Human aldose reductase unfolds through an intermediate. [version 1; peer review: 3 approved with reservations]

Background: Human aldose reductase (hAR) converts glucose to sorbitol under hyperglycemic conditions. Aldose reductase is first and rate limiting enzyme of polyol pathway. Under hyperglycemia, increased flux of glucose through this pathway has been implicated in development of secondary complication in diabetes. Due to this clinical implication, aldose reductase attracted considerable attention from drug discovery perspective. In spite of extensive characterization of the biochemical and structural context, little is known about the unfolding behavior of aldose reductase. This study reports equilibrium unfolding studies of human aldose reductase. Methods: We carried out thermal and chemical induced equilibrium unfolding studies of human aldose reductase monitored by circular dichroism and tryptophan and ANS fluorescence spectroscopy. Results: Thermal unfolding studies present a classical picture of two state unfolding from native to unfolded state. The data was used to derive thermodynamic parameters and study thermostability of aldose reductase. Urea and GuHCl induced equilibrium unfolding studies led us to discover an intermediate state, which gets populated at 3.5-4.0 M and 0.7-2 M of urea and GuHCl, respectively. Thermodynamic parameters from chemical induced unfolding are in agreement with those obtained from thermal unfolding. Conclusion: This study revealed that aldose reductase unfolds from native to unfolded state via an intermediate. Assessment of thermodynamic stability of native, intermediate and unfolded state shows that three states are separated by significant energy barriers that ensure cooperativity of unfolding. As hAR functions in cells which are under osmotic and oxidative stress, these in vitro findings may have implications for its native conformation under physiological state.