Hemoglobin Interactions with αB Crystallin: A Direct Test of Sensitivity to Protein Instability

As a small stress response protein, human alpha B crystallin, detects protein destabilization that can alter structure and function to cause self assembly of fibrils or aggregates in diseases of aging. The sensitivity of alpha B crystallin to protein instability was evaluated using wild-type hemoglobin (HbA) and hemoglobin S (HbS), the glutamate-6-valine mutant that forms elongated, filamentous aggregates in sickling red blood cells. The progressive thermal unfolding and aggregation of HbA and HbS in solution at 37 degrees C, 50 degrees C and 55 degrees C was measured as increased light scattering. UV circular dichroism (UVCD) was used to evaluate conformational changes in HbA and HbS with time at the selected temperatures. The changes in interactions between alpha B crystallin and HbA or HbS with temperature were analyzed using differential centrifugation and SDS PAGE at 37 degrees C, 50 degrees C and 55 degrees C. After only 5 minutes at the selected temperatures, differences in the aggregation or conformation of HbA and HbS were not observed, but alpha B crystallin bound approximately 6% and 25% more HbS than HbA at 37 degrees C, and 50 degrees C respectively. The results confirmed (a) the remarkable sensitivity of alpha B crystallin to structural instabilities at the very earliest stages of thermal unfolding and (b) an ability to distinguish the self assembling mutant form of HbS from the wild type HbA in solution.