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

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Abstract

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.

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Acknowledgements

Financial support by DST-IRHPA and UGC-INNOVATIVE Programme is acknowledged. R.K thanks the UGC for providing the financial assistance.

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Authors and Affiliations

  1. National Centre for Ultrafast Processes, University of Madras, Taramani Campus, Chennai, 600 113, India

    R. Kumaran & P. Ramamurthy

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  1. R. Kumaran
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  2. P. Ramamurthy
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Correspondence to P. Ramamurthy.

Electronic supplementary material

Below is the link to the electronic supplementary material.

S1

Absorption spectra of BSA as a function of [DMU]. (1) BSA alone, (2) BSA + DMU 1.0 M, (3) BSA + DMU 2.0 M, (4) BSA + DMU 4.0 M. (JPEG 63 kb)

S2

Absorption spectra of BSA as a function of [TMU]. (1) BSA alone, (2) BSA + TMU 0.2 M, (3) BSA + TMU 0.4 M, (4) BSA + TMU 0.8 M. (JPEG 66 kb)

S3

Absorption spectra of BSA as a function of [EU]. (1) BSA alone, (2) BSA + EU 0.5 M, (3) BSA + EU 1.0 M, (4) BSA + EU 2.0 M. (JPEG 64 kb)

S4

Absorption spectra of BSA as a function of [BU]. (1) BSA alone, (2) BSA + BU 0.5 M, (3) BSA + BU 1.0 M, (4) BSA + BU 2.0 M. (JPEG 64 kb)

S5

Emission spectra of BSA in the absence and presence of [DMU]. λex 295 nm. (1) 0.0 M, (2) 1.0 M, (3) 2.0 M, (4) 4.0 M, (5) 5.0 M, (6) 6.0 M, (7) 7.0 M, (8) 8.0 M. (JPEG 82 kb)

S6

3D Contour spectral studies of BSA with urea and symmetrical alkyl urea derivatives in aqueous solution. Excitation wavelength scan: 200–500 nm. Emission wavelength scan: 300–600 nm. a) BSA, b) BSA + Urea 1.2 M, c) BSA + DMU 1.2 M, d) BSA + TMU 1.2 M. (JPEG 171 kb)

S7

3D Contour spectral studies of BSA with unsymmetrical alkyl urea derivatives in aqueous solution. Excitation wavelength scan: 200–500 nm. Emission wavelength scan: 300–600 nm. a) BSA + MU 1.2 M, b) BSA + EU 1.2 M, c) BSA + BU 1.2 M. (JPEG 159 kb)

S8

Fluorescence decay of BSA in the absence and presence of [EU]. (1) Laser Profile, (2) 0.0 M, (3) 0.6 M, (4)1.2 M, (5) 2.4 M, (6) 4.8 M. (JPEG 79 kb)

S9

Fluorescence decay of BSA in the absence and presence of BU. (1) Laser Profile, (2) 0.0 M, (3) 0.6 M, (4)1.2 M, (5) 2.4 M,(6) 4.8 M. (JPEG 86 kb)

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Kumaran, R., Ramamurthy, P. Denaturation Mechanism of BSA by Urea Derivatives: Evidence for Hydrogen-Bonding Mode from Fluorescence Tools. J Fluoresc 21, 1499–1508 (2011). https://doi.org/10.1007/s10895-011-0836-0

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