DETERMINATION OF NATIVE ZINC CONTENT OF ALPHA-2-MACROGLOBULIN IN NORMAL, HYPERZINCEMIC AND HYPOZINCEMIC SERA BY SUCROSE DENSITY GRADIENT CENTRIFUGATION

Fractions of human serum proteins obtained by sucrose density gradient centrifugation were analyzed for zinc content by atomic absorption spectrophotometry and colorimetry. Zinc was found in two distinct fractions: one fraction appeared at the albumin sedimenting area, and the second fraction appeared with alpha-2-macroglobulin (alpha-2-M). Approximately 30% of total serum zinc was associated with the alpha-2-M fraction. The high purity of this alpha-2-M containing fraction was reflected by the excellent correlation of results obtained from the determination of the protein content of these fractions by Bradford method and radial immunodiffusion analysis. Further evidence of the purity of alpha-2-M in these fractions was obtained by immunoelectrophoresis. Moreover, these alpha-2-M containing fractions did not have any significant amount of trace elements that could possibly interfere with zinc determination by the dithizone method. The suitability and reliability of this method for the determination of the native zinc content of serum alpha-2-M is supported by the experimental findings that this procedure does not cause exogenous zinc contamination and is not disruptive to protein-metal interaction. Our results also indicate that native alpha-2-M contains approximately 80-100 μg Zn/g protein and that the molar ratio between zinc and alpha-2-M is approximately 1.0. This ratio was also maintained in hypo- and hyper-zincemic sera obtained from patients with multiple myeloma and lymphocytic lymphoma. © 1979.