A CAPILLARY ELECTROPHORESIS METHOD FOR STUDYING APO, HOLO, RECOMBINANT, AND SUBUNIT DISSOCIATED FERRITINS

A capillary electrophoresis (CE) method is described for detecting and quantitating apo and holo ferritins from horse spleen (HoSF), rat liv er (RLF), recombinant human light chain (rLF), recombinant human heavy chain (rHF), site-directed variants of human light chain, and Azotoba cter vinelandii bacterial ferritin (AVBF). This procedure is carried o ut at pH 8.2, where the ferritin molecules are associated into their 2 4-mers. Protein mobilities as expressed as elution times were clearly resolved and could be used to distinguish one ferritin type from anoth er, providing a means for detecting and quantitating various ferritin species in purified or partially purified states. Measurements of thes e and other ferritins were also conducted at pH 2.0, where dissociatio n into their respective subunits occurs. For HoSF and RLF, the individ ual L and H subunits were resolved and their relative concentrations w ere determined by integrating the areas of the elution peaks. HoSF gav e 89.8% L and 10.2% H and RLF gave 70.7% L and 29.3% H, while rLF, rHF , and AVBF gave only a single subunit, all in agreement with reported values obtained by polyacrylamide gel electrophoresis. CE of HoSF, con taining increasing amounts of iron in the interior, in general, showed that protein mobilities increased, reached a plateau, and then slowly decreased with increasing core size, although buffer effects altered this CE behavior to some extent. Such results indicate that species fo rmed early during core formation have individual iron atoms present an d differ from those formed later in which the oligomeric iron core has formed. The binding of various metal ions to apo HoSF is readily dete rmined by CE and provides a means for studying metal ion-ferritin inte raction. Dramatic changes in the elution times were noted in different buffer systems, indicating that strong buffer interactions were occur ring during metal binding to HoSF. (C) 1994 Academic Press, Inc.