Acid-induced unfolding of flounder hemoglobin: evidence for a molten globular state with enhanced pro-oxidative activity.

The acid-induced unfolding of flounder oxyhemoglobin was investigated and the effect on pro-oxidative activity assessed. Hemoglobin exhibited multistep unfolding transitions as pH was lowered, with the major transition between pH 3.5 and 4 5. The protein was maximally acid-unfolded (but not fully unfolded) at approximately pH 2.5, and further titration with HCl led to a partially refolded protein due to a stabilizing effect of Cl(-) anions. At low pH, the protein retained a sizable amount of secondary structure and had increased ANS binding, suggesting a molten globular form at low pH. Dramatic changes in the heme environment occurred concurrently with the changes in protein conformation. These changes resulted in an enhancement in the pro-oxidative activity of the protein. The results show that an increase in flounder hemoglobin pro-oxidation was correlated with the extent of its unfolding, and they provide useful insight into what may occur with hemoglobin in processes where highly acidic conditions are employed.