Instability of purified 9-kDa porcine intestinal calcium-binding protein in the absence of calcium

Abstract

Purified 9-kDa porcine intestinal calcium-binding protein (ICaBP, Calbindin D 9K) is unstable when stored at 4 °C in the absence of Ca(II). Cleavage of the polypeptide occurs producing ~5.2- and 3.7-kDa fragments. The former dimerizes giving a species which migrates on sodium dodecylsulfate-urea gels with an M r 13,700, in contrast to the observed M r 11,000 for native ICaBP. The fragmentation also results in an irreversible loss of high affinity Ca(II) binding and an alteration of the ultraviolet absorption spectrum. The presence of Ca(II) bound to ICaBP prevents fragmentation. The dimer can be isolated from aged preparations of apo-ICaBP and is stable on further storage with or without Ca(II) present. The observed molecular weights of the fragments along with the amino acid analysis and ultraviolet spectra of the dimer suggest cleavage of the polypeptide chain of ICaBP in the vicinity of residue 49.