Purification and characterization of a novel 12,000-Da calcium binding protein from smooth muscle

Abstract

A new low molecular weight calcium binding protein, designated 12-kDa CaBP, has been isolated from chicken gizzard using a phenyl-Sepharose affinity column followed by ion-exchange and gel filtration chromatographies. The isolated protein was homogeneous and has a molecular weight of 12,000 based on sodium dodecyl sulfate-gel electrophoresis. The amino acid composition of this protein is similar to but distinct from other known low molecular weight Ca 2+ binding proteins. Ca 2+ binding assays using Arsenazo III (Sigma) indicated that the protein binds 1 mol of Ca 2+/mol of protein. The 12-kDa CaBP underwent a conformational change upon binding Ca 2+, as revealed by uv difference spectroscopy and circular dichroism studies in the aromatic and far-ultraviolet range. Addition of Ca 2+ to the 12-kDa CaBP labeled with 2- p-toluidinylnaphthalene-6-sulfonate (TNS) resulted in a sevenfold increase in fluorescence intensity, accompanied by a blue shift of the emission maximum from 463 to 445 nm. Hence, the probe in the presence of Ca 2+ moves to a more nonpolar microenvironment. Like calmodulin and other related Ca 2+ binding proteins, this protein also exposes a hydrophobic site upon binding calcium. Fluorescence titration with Ca 2+ using TNS-labeled protein revealed the presence of a single high affinity calcium binding site ( k d ≈ 1 × 10 −6 M).