Purification and Characterization of a Novel 21 kD Calcium-binding Protein from Rabbit Appendix Lymphocytes

A novel 21 kD calcium-binding protein from rabbit appendix B lymphocytes has been purified and characterized. Through heat-denaturation, using Phenyl-Sepharose and DEAE-Sepharose chromatography, we obtained 5.3 mg SDS-PAGE homogeneous CaBP(21) from 1 kg lymphocyte cells. Amino acid analysis showed the acidic amino acid content (Asp + Glu) to be 26% after HCL hydrolysis. The blocking of the N-terminus of CaBP(21) prevents the de novo Edman degradation, like most of the other calcium-binding proteins. CaBP(21) has 46% of hydrophobic amino acid (with Gly, without Trp) content, 10% of basic amino acid content and 44% of acidic and polar amino acids. Peptide mapping and SDS-PAGE combined Sephadex G-25 gel filtration proves that CaBP(21) consists of two identical or similar subunits. Ca(2+)-binding assays using Arsenazo III indicated one protein to bind 4 Ca(2+) with dissociation constant (K(d)) for Ca(2+) about 10(-5) mol/L.