Keyhole limpet hemocyanin (KLH), II: Characteristic reassociation properties of purified KLH1 and KLH2

Abstract

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° 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 have also been prepared by platinum-carbon shadowing, following freeze-cleavage. The two hemocyanins reassociate to produce characteristic oligomeric and polymeric forms. Subunits of purified KLH1 reassociate to produce a small number of didecamers, short multidecamers (ca 33 nm diameter) and much larger quantity of a ca 25 nm diameter flexible/undulatory tubular form of varying length. These tubules exhibit characteristic oblique features, indicative of an ‘open’ helical structure which appears to be a loosly or incompletely annealed twisted ribbon of subunits. After a period of days the tubules aggregate in parallel to produce 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 unstable; 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 reassociate to produce ca 25 nm diameter ‘closed’ tubules, which do not exhibit the oblique ‘open’ features shown by the KLH1 tubules; however, the ends of these ‘closed’ tubule are often oblique. In addition to the tubular form, KLH2 reassociation also generates a somewhat small proportion of ca 33 nm diameter multidecamers, often containing many decamers and more than one ‘nucleating’ didecamer. On transfer to low calcium 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 multidecamers (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 surface, indicating internalization of subunit domains that are not required for the construction of the tubular wall, in accord with current understanding of the subunit organization within the native molecules.