Abstract
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]
) and difference absorption coefficient at 406nm (Δε
) 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]
and Δε
) suggests that folding/unfolding of TIM and β-subunit proteins is a two-state process. Denaturation curves of [θ]
and Δε
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.