Purification, preliminary X-ray crystallography and biophysical studies of triose phosphate isomerase-β-globin subunit complex.

Triose phosphate isomerase (TIM) is a cytoplasmic enzyme of prime importance in the mammalian glycolytic pathway. It has a major role in the conversion of dihydroxyacetone phosphate into glyceraldehyde-3-phosphate. We have successfully purified a stable complex of TIM with β-globin subunit from the sheep kidney using a simple two-step chromatography procedure. It is seen for the first time that TIM is forming a stable complex with β-globin. The purified protein-protein complex was crystallized and preliminary diffraction data were collected at 2.1Å resolution. We further studied guanidinium chloride (GdmCl)-induced denaturation of TIM-β-globin complex by monitoring changes in the mean residue ellipticity at 222nm ([θ] ₂₂₂ ) and difference absorption coefficient at 406nm (Δε ₄₀₆ ) at pH 7.5 and 25°C. We have observed that GdmCl-induced denaturation is reversible. Coincidence of normalized transition curves of both physical properties ([θ] ₂₂₂ and Δε ₄₀₆ ) suggests that folding/unfolding of TIM and β-subunit proteins is a two-state process. Denaturation curves of [θ] ₂₂₂ and Δε ₄₀₆ were used to estimate the stability parameters of the protein-protein complex. This is the first report on the isolation, purification, crystallization and biophysical characterization of the naturally occurring complex of TIM with the β-globin subunit.
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