6533b85bfe1ef96bd12bbded

RESEARCH PRODUCT

Keyhole limpet haemocyanin (KLH): Purification of intact KLH1 through selective dissociation of KLH2

subject

description

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 form of didecamers and small clusters of PEG at pH 5.9 (pH 6.2 at 4°C). Assessment of the achieved separation of KLH1 and KLH2 was monitored by gel electrophoresis under native conditions and by electron microscopy of negatively stained specimens. Integrity of the multi-domain KLH1 and KLH2 subunits was assessed by SDS-PAGE. The significance of this separation for the overall understanding of the two types of KLH is that it provides for the first time purified KLH1 didecamers. Reassociation of the pH 5.9 dissociated KLH2 was achieved by prolonged dialysis against calcium-supplemented stabilizing buffer at pH 7.2 and resulted in multidecamers and slightly smaller diameter helical tubes.