KEYHOLE LIMPET HEMOCYANIN (KLH) .2. 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 polyacryla mide gel electrophoresis from Immucothel(R), have been used for macrom olecular reassociation studies. In-vitro reassociation has been achiev ed with a standardized system using a Tris-saline stabilizing buffer a t pH 7.4 containing 100 mM calcium and magnesium chloride at 4 degrees C. The relatively slow progress of reassociation has been monitored a nd the varying oligomeric forms of KLH1 and KLH2 produced have been st udied by transmission electron microscopy, using specimens negatively stained with 5% ammonium molybdate containing 1% trehalose. Specimens have also been prepared by platinum-carbon shadowing, following freeze -cleavage. The two hemocyanins reassociate to produce characteristic o ligomeric and polymeric forms. Subunits of purified KLH1 reassociate t o produce a small number of didecamers, short multidecamers (ca 33 nm diameter) and much larger quantity of a ca 25 nm diameter flexible/und ulatory tubular form of varying length. These tubules exhibit characte ristic oblique features, indicative of an 'open' helical structure whi ch appears to be a loosly or incompletely annealed twisted ribbon of s ubunits. After a period of days the tubules aggregate in parallel to p roduce large paracrystalline bundles, which do not have a tendency to associate end-to-end. Following transfer of this reassociated KLH1 to low calcium magnesium stabilizing buffer, the tubular bundles are unst able; they slowly break down into shorter lengths, fragments, subunit groups and individual subunits, which subsequently regenerate decamers , didecamers and some multidecamers. Subunits of purified KLH2 reassoc iate to produce ca 25 nm diameter 'closed' tubules, which do not exhib it the oblique 'open' features shown by the KLH1 tubules; however, the ends of these 'closed' tubules are often oblique. In addition to the tubular form. KLH2, reassociation also generates a somewhat small prop ortion of ra 33 nm diameter multidecamers, often containing many decam ers and more than one 'nucleating' didecamer. On transfer to low calci um magnesium stabilizing buffer the KLH2 tubules are remarkably stable , but the number of multidecamers slowly increases with time. There is a significant structural difference between the short KLH1 multidecam ers (only detected following in-vitro reassociation) and those of KLH2 quite apart from their length. Study of the metal shadowed specimens confirmed the difference between the KLH1 and KLH2 tubular forms, with relatively smooth helical surface ridges and a rougher internal surfa ce, indicating internalization of subunit domains that are not require d for the construction of the tubular wall, in accord with current und erstanding of the subunit organization within the native molecules. (C ) 1997 Elsevier Science Ltd.