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The reassociation of Lumbricus terrestris hemoglobin dissociated at alkaline pH
Abstract:
The reassociation of the extracellular hemoglobin of Lumbricus terrestris (Mr approximately 3.9 X 10(6) ) at neutral pH, subsequent to its dissociation at pH above 8.0, was examined using gel filtration, ultracentrifugation, and scanning transmission electron microscopy. Gel filtration on Sephacryl S-200 at pH 6.8 of the hemoglobin exposed to pH above 8 showed the presence of four peaks: Ia, consisting of whole molecules, undissociated and reassociated, and smaller heme-containing fragments Ib (Mr approximately 3.0 X 10(5) ), II (Mr approximately 6.5 X 10(4) ), and III (Mr approximately 1.8 X 10(4) ). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that although the pattern of Ia was identical with that of the native hemoglobin, consisting of six polypeptide chains (I-VI), Ib appeared to have less of chains V and VI; II consisted of polypeptide chains II-VI, and III was identified as chain I. Lumbricus hemoglobin exposed to pH over the range 8.4 to 10.2 was subjected to gel filtration on Sepharose CL-6B and resolved into undissociated and dissociated fractions. The combined dissociated fractions, when brought back to pH 6.8 and subjected to gel filtration on Sepharose CL-6B and Sephacryl S-200, demonstrated that the extent of reassociation into whole molecules (IaR) varied from about 50% at pH 8 to 30% at pH 10.2. IaR possessed a sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern identical with that of the native hemoglobin. Ia, IaR, and Ib dissociated when exposed to alkaline pH; upon return to neutral pH, both Ia and IaR reassociated partially to whole molecules but Ib did not. These results suggest that reassociation comprises two pathways: one leading to the formation of IaR and a "dead-end" pathway, along which reassociation stops at the level of Ib. Digital image processing of scanning transmission electron micrographs of negatively stained native hemoglobin and IaR showed that the two molecules were very similar.