Denaturation Mechanism of BSA by Urea Derivatives: Evidence for Hydrogen-Bonding Mode from Fluorescence Tools

Urea and alkyl urea derivatives, which posses a free N-H moiety in the urea molecular framework is responsible for the fluorescence quenching of BSA. Fluorescence quenching accompanied with a blue initially and subsequently a red shift in the emission maximum of BSA is observed on the addition of urea derivatives containing N-H moieties. On the contrary, a fluorescence enhancement accompanied with a shift in the emission maximum towards the blue region is observed on the addition of tetramethylurea (TMU). Urea derivatives, which posses a free N-H moiety acts as a perfect denaturant by direct hydrogen-bonding interaction with BSA resulting in the unfolding process. The unfolding of the buried tryptophan moieties to the aqueous phase does not occur, when all the N-H moieties in the urea are methyl substituted (TMU). Fluorescence spectral techniques reveal that the direct hydrogen-bonding interaction of the N-H moiety of urea molecular framework with the carbonyl oxygen moieties of BSA results in the unfolding of the tryptophan moieties to the aqueous phase, while that of the carbonyl oxygen of urea with the N-H moieties of BSA is definitely not involved in the denaturation process. Steady state and time-resolved fluorescence studies illustrate that the extent of protein folding occurs at a relatively lower concentration of unsymmetrical alkyl urea derivatives (butyl urea (BU) and ethyl urea (EU)), compared to that of urea.