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Updated: Jul 29, 2026
Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis
Published on: October 28, 2016
Re-examining the oligomerization state of macrophage migration inhibitory factor (MIF) in solution
John S Philo1, Tzung-Horng Yang, Michael LaBarre
1Alliance Protein Laboratories, 3957 Corte Cancion, Thousand Oaks, CA 91360, USA. jphilo@mailway.com
Abstract:
The state of oligomerization of macrophage migration inhibitory factor (MIF, also known as glycosylation inhibiting factor, GIF) in solution has been variously reported as monomer, dimer, trimer, or mixtures of all three. Several crystal structures show MIF to be a trimer. Sedimentation velocity shows a recombinant human MIF sample is quite homogeneous, with 98% as a species with s(20,w)=3.07 S and D(20,w)=8.29 x 10(-7) cm(2)/s. Using the partial specific volume calculated from the amino acid composition these values imply a mass of 33.56 kDa, well above that of dimer, but also 9% below the trimer mass of 37.035 kDa. Sedimentation equilibrium data at loading concentrations from 0.01 to 1 mg/ml show unequivocally that the self-association is extremely tight. However, the apparent mass is 33.53 kDa [95% confidence 33.25-33.82], again 9% below that expected for 100% trimer. To examine the possibility this protein has an unusual partial specific volume, sedimentation equilibrium was also done in H(2)O/D(2)O mixtures, giving 0.765+/-0.017 ml/g rather than the calculated 0.735 ml/g. With this revised partial specific volume, the equilibrium and velocity data each give M=37.9+/-2.8 kDa, fully consistent with a strongly-associated trimeric quaternary structure.
Insights
Macrophage migration inhibitory factor (MIF) forms stable trimers in solution. Sedimentation studies, using a revised partial specific volume, confirm MIF
Area of Science:
- Biochemistry
- Structural Biology
- Protein Chemistry
Background:
- The oligomeric state of macrophage migration inhibitory factor (MIF), also known as glycosylation inhibiting factor (GIF), in solution remains debated, with reports of monomer, dimer, and trimer forms.
- Crystal structures consistently depict MIF as a trimer, yet solution studies have yielded conflicting results regarding its quaternary structure.
Purpose of the Study:
- To definitively determine the oligomeric state and quaternary structure of recombinant human MIF in solution.
- To reconcile discrepancies between crystallographic and solution-based data regarding MIF's oligomerization.
Main Methods:
- Sedimentation velocity analytical ultracentrifugation to assess homogeneity and estimate molecular mass.
- Sedimentation equilibrium analytical ultracentrifugation to investigate self-association and determine molecular mass under equilibrium conditions.
- Utilizing H2O/D2O mixtures in sedimentation equilibrium to accurately determine the partial specific volume of MIF.
Main Results:
- Sedimentation velocity indicated a homogeneous sample, but calculated mass was inconsistent with a trimer.
- Sedimentation equilibrium data suggested tight self-association, but the apparent mass was consistently lower than expected for a trimer.
- Revised partial specific volume, determined using H2O/D2O mixtures, resolved the mass discrepancy, yielding values consistent with a trimer.
Conclusions:
- Macrophage migration inhibitory factor (MIF) exhibits a strongly associated trimeric quaternary structure in solution.
- Accurate determination of partial specific volume is critical for interpreting sedimentation data of MIF.
- The findings resolve previous ambiguities and confirm the trimeric state of MIF in solution, aligning with crystallographic data.
