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.
Highlights
Lumbricus terrestris ( M r -3.9 X lo6)at neutral pH, of 12 pentagonal-shaped subunits [1, 2]
VI), Ib appeared to have less of chains V and VI; I1 globin dissociates at pHabove 8 into heme-containingspecies consisted of polypeptide chains11-VI, and I11was iden- with sedimentation coefficients of9-10 S, 3.5 S, and 2.3 S
The patterns of IaR and the native whether reassociation to whole molecules occurs when freshly hemoglobin were identical, and peak Ib again contained less prepared peaks Ib, 11, and 111 of Lumbricus hemoglobin are of chains V and VI; peak I1 consisted of polypeptide chains
Summary
8.4, and 0.05 M sodium tetraborate, 1mM EDTA, pH 8.7,9.0,9.3,9.6, be the undissociated whole molecules and thefractions corresponding and 10.2, was carried out using a column of Sepharose CL-GB Molecular weight standards for the Sephacryl S-200 column were: sociation of freshly prepared Lumbricus hemoglobin was estimated bovine serum albumin (66,000), ovalbumin (43,000), carbonic anhy- by carrying out thefollowing sequence of operations: gel filtration on drase (29,000), and sperm whale myoglobin [17,800]. The protein solutions were concentrated by ultrafiltration using leaving an area of 128 X 512 pixels on the right side of the monitor an Amicon UM05 membrane; 0.1 M sodium phosphate buffers were free for processing.
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