. difference spectroscopy and fluorescence measurements. At pH 7.5, S-100b protein binds two Cd2+ ions per monomer with a Kd value of 3 x 10(-5) M. Addition of Cd2+ resulted in perturbing the single tyrosine residue (Tyr17) in the protein as indicated by u.v. difference spectroscopy and aromatic c.d. measurements. In the presence of Cd2+, the tyrosine residue moves to a more non-polar environment, since a red shift was observed in the u.v. difference spectrum. When the protein was excited at 278 nm, the tyrosine fluorescence emission maximum was centred at 306 nm. Cd2+ addition resulted in an increase in intrinsic fluorescence intensity. Fluorescence titration with Cd2+ indicated the protein binds Cd2+ with a Kd value of 3 x 10(-5) M. 2-p-Toluidinylnaphthalene-6-sulphonate-labelled protein, when excited at 345 nm, had a fluorescence emission maximum at 440 nm. Addition of Cd2+ to labelled protein resulted in a 5-fold increase in fluorescence intensity accompanied by a 5 nm blue shift in the emission maximum, suggesting that the probe, in the presence of Cd2+, moves to a hydrophobic domain. U.v. difference spectroscopic studies indicated a unique Cd2(+)-binding site on the protein, since Cd2+ addition yielded a large positive absorption band in the 240 nm region that is not found with either Ca2+ or Zn2- ions. Similar absorption bands have been observed in Cd-protein complexes such as Cd-metallothionein [Vasak, Kagi & Hill (1981) Biochemistry 20, 2852-2856] and also in model complexes of Cd2+ with 2-mercaptoethanol. This absorption band is believed to arise as a result of charge-transfer transitions between the thiolate and Cd2+. Of the two Cd2- -binding sites on the beta-chain, one must be located at the N-terminal end near the single tyrosine residue, since Cd2- and Zn2+ produced similar effects on the intrinsic protein fluorescence. The other Cd2+ site which is unique to Cd2+ must be Cys84, located at the C-terminal end.
Donato
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Department of Biochemistry, University of Alberta, Edmonton, Canada.
Mani
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RS
Kay
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D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002104:Cadmium / Q000378:metabolism* / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002413:Cations, Divalent; D002417:Cattle; D002942:Circular Dichroism; D014644:Genetic Variation; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors; D011485:Protein Binding; D011487:Protein Conformation; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D015032:Zinc / Q000494:pharmacology
D002135:Calcium-Binding Proteins; D002413:Cations, Divalent; D046911:Macromolecular Substances; D009414:Nerve Growth Factors; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; D002104:Cadmium; D015032:Zinc; D002118:Calcium