Sedimentation equilibrium measurements of recombinant DNA-derived human interferon .gamma.

Abstract

Recombinant DNA derived human interferon gamma (IFN-gamma) from Escherichia coli was examined by equilibrium ultracentrifugation. Short-column equilibrium experiments at pH 6.9 in 0.1 M ammonium acetate buffer gave a z-average molecular weight of 33,500 +/- 1400 at infinite dilution, corresponding to 1.98 +/- 0.08 times the formula weight. Long- (2.6 mm) column experiments at pH 7.5 in 0.04 M imidazole buffer gave a molecular weight of 33,400 +/- 500. Under the latter conditions IFN-gamma behaves somewhat nonideally, with the departure from ideality accounted for by an effective (Donnan) charge of about 6+. No association of this dimer to form tetramer or higher polymers was observed, with the association constant for formation of tetramer from dimer K24 found to be less than 34 L mol-1. Similarly, no dissociation to monomers was observable, with the dissociation constant to monomer K21 being less than 5 X 10(-8) mol L-1. At pH 3.55 in 0.02 M buffer (acetate plus acetic acid), there was virtually complete dissociation of the dimer to monomer. Extreme nonideality was seen in this low ionic strength system, and the effective charge on the protein was estimated to be about 11+. The reduced molecular weight M(1 -upsilon rho) of the monomer was found to be about 4.09 +/- 0.20 kg mol-1; this corresponds to a molecular weight of 16,410 +/- 820, with the Scatchard definition of components. A small amount of a polymer with a molecular weight of about 0.5 X 10(6) was detected under these conditions.

Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition

“…Figure 3C shows the residuals with an rms error of 0.0056 fringes, which is within the usual noise of the optical system. (Some very low level of remaining apparently non-random noise is observed, but in our hands this is common for modeling interference SE data; the residual randomness and the rms errors compare well with those observed in other laboratories (48) or (49)). The consistency of the interference data with the absorbance data in Figure 1 supports the validity of the algebraic signal decomposition.…”

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

“…It has been shown previously that rhIFN-g undergoes aggregation in buffers undergoing acidi®cation during lyophilization. 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.…”

A transient expansion of the native state precedes aggregation of recombinant human interferon-γ

“…This is in remarkable agreement with the surface tension treatment, which predicted an expansion to a diameter of 3.86 nm. Because native rhIFN-␥ exists as a dimer (K d is Ͻ50 nM) (38), it might be expected that the surface area increase preceding aggregation may involve a dimer-monomer transition. However, based on the large apparent surface area of the dimer interface (35 to 40% of the total surface area of the dimer), it appears that the dimeric nature of the protein remains intact before aggregation.…”