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

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Abstract
The extracellular Hb from the polychaete E. aphroditois, existed as a mixture of a heavy major component .**GRAPHIC**. = 56.96 .+-. 0.125) and a light minor component .**GRAPHIC**. = 10.00 .+-. 0.13S), the latter probably being a dissociation product of the former. The molecular weight of the purified heavier component, as determined by sedimentation equilibrium, was 3.44 .times. 106 .+-. 0.04 .times. 106. The molecule had the EM appearance typical of annelid Hb, consisting of a stack of 2 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 was reported, and the isoelectric point was .apprx. pH 7.8. The heme content was 2.31 .+-. 0.01%, corresponding to a minimal MW of 26,700. Detergent/gel electrophoresis revealed the presence of at least 4 bands with MW in the range 14,600-31,000. 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 (< pH 5.0) a 16S and a 1.9S component were found. The absorption and circular-dichroic spectra were reported and the .alpha.-helical content, calculated from the ellipticity at 222 nm, was .apprx. 40%. The molecule bound O2 co-operatively with a maximum value of the Hili coefficient, h, of 3.9. Over the pH range 7.0-8.0 there was a positive Bohr effect.

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