Effect of calcium on the monomer-dimer association of calcium-binding protein in the serum of laying hens

Summary A major calcium-binding protein of laying hen plasma was fractionated and partially characterized. After lipid flotation, approximately 80% of the total binding activity was localized in a high-density plasma fraction. Chromatography on Sepha-dex G-200 or Sepharose 6B gave a bimodal elution pattern. Sucrose gradient centrifugation yielded a similar bimodal pattern; the fast- and slow-sedimenting ultraviolet (UV) absorption peaks appeared identical to the first and second chromatographic elution peaks, respectively. Equilibrium dialysis localized the calcium-binding activity to the faster-sedimenting material. An orcinol-sulfuric acid test indicated a total carbohydrate content of 5% for the fast peak. Analytical velocity ultracentrifugation measurements of this calcium-binding component yielded varying amounts of two entities with sedimentation coefficients (S20,w) of approximately 16 and 8 S. The 16 S material was shown by equilbrium centrifugation (meniscus depletion) to have a 5.4 × 105 M r. Dialysis of the 16 S moiety against citrate or ethylene diamine tetraacetic acid (EDTA) buffer produced 8S material having a 2.7 × 105 M r and this monomer-dimer association was partially reversible by subsequent dialysis against calcium ions. The calcium-binding molecule appears to be vitellogenin. Calcium-binding protein was found at elevated levels in plasma from long-term estrogenized birds but not from nonlaying birds. This research was supported in part by U.S. Public Health Service Grants R01-AM ES 05970 and R01 AM ES 17217.