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 liver (RLF), recombinant human light chain (rLF), recombinant human heavy chain (rHF), site-directed variants of human light chain, and Azotobacter vinelandii bacterial ferritin (AVBF). This procedure is carried out at pH 8.2, where the ferritin molecules are associated into their 24-mers. Protein mobilities as expressed as elution times were clearly resolved and could be used to distinguish one ferritin type from another, providing a means for detecting and quantitating various ferritin species in purified or partially purified states. Measurements of these and other ferritins were also conducted at pH 2.0, where dissociation into their respective subunits occurs. For HoSF and RLF, the individual L and H subunits were resolved and their relative concentrations were determined by integrating the areas of the elution peaks. HoSF gave 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, containing 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 formed early during core formation have individual iron atoms present and differ from those formed later in which the oligomeric iron core has formed.(ABSTRACT TRUNCATED AT 250 WORDS)
Zhao
Z
Z
Department of Chemistry, Brigham Young University, Provo, Utah 84602.
Malik
A
A
Lee
M L
ML
Watt
G D
GD
D000818:Animals; D001052:Apoferritins / Q000737:chemistry*; D016948:Azotobacter vinelandii; D002197:Capillary Action; D002413:Cations, Divalent; D004586:Electrophoresis / Q000379:methods*; D005293:Ferritins / Q000031:analogs & derivatives / Q000737:chemistry*; D006736:Horses; D006801:Humans; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D015203:Reproducibility of Results
D002413:Cations, Divalent; D011994:Recombinant Proteins; D005293:Ferritins; D001052:Apoferritins