Isolation, characterization and oxygen equilibrium of an extracellular haemoglobin from Eunice aphroditois (Pallas)

Abstract

The extracellular haemoglobin from the polychaeta,Eunice aphroditois, existed as a mixture of a heavy major component (so20, w = 56.96 +/- 0.125) and a light minor component (so20, w = 10.00 +/- 0.13S), the latter probably being a dissociation product of the former. The molecular weight of the purified heavier component, as detetermined by sedimentation equilibrium, was 3.44 x 10(6) +/- 0.04x10(6). The molecule had the electron-microscopic appearance typical of annelid haemoglobins, consisting of a stack of two hexagonal plates, with dimensions 26.32 +/- 0.27 nm across the flats of the hexagon, height of stack 17.86 +/- 0.34 nm. The sugar composition is reported, and the isoelectric point was approx. pH7.8. The haem content was 2.31 +/- 0.01%, corresponding to a minimal mol.wt. of 26700. Detergent/gel electrophoresis revealed the presence of at least four bands with molecular weights in the range 14600-31000. Five N-terminal amino acids were found. In addition to the 10S component, which co-existed with the 57S component at all pH values in the range 4.0-10.6, at low pH values (less than pH.5.0) A 16S and a 1.9 S component were found. The absorption and circular-dichroic spectra are reported, and the alpha-helical content, calculated from the ellipticity at 222 nm, was about 40%. The molecule bound O2 co-operatively with a maximum value of the Hill coefficient, h, of 3.9. Over the pH range 7.0-8.0 there was a positive Bohr effect.

Dissociation of Cu–Zn superoxide dismutase into monomers by urea

“…Sword fish copper-zinc superoxide dismutase has been suggested [6] to dissociate into monomers in 8 M urea on the basis of chromatographic evidence. Our gel filtration experiments show that the same chromatographic behaviour as the sword fish enzyme is observed with the bovine and yeast superoxide dismutases, that is two molecules structurally most distant from each other within the same class of homologous proteins [ 1 l] .…”

“…The effect of urea seems to be limited to destabilization of the dimeric structure, as other properties of these enzymes appear to be almost unaffected [6,12,13]. Urea at 8 M, at variance with SDS,leads to monomer formation independently of the enzyme source and other complementary treatments, such as the presence of /3-mercaptoethanol or the removal of metals [4,14].…”

Fluorescence lifetime distribution of folded and unfolded proteins

“…HSOD was purified from human erythrocytes [20]. Metal-depleted HSOD was prepared by dialyzing the protein against 1 mM EDTA in 0.05 M sodium acetate buffer, pH 3.5.…”

Oxygen and carbon monoxide binding to myoglobin from the dolphin fish Coryphaena hippurus

“…In this communication we report a complete characterization of the equilibrium and kinetics of 02 and CO binding to the myoglobin from the dolphin fish, Coryphaena hippurus, recently purified by Bannister and Bannister [ 1 ]. This work seemed of interest from a comparative standpoint, since no kinetic information on fish myoglobin is as yet available.…”