Dissociation Equilibrium of Human Recombinant Interferon γ

Abstract

The biologically active form of interferon gamma is a dimer composed of two noncovalently bound identical polypeptide chains of 17 kDa each. In this study, it was found that dissociation of the dimer into monomers significantly reduced the fluorescence quantum yield and the efficiency of the intermolecular Tyr to Trp radiationless energy transfer. The same process caused significant changes in the fluorescence decay and in the fluorescence anisotropy decay. The kinetic and thermodynamic parameters of the dimer-monomer equilibrium were determined by fluorescence measurements at different temperatures and by a theoretical mathematical model. Dissociation of the dimers into monomers was an endothermic process and was favored by concentrations of the protein lower than 1 microM and by increasing the temperature. It was accompanied by formation of aggregates, a slow and partially reversible process leading to inactivation of the interferon. It is suggested that certain monomeric conformers are competent for aggregation.

A new mechanism for decreasing aggregation of recombinant human interferon‐γ by a surfactant: Slowed dissolution of lyophilized formulations in a solution containing 0.03% polysorbate 20

“…40 rhIFN-g dissociates into monomers at low pH, 7,12 and the monomeric species has been linked to aggregation of the protein. 28,41,42 However, the magnitudes of the pH changes in this study did not correlate to aggregation levels after reconstitution. In fact, the commercial product is formulated at pH 5.0, though a buffer with low ionic strength (5 mM sodium succinate) is used.…”

Monitoring of Salt Tolerance Responses of Different Maize Inbred Lines Using Protein Profile and Protein Spectrofluorescence

“…4). This fluorescent emission value is very close to the emission peak of tryptophan amino acid (Boteva et al, 1996;Kabiri et al, 2012). The tryptophan residue in protein has emission spectrum a ranged from 310~350 nm should reflect the average environment of the tryptophan.…”

Conformational Stability of Human Interferon‐Gamma on Association with and Dissociation from Liposomes

“…This result is in accord with previous studies. 21,38 The individual lifetime values of liposome-desorbed hIFN␥ were slightly increased and the contribution of the short lifetime was increased (p 1 ‫ס‬ 41.1%), as compared with untreated hIFN␥ in SBS or SBS/ SBN buffer (p 1 ‫ס‬ 15.8-16.2%). However, the confidence intervals of both the pre-exponential factors and the lifetimes overlapped with those of untreated hIFN␥, and the average lifetimes of all free hIFN␥ formulations were comparable ( Table 2).…”

“…20 Dissociation of the dimers into monomers is an endothermic process, favored by concentrations of the protein <1 M (17 g/mL) and increasing temperature. 21 Dissociation of the dimer at higher concentrations requires treatment with strong denaturants, such as guanidinium hydrochloride or sodium dodecyl sulfate, or reduction of the pH to <2. 22 IFN␥ has an isoelectric point of ∼10, as calculated from its amino acid content, contains no disulfide bonds, and has one tryptophan residue at position 36.…”