0000000000656951
AUTHOR
Sabine M. Söhngen
Mass determination, subunit organization and control of oligomerization states of keyhole limpet hemocyanin (KLH).
Analytical dark-field scanning transmission electron microscopy (STEM) of freeze-dried unstained specimens of keyhole limpet hemocyanin (KLH; from Megathura crenulata, a prosobranch gastropod) gave a molecular mass of 400 kDa for the subunit of KLH1 and of 345 kDa for the subunit of KLH2, which confirms our published values from SDS/PAGE. Within the 400-kDa KLH1 subunit we identified, by limited proteolysis, isolation of fragments and N-terminal sequencing, eight distinct 45-60 kDa functional domains (termed 1a through 1h) and determined their sequential arrangement. The KLH1 domains differ biochemically and immunologically from each other and from the previously characterized seven domains…
Keyhole limpet hemocyanin (KLH), II: Characteristic reassociation properties of purified KLH1 and KLH2.
Subunits of the two types of keyhole limpet hemocyanin (KLH1 and KLH2), purified by gel filtration chromatography and preparative polyacrylamide gel electrophoresis from Immucothel, have been used for macromolecular reassociation studies. In-vitro reassociation has been achieved with a standardized system using a Tris-saline stabilizing buffer at pH 7.4 containing 100 mM calcium and magnesium chloride at 4 degrees C. The relatively slow progress of reassociation has been monitored and the varying oligomeric forms of KLH1 and KLH2 produced have been studied by transmission electron microscopy, using specimens negatively stained with 5% ammonium molybdate containing 1% trehalose. Specimens ha…
Keyhole limpet haemocyanin (KLH): Purification of intact KLH1 through selective dissociation of KLH2
Abstract Keyhole limpet haemocyanin (KLH) from almost all newly captive animals contains a mixture of KLH1 and KLH2. We show that the dissociation of KLH2 can be produced during EM specimen preparation by the negative staining-carbon film (NS-CF) procedure and in solution by ammonium molybdate-PEG solutions at slightly acidic pHs. The KLH2 multidecamers split apart in the pH range 7.5-6.5 and in the pH range 6.5-6.0 the individual decamers break open and start to dissociate. At pH 5.9 the dissociation of KLH2 yields predominantly a mixture of single subunits and what appear to be subunit dimers. Over the pH range 7.0-5.7 the KLH1 didecamer remains stable. Separation of intact KLH1, in the f…