Some Physico-Biochemical Properties of the Hemocyanin from Haliotis Iris

Abstract

The hemocyanin from gastropod Haliotis iris is present in the hemolymph as a large molecule with molecular weight about 9 x 106 and sediments as a single peak with SO20,W = 103S. Electron microscopy shows molecules with a cylindrical structure. In the pH range 7 to 9.6, 9S, 11.9S and 60S particles are observed in proportions dependent on presence of Ca2+ or EDTA. The major band observed by polyacrylamide gel electrophoresis in the presence of SDS corresponded to Mr = 330,000. Ion exchange chromatography yields three fractions, which on SDS gel electrophoresis give bands corresponding to masses 320,000 and 200,000; 430,000; 320,000 respectively. Proteolytic digestion, carried out with trypsin on whole hemocyanin yielded two particles; one a long tubular aggregate with Mr 20-30 times larger than the native molecule, formed by end-to-end polymerization, and the second fragment which dissociates upon SDS gel electrophoresis into 50,000 dalton polypeptide chains. Electron micrographs of the protein subunits reassociated in the presence of Ca2+ show a cylindrical structure typical of the native protein, and the absorption spectrum of the reassociated protein is the same as that of the native hemocyanin. Oxygen binding experiments with the native hemocyanin, half molecules and twentieth particles, all show cooperative binding, a small reverse Bohr effect and an increase in oxygen affinity with increasing temperature. Both, high and low molecular weight fractions from trypsin digest of hemocyanin retain oxygen binding capacity, and Fraction II posseses much higher cooperativity compared with Fraction 1. From amino acid analyses of the native hemocyanin and Fraction II, it was concluded that there were no gross differences in chemical composition between the two.