Isolation, characterization and metal-ion-binding properties of the alpha-subunit from S-100a protein.
The brain-specific S-100 protein is a mixture of two predominant components, S-100a and S-100b, with subunit compositions of alpha beta and beta beta respectively. In the present study, the alpha-subunit, isolated from S-100a by using anion-exchange chromatography in the presence of 8 M-urea, was homogeneous by the criteria of SDS/polyacrylamide-gel, urea/SDS/polyacrylamide-gel and non-SDS/polyacrylamide-gel electrophoresis. The alpha-subunit underwent a conformational change upon binding Ca2+ and Zn2+ at pH 7.5, as revealed by u.v. difference spectroscopy, c.d. and fluorescence measurements. Far-u.v. c.d. studies indicated the apparent alpha-helical content to fall when the protein bound either Ca2+ or Zn2+. Addition of Ca2+ to the alpha-subunit resulted in exposing to the solvent the single tryptophan residue and one or more tyrosine and phenylalanine residues. Zn2+ induced only a small conformational change, and among the aromatic chromophores only tyrosine residues were affected to a small extent. Ca2+ was able to bind to the alpha-subunit in the presence of Zn2+, and the two metal-ion-binding sites appeared to be different. When the apoprotein was excited at 280 nm, the fluorescence emission maximum was located at 337 nm. In the presence of Ca2+, the emission maximum occurred at 340 nm and was accompanied by a nearly 25% increase in fluorescence intensity. Fluorescence titration with Ca2+ at pH 7.5 revealed only one class of binding site, with a Kd value of 1.26 X 10(-4) M. The effect of K+ on the protein was slightly antagonistic to that of Ca2+, as indicated by u.v. difference spectroscopy and fluorescence titration.
D000596:Amino Acids; D015415:Biomarkers; D002118:Calcium / Q000378:metabolism; D002942:Circular Dichroism; D008623:Mercaptoethanol / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D009418:S100 Proteins / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D015032:Zinc / Q000378:metabolism
D000596:Amino Acids; D015415:Biomarkers; C030564:S-100 calcium-binding protein alpha subunit; D009418:S100 Proteins; D008623:Mercaptoethanol; D015032:Zinc; D011188:Potassium; D002118:Calcium