Affinity mode of capillary isoelectric focusing for the characterization of the biotin-binding protein actinavidin.
Different modes of capillary isoelectric focusing (CIEF) with salt mobilization and zwitterionic additive to cathodic mobilizer were applied to characterize the biotin-binding protein actinavidin and its affinity properties. The analysis is performed in a neutral coated capillary with completely eliminated electroosmotic flow. Synthetic pI standards with absorption in the visible region were used in all runs and pI determinations were based on a fitted nonlinear calibration graph. CIEF of highly purified actinavidin in native conditions was revealed in about 12 peaks in pI range 6.2-7.5, with three major forms having pI 6.80, 6.86, 6.90. CIEF of a mixture of actinavidin and increasing concentrations of two affinity ligands (biotin and biotinylated oligonucleotide) demonstrated drastic changes in the number of protein isoforms. In the latter case it resulted in only one peak (pI 5.05) when the ligand was in excess. This method, which can be named affinity CIEF, was found to be well-suited for studying structural changes, occurring in receptor proteins upon binding with a ligand. CIEF of the protein, performed under denaturing conditions (6 M urea in a solution of carrier ampholytes) is also reported. It was revealed in three isoforms with a pI more acidic than that of native actinavidin. It is demonstrated that careful selection of denaturing conditions was necessary for the reproducible results.
Okun
V M
VM
Institute of Analytical Instrumentation, Russian Academy of Science, St. Petersburg. vadim@def.mail.iephb.ru
D000345:Affinity Labels; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D001710:Biotin / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D007525:Isoelectric Focusing / Q000379:methods*; D007526:Isoelectric Point; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011489:Protein Denaturation; D013302:Streptomyces / Q000737:chemistry
D000345:Affinity Labels; D001426:Bacterial Proteins; D002352:Carrier Proteins; D009838:Oligodeoxyribonucleotides; C017071:biotin-binding proteins; D001710:Biotin