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Purification and spectral studies on the Ca2+-binding properties of 67 kDa calcimedin.

ABSTRACT: The 67 kDa calcimedin, isolated by using a phenyl-Sepharose affinity column followed by DEAE-cellulose and gel-filtration chromatographies, was homogeneous by the criterion of SDS/polyacrylamide-gel electrophoresis. In non-SDS gels, the protein moved faster in the presence of EDTA, suggesting that Ca2+ binding affects its mobility in a manner similar to other Ca2+-binding proteins such as calmodulin and S-100 proteins. The 67 kDa protein underwent a conformational change upon binding Ca2+, as revealed by u.v. difference spectroscopy and near-u.v. c.d. measurements. Tryptophan and tyrosine residues were perturbed upon Ca2+ binding, moving to a more non-polar environment in the presence of Ca2+. Upon excitation of the protein at 280 nm, the fluorescence emission maximum was centered around 325 nm, suggesting that the tryptophan residues are located in a fairly hydrophobic region. Ca2+ addition did not induce a significant change in the intrinsic protein fluorescence intensity at 325 nm. Addition of Ca2+ to the 67 kDa protein labelled with 2-p-toluidinylnaphthalene-6-sulphone (TNS) resulted in a 25% increase in fluorescence intensity, accompanied by a blue shift of the emission maximum from 442 to 432 nm. Hence, the probe in the presence of Ca2+ moves to a more non-polar microenvironment, like calmodulin and other Ca2+-binding proteins. Fluorescence titration with Ca2+ using TNS-labelled protein revealed one class of binding site, with a Kd value of 2 x 10(-5) M.

SUBMITTER: Mani RS

PROVIDER: S-EPMC1138588 | biostudies-other | 1989 May

REPOSITORIES: biostudies-other

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Project description: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.

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Purification and spectral studies on the Ca2+-binding properties of 67 kDa calcimedin.

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