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John I. Clark, Scott A. Houck, Tyler J. Clark; αB Crystallin Interactions With Hemoglobin: A Direct Test of Sensitivity to Protein instability. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2280.
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Early detection of unstable proteins is critical for the protective function of the small stress protein, human αB crystallin. These experiments tested the hypothesis that αB crystallin recognition and binding occurs at the earliest stages of protein instability.
Human αB crystallin was prepared as described previously (Muchowski et al. 1997 J.Biol.Chem 272:2478). Sensitivity and binding were evaluated using wild type hemoglobin (HbA) and mutant sickle cell hemoglobin (HbS) which forms elongated filamentous aggregates in sickle cell disease. UVCD (ultraviolet circular dichroism) determined the change in conformation of HbA and HbS and light scattering measured aggregation at 37, 50 and 55ºC. Interactions between proteins at selected temperatures were analyzed using SDS-PAGE following centrifugation and filtration to separate soluble and insoluble constituents of mixtures of αB with HbA or HbS.
The similarity between the 3D structure of HbA and HbS was confirmed using UVCD. With increasing temperatures, HbS unfolded more readily than HbA and aggregated earlier. After only 5 minutes at the selected temperature, prior to significant changes in UVCD of HbA or HbS or increased light scattering, increased interactions were determined using SDS PAGE. For example, at 50ºC, recognition of thermal instability by αB crystallin was approximately 64% greater for HbS than for HbA.
The results determined experimentally that human αB crystallin is remarkably sensitive to very early stages of thermal destabilization of proteins in the absence of compromised conformation.
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