KEYHOLE LIMPET HEMOCYANIN (KLH) - PURIFICATION OF INTACT KLH1 THROUGHSELECTIVE DISSOCIATION OF KLH2

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 s taining-carbon film (NS-CF) procedure and in solution by ammonium moly bdate-PEG solutions at slightly acidic pHs. The KLH2 multidecamers spl it apart in the pH range 7.5-6.5 and in the pH range 6.5-6.0 the indiv idual decamers break open and start to dissociate. At pH 5.9 the disso ciation 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 d idecamer remains stable. Separation of intact KLH1, in the form of did ecamers and small clusters of didecamers, from dissociated KLH2 has be en achieved by gel filtration chromatography in the presence of ammoni um molybdate-PEG at pH 5.9 (pH 6.2 at 4 degrees C). Assessment of the achieved separation of KLH1 and KLH2 was monitored by gel electrophore sis under native conditions and by electron microscopy of negatively s tained 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-su pplemented stabilizing buffer at pK 7.2 and resulted in multidecamers and slightly smaller diameter helical tubes.