The isolation and partial characterization of α 2-macroglobulin from the serum of the ostrich ( Struthio camelus)

Abstract

1. 1. α 2-Macroglobulin (α 2M) activity is present in the serum of the ostrich, Struthio camelus. The chromogenic synthetic peptide substrates BAPNA and ATNA were hydrolysed by trypsin and chymotrypsin, respectively, in the presence of ostrich serum and the α 2M in ostrich serum protected trypsin from being inhibited by soybean trypsin inhibitor. Ostrich α 2M proved to be a potent inhibitor of bovine pancreatic trypsin and chymotrypsin. 2. 2. α 2M was purified to apparent homogeneity by PEG precipitation, DEAE-Toyopearl 650M, Bio-Gel A-5m and Zn 2+-affinity chromatography. 3. 3. Ostrich α 2M migrated as a single band ( M r 779,000) during non-denaturing gradient gel electrophoresis and showed increased mobility after reaction with trypsin. Denaturation dissociated ostrich α 2 M into half-molecules. Denaturation with reduction further dissociated the protein into quarter-subunits. 4. 4. Isoelectric focusing revealed a pI of 5.3. 5. 5. The amino acid composition of ostrich α 2M is typical of an α 2M, comparing favourably with those of other animal species. The carbohydrate composition of the purified protein, in percentage dry weight of the molecule, was galactose: mannose (1:1), 4.55; N-acetylglucosamine, 2.35; N-acetylneuraminic acid, 0.58; and fucose, 0.77. 6. 6. α 2M was assessed immunologically by Ouchterlony double-diffusion and Western blot analysis with polyvalent antisera directed against ostrich α 2M. 7. 7. Ostrich α 2M seems to show many physical, chemical and kinetic properties similar to those of other known α 2Ms, but is expected to differ from other αMs when considering the primary structure of the bait region, the area differing among α Ms from different species and determining its specificity.