title: 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.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Canada.
authors.forename: R S
authors.initials: RS
authors.lastname: Mani
authors.forename: C M
authors.initials: CM
authors.lastname: Kay
mesh_terms: D000818:Animals; D017302:Annexins; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002942:Circular Dichroism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D017302:Annexins; D002135:Calcium-Binding Proteins; D002118:Calcium