Analysis of Cibacron blue F3G-A interaction with therapeutic proteins by MALDI-TOF mass spectrometry

The formation of the complexes between Cibacron blue F3G-A and two therapeutic proteins, recombinant human interferon-alpha2b and recombinant human growth hormone, was investigated. The method of time-resolved limited proteolysis coupled with MALDI-TOF mass spectrometry was used. The analysis of peptide maps revealed that A(17)HR(19) and L(20)HQLAFDTYQEFEEAYIPK(38) of hGH, and R(14)TLMLLAQMR(23) and D(33)RHDFGFPQEEFGNQFQK(50) of hIFN-alpha2b, exhibit affinity to Cibacron blue F3G-A. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Comparative studies of recombinant human granulocyte-colony stimulating factor, its Ser-17 and (His)6-tagged forms interaction with metal ions by means of immobilized metal ion affinity partitioning. Effect of chelated nickel and mercuric ions on extraction and refolding of proteins from inclusion bodies

The chelation capability of the reactive dye Light Resistant Yellow 2KT towards metal ions, particularly mercury(II) was evaluated in the pH range 5.0-7.0, and it was shown that the dye-Hg(II) complex has a free site for the interaction with human recombinant granulocyte-colony stimulating factor (rhG-CSF) from Escherichia coli. Affinity partitioning of three rhG-CSF forms--native, rhG-CSF[Cys17--->Ser17] and (His)6-rhG-CSF was studied in aqueous two-phase systems, which contained metal ions--Cu(II), Ni(II) and Hg(II)--chelated by dye-poly(ethylene glycol) at pH 5.0 and 7.0, in the presence or absence of many selected agents. It was determined, that chelated Ni(II) ions exhibited stronger interaction with the hexahistidine-tagged protein form, while the extraction power of Cu(II) ions was found to be of comparable order of magnitude for all three protein forms at pH 7.0. A comparative study of rhG-CSF and both its forms partitioning in the presence of chelated Hg(II) ions at pH 7.0 and 5.0 revealed possible direct interaction between Hg(II) ions and unpaired Cys-17 of rhG-CSF. The partitioning of three rhG-CSF forms inclusion body extract was studied in the presence of chelated Ni(II) and Hg(II) ions thus explaining the efficiency of targeted proteins renaturation gained upon their inclusion body forms interactions with chelated metal ions.     

Examination of dye-protein interaction by gel-permeation chromatography

The interaction of Cibacron blue F3G-A with two therapeutic proteins, recombinant human growth hormone and recombinant human interferon-alpha2b, has been examined by applying gel-permeation chromatography in combination with the absorption difference spectroscopy. The complexes of these proteins with Cibacron blue F3G-A have been isolated, and their absorbance spectra have been registered. The influence of Cibacron blue F3G-A on the oligomeric state of proteins has been investigated. It was found that Cibacron blue F3G-A promotes the generation of interferon-alpha2b dimers at pH 5.0.     

Interaction of serum albumin with vinyl sulfonate azo dye

The interaction between bovine serum albumin and the mono azo reactive dye Orange ZT has been investigated using absorption difference spectroscopy. The influence of pH and ionic strength of the solution on the stability of the dye-protein complex has been determined. At 25°C, the complex dissociation constants were equal to 24.0, 28.0, 7.0, 11.0, 17.6 and 46.0 μM at pH 7.0, 6.5, 6.0, 5.5, 5.0 and 4.3, respectively. In the presence of 0.1, 0.2, 0.3 M KCl, at pH 6.0 and 25°C, the complex dissociation constants were 8.8, 20.0, 18.0 μM, respectively. The protein-dye complex dissociation constants show that Orange ZT could be used as an affinity ligand for protein purification.      

Determination of the Dissociation Constant and Stoichiometry of a Complex of the Protein Interferon α-2b with Cibacron Blue F3G-A

The nonlinear least-squares technique was used for determining the complexation parameters of the recombinant human cytokine interferon -2b with a pseudobiospecific ligand, Cibacron Blue F3G-A. The dissociation constant of the complex and the number of ligand–protein binding sites were found.     

Chelated mercury as a ligand in immobilized metal ion affinity chromatography of proteins

Chelation of mercuric ions by an iminodiacetate-Sepharose gel was evaluated. The retentive properties of iminodiacetate-Sepharose gel column was studied towards proteins varying the composition of eluting systems from 2-mercaptoethanol to NaCl and imidazole, determining also the extent of mercury leaching. It was demonstrated that chelated mercury contained free sites for interaction with proteins such as bromelain and recombinant human granulocyte colony stimulating factor from E. coli. The extraction of the latter by chromatography of its inclusion bodies solution on Hg(II)-loaded Sepharose-iminodiacetate gel was also evaluated.