Carboxylic acids of different nature induces aggregation of hemoglobin

Misfolded proteins that escape cellular quality control check lay the foundation for several progressively widespread neurodegenerative diseases, diabetes and others. Here, crotonic and citric acid are employed to study aggregation behaviour of hemoglobin (Hb). A systematic investigation on varying concentrations of acids from 0 to 60 mM on Hb gives an idea that transition is taking place in the vicinity of 10-30 mM. Hb showed increased intrinsic Trp fluorescence in the presence of both acids. A red shift of 10 nm in presence of citric acid contrary to a blue shift of 5 nm in presence of crotonic acid is observed. ANS and ThT fluorescence marked aggregation at 50 mM, supported by Congo red and Soret absorbance spectroscopy. CD, RLS and DLS studies also validate the findings. Molecular docking analysis exhibited the binding mode of Hb with acids. Aggregates were dense, beaded structure as visualised under TEM. Crotonic and citric acid at 20 and 30 mM, respectively, induced structural changes in Hb which transmutes to aggregate at higher concentration. These alterations remained almost constant and no significant changes were observed on increasing concentration further. Also, crotonic acid is more noxious, as it instigates conformational alterations at lower concentration than citric acid.