The quaternary structure of Phascolosoma agassizii coelomic hemerythrin

The coelomic hemerythrin of Phascolosoma agassizii gave a molecular weight of 32,200 on Sephadex gel chromatography and an s 20,ω of 3.65 S in sedimentation velocity experiments. A combination of the sedimentation coefficient and the Stoke's radius determined by gel chromatography permitted the calculation of a molecular weight of 40,600. A subunit molecular weight of 12,800 was obtained by SDS-polyacrylamide gel electrophoresis. Iron analyses indicated the hemerythrin contained 0.885% Fe. Assuming 2Fe/subunit, a minimum subunit molecular weight of 12,600 was calculated. Crosslinking experiments of native protein with dimethyl suberimidate yielded three bands on SDS-polyacrylamide gel electrophoresis. The molecular weights of these species were 13,000, 26,500, and 40,500. Nine bands which stainded for protein and iron were observed in disc gel electrophoresis of the native hemerythrin. Electrophoresis in gels containing 8 m urea showed three major and one minor band. Individual native hemerythrin bands, isolated by preparative disc electrophoresis, gave one, two, or three bands when reelectrophoresed on 8 m urea gels. Based on this evidence, it was concluded that P. agassizii coelomic hemerythrin exist as a trimeric protein. A model is proposed for the subunit composition of the native protein which accounts for the nine bands observed in disc gel electrophoresis.