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 la through 1h) and determined their sequential arrangement, The KLH1 domains differ biochemically and immunologically from each other and from the previously characterized seven domains of KLH2 (termed 2a through 2g). Our partial amino acid sequences suggest that a domain, equivalent to the C-terminal domain Ih, is missing in KLH2. This deficiency is believed to be genuine and not an artifact of the subunit preparation procedure, since STEM measurements of the native didecamers yielded a mass difference of about 800 kDa between KLH1 and KLH2 (8.3 MDa versus 7.5 MDa), correlating with 20 copies of a functional th domain. It was also shown that the KLH1 didecamer can be rapidly split (minutes) into on almost homogeneous population of stable decamers by increasing the pH of the Tris/saline stabilizing buffer (routinely pH 7.4), which contains 5 mM CaCl2 and 5 mM MgCl2, to pH 8.5. Reformation of the didecamers occurred mon slowly (days) upon dialysis against the pH 7.4 stabilizing buffer. Addition of 100 mM calcium and 100 mM magnesium ions to the FH 7.4 stabilizing buffer leads to the more rapid (overnight) formation of didecamers together with a significant number of previously unobserved KLH1 multidecamers, which could be structurally distinguished from the established multidecamers of KLH2.