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Pressure- and Urea-Induced Denaturation of Bovine Serum Albumin: Considerations about Protein Heterogeneity

DOI: 10.4236/ojbiphy.2012.21002    6,466 Downloads   17,286 Views   Citations


Urea denatures proteins at different concentrations, depending on the experimental conditions and the protein. We in-vestigated the pressure-induced denaturation of bovine serum albumin (BSA) in the presence of subdenaturing concen-trations of urea based on a two-state equilibrium. Pressure-induced denaturation was enhanced at urea concentrations ([U]) of 3.5 M to 8.0 M, with the free energy of denaturation at atmospheric pressure ranging from +5.0 to –2.5 kJ/mol of BSA. The m values appeared to be biphasic, with m1 and m2 of 0.92 and 2.35 kJ mol–1?M–1, respectively. Plots of versus ln[U] yielded values of u, the apparent stoichiometric coefficient, of 1.68 and 6.67 mol of urea/mol of BSA for m1 and m2, respectively. These values were compared with the m and u values of other monomeric proteins reported in or calculated from the literature. The very low values of u systematically observed for proteins were suggestive of heterogeneity in the free energy of denaturation. Thus, a u value of 140 mol of urea/mol of BSA may indicate the existence of a heterogeneous molecular population with respect to the free energy of dena-turation.

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D. Norberto, J. Vieira, A. de Souza, J. Bispo and C. Bonafe, "Pressure- and Urea-Induced Denaturation of Bovine Serum Albumin: Considerations about Protein Heterogeneity," Open Journal of Biophysics, Vol. 2 No. 1, 2012, pp. 4-14. doi: 10.4236/ojbiphy.2012.21002.


[1] C. Camilloni, A. G. Rocco, I. Eberini, E. Gianazza, R. A. Broglia and G. Tiana, “Urea and Guanidinium Chloride Denature Protein L in Different Ways in Molecular Dynamics Simulations,” Biophysical Journal, Vol. 94, Mo. 12, 2008, pp. 4654-4661.
[2] K. L. Schweiker, V. W. Fitz and G. I. Makhatadze, “Universal Convergence of the Specific Volume Changes of Globular Proteins upon Unfolding,” Biochemistry, Vol. 48, No. 46, 2009, pp. 10846-10851. doi:10.1021/bi901220u
[3] C. F. S. Bonafe, C. M. R. Vital, R. C. B. Telles, M. C. Gon?alves, M. S. A. Matsuura, F. B. T. Pessine, D. R. C. Freitas and J. Vega, “Tobacco Mosaic Virus Disassembly by High Hydrostatic Pressure in Combination with Urea and Low Temperature,” Biochemistry, Vol. 37, No. 31, 1998, pp. 11097-11105. doi:10.1021/bi980349n
[4] J. L. R. Santos, R. Aparicio, I. Joekes, J. L. Silva, J. A. C. Bispo and C. F. S. Bonafe, “Different Urea Stoichiometries between the Dissociation and Denaturation of Tobacco Mosaic Virus as Probed by Hydrostatic Pressure,” Biophysical Chemistry, Vol. 134, No. 3, 2008, pp. 214-224. doi:10.1016/j.bpc.2008.02.010
[5] C. F. S. Bonafe, M. Glaser, E. W. Voss, G. Weber and J. L. Silva, “Virus Inactivation by Anilinonaphthalene Sulfonate Compounds and Comparison with Other Ligands,” Biochemical and Biophysical Research Communications, Vol. 275, No. 3, 2000, pp. 955-961. doi:10.1006/bbrc.2000.3402
[6] C. F. S. Bonafe, M. Villas-Boas, M. C. Suarez and J. L. Silva, “Reassembly of a Large Multisubunit Protein Promoted by Nonprotein Factors. Effects of Calcium and Glycerol on the Association of Extracellular Hemoglobin,” Journal Biological Chemistry, Vol. 266, 1991, pp. 13210-13216.
[7] J. L. R. Santos, J. A. C. Bispo, G. F. Landini and C. F. S. Bonafe, “Proton Dependence of Tobacco Mosaic Virus Dissociation by Pressure,” Biophysical Chemistry, Vol. 111, No. 1, 2004, pp. 53-61. doi:10.1016/j.bpc.2004.04.003
[8] J. A. C. Bispo, J. L. R. Santos, G. F. Landini, J. M. Gon?alves and C. F. S. Bonafe, “pH Dependence of the Dissociation of Multimeric Hemoglobin Probed by High Hydrostatic Pressure,” Biophysical Chemistry, Vol. 125, 2007, No. 2-3, pp. 341-349.
[9] R. Simpson and W. Kauzmann, “The Kinetics of Protein Denaturation, 1. The Behavior of the Optical Rotation of Ovalbumin in Urea Solutions,” Journal of the American Chemical Society, Vol. 75, 1953, pp. 5139-5152. doi:10.1021/ja01117a001
[10] C. Tanford, “Physical Chemistry of Macromolecules,” John Wiley & Sons, New York, 1961.
[11] J. Wyman, “Linked Functions and Reciprocal Effects in Hemoglobin—A Second Look,” Advances in Protein Chemistry, Vol. 19, 1964, pp. 223-286.
[12] J. A. Schellman, “Solvent Denaturation,” Biopolymers, Vol. 17, 1978, pp. 1305-1322. doi:10.1002/bip.1978.360170515
[13] J. A. Schellman, “Macromolecular Binding,” Biopolymers, Vol. 14, No. 5, 1975, pp. 999-1018. doi:10.1002/bip.1975.360140509
[14] I. V. Baskakov and D. W. Bolen, “Monitoring the Sizes of Denatured Ensembles of Staphylococcal Nuclease Proteins: Implications Regarding M Values, Intermediates, and Thermodynamics,” Biochemistry, Vol. 37, 1998, pp. 18010-18017. doi:10.1021/bi981849j
[15] Y. Kita, T. Arakawa, T. Y. Lin and S. N. Timasheff, “Contribution of the Surface Free Energy Perturbation to Protein-Solvent Interactions,” Biochemistry, Vol. 33, No. 50, 1994, pp. 15178-15189. doi:10.1021/bi00254a029
[16] M. Auton and D. W. Bolen, “Predicting the Energetics of Osmolyte-Induced Protein Folding/Unfolding,” Proceedings of the National Academy of Sciences, Vol. 102, No. 42, 2005, pp. 15065-15068. doi:10.1073/pnas.0507053102
[17] R. F. Greene and C. N. Pace, “Urea and Guanidine Hydrochloride Denaturation of Ribonuclease, lysozyme, a-Chymotrypsin, and β-Lactoglobulin,” Journal of Biological Chemistry, Vol. 249, No. 17, 1974, pp. 5388-5393.
[18] J. A. Schellman, “The Thermodynamic Stability of Proteins,” Annual Review of Biophysics and Biophysical Chemistry, Vol. 16, No. 1, 1987, pp. 115-137. doi:10.1146/
[19] A. A. Paladini and G. Weber, “Absolute Measurements of Fluorescence Polarization at High Pressures,” Review of Scientific Instruments, Vol. 52, No. 3, 1981, pp. 419-427. doi:10.1063/1.1136596
[20] J. A. Schellman, “Protein Stability in Mixed Solvents: A Balance of Contact Interaction and Excluded Volume,” Biophysical Journal, Vol. 85, No. 1, 2003, pp. 108-125. doi:10.1016/S0006-3495(03)74459-2
[21] E. F. Casassa and H. Eisenberg, “Thermodynamic Analysis of Multicomponent Solutions,” Advances in Protein Chemistry, Vol. 19, 1964, pp. 287-395. doi:10.1016/S0065-3233(08)60191-6
[22] E. P. Hade and C. Tanford, “Isopiestic Compositions as a Measure of Preferential Interactions of Macromolecules in Two-Component Solvents. Application to Proteins in Concentrated Aqueous Cesium Chloride and Guanidine Hydrochloride,” Journal of the American Chemical Society, USA, Vol. 89, No. 19, 1967, pp. 5034-5040. doi:10.1021/ja00995a036
[23] I. V. Baskakov and D. W. Bolen, “The Paradox between M Values and Delta Cp’S for Denaturation of Ribonuclease T1 with Disulfide Bonds Intact and Broken,” Protein Science, Vol. 8, No. 6, 1999, pp. 1314-1319. doi:10.1110/ps.8.6.1314
[24] P. Banerjee, S. Pramanik, A. Sarkar and S. C. Bhattacharya, “Deciphering the Fluorescence Resonance Energy Transfer Signature of 3-Pyrazolyl 2-Pyrazoline in Transport Proteinous Environment,” Journal of Physical Chemistry B, Vol. 113, No. 33, 2009, pp 11429-11436. doi:10.1021/jp811479r
[25] H. Wu, P. Wang, X. Hu, Z. Dai and X. Zou, “Site-Selective Probe for Investigating the Asynchronous Unfolding of Domains in Bovine Serum Albumin,” Talanta, Vol. 84, No. 3, 2011, pp. 881-886. doi:10.1016/j.talanta.2011.02.027
[26] R. Kumaran and P. Ramamurthy, “Denaturation Mechanism of Bsa by Urea Derivatives: Evidence for Hydrogen-Bonding Mode from Fluorescence Tools,” Journal of Fluorescence, Vol. 21, No. 4, 2011, pp. 1499-1508. doi:10.1007/s10895-011-0836-0
[27] D. Barrick, “What Have We Learned from the Studies of Two-State Folders, and What Are the Unanswered Questions about Two-State Protein Folding?” Physical Biology, Vol. 6, No. 1, 2009, pp. 1-9. doi:10.1088/1478-3975/6/1/015001
[28] D. Barrick, D. U. Ferreiro and E. A. Komives, “Folding Landscapes of Ankyrin Repeat Proteins: Experiments Meet Theory,” Current Opinion in Structural Biology, Vol. 18, No. 1, 2008, pp. 27-34. doi:10.1016/
[29] C. N. Pace, E. J. Hebert, K. L. Shaw, D. Schell, V. Both, D. Krajcikova, J. Sevcik, K. S. Wilson, Z. Dauter, R. W. Hartley and G. R. Grimsley, “Conformational Stability and Thermodynamics of Folding of Ribonucleases Sa, Sa2 and Sa3,” Journal of Molecular Biology, Vol. 279, No. 1, 1998, pp. 271-286. doi:10.1006/jmbi.1998.1760
[30] R. F. Latypov, T. S. Harvey, D. Liu, P. V. Bondarenko, T. Kohno, R. A. Fachini, R. D. Rosenfeld, R. R. Ketchem, D. N. Brems and A. A. Raibekas, “Biophysical Characterization of Structural Properties and Folding of Interleukin-1 Receptor Antagonist,” Journal of Molecular Biology, Vol. 368, No. 4, 2007, pp. 1187-1201. doi:10.1016/j.jmb.2007.02.031
[31] B. Farruggia and G. A. Picó, “Thermodynamic Features of the Chemical and Thermal Denaturations of Human Serum Albumin,” International Journal of Biological Macromolecules, Vol. 26, No. 5, 1999, pp. 317-323. doi:10.1016/S0141-8130(99)00054-9
[32] J. H. Carra, E. A. Anderson and P. L. Privalov, “Thermodynamics of Staphylococcal Nuclease Denaturation. I. the Acid-Denatured State,” Protein Science, Vol. 3, No. 6, 1994, pp. 944-951. doi:10.1002/pro.5560030609
[33] P. Sen, B. Ahmad and R. H. Khan, “Formation of a Molten Globule Like State in Bovine Serum Albumin at Alkaline pH,” European Biophysical Journal, Vol. 37, No. 8, 2008, pp. 1303-1308. doi:10.1007/s00249-008-0335-7
[34] M. Y. Khan, S. K. Agarwal and S. Hangloo, “Urea- Induced Structural Transformations in Bovine Serum Albumin,” The Journal of Biochemistry, Vol. 102, No. 2, 1987, pp. 313-317.
[35] C. A. Royer, “Revisiting Volume Changes in Pressure-Induced Protein Unfolding,” Biochimica et Biophysica Acta, Vol. 1595, No. 1-2, 2002, pp. 201-209. doi:10.1016/S0167-4838(01)00344-2
[36] I. Luque, S. Leavitt and E. Freire, “The Linkage between Protein Folding and Functional Cooperativity: Two Sides of the Same Coin?” Annual Review of Biophysics & Biomolecular Structure, Vol. 31, 2002, pp. 235-256. doi:10.1146/annurev.biophys.31.082901.134215
[37] N. Tanaka, H. Nishizawa and S. Kunugi, “Structure of Pressure-Induced Denatured State of Human Serum Albumin: A Comparison with the Intermediate in Urea- Induced Denaturation,” Biochimica et Biophysica Acta, Vol. 1338, No. 1, 1997, pp. 13-20. doi:10.1016/S0167-4838(96)00175-6
[38] B. Ahmad, M .K. A. Khan, S. K. Haq and R. H. Khan, “Intermediate Formation at Lower Urea Concentration in B Isomer of Human Serum Albumin: A Case Study Using Domain Specific Ligands,” Biochemical and Biophysical Research Communications, Vol. 314, No. 1, 2004, pp. 166-173. doi:10.1016/j.bbrc.2003.12.069
[39] B. Ahmad, Ankita and R. H. Khan, “Urea Induced Unfolding of F Isomer of Human Serum Albumin: A Case Study Using Multiple Probes,” Archives of Biochemistry and Biophysics, Vol. 437, No. 2, 2005, pp. 159-167. doi:10.1016/
[40] D. A. Egan, T. M. Logan, H. Liang, E. Matayoshi, S. W. Fesik and T. F. Holzman, “Equilibrium Denaturation Of Recombinant Human FK Binding Protein in Urea,” Biochemistry, Vol. 32, No. 8, 1993, pp. 1920-1927. doi:10.1021/bi00059a006
[41] C. N. Pace, “Determination and Analysis of Urea and Guanidine Hydrochloride Denaturation Curves,” Methods in Enzymology, Vol. 131, 1986, pp. 266-280. doi:10.1016/0076-6879(86)31045-0
[42] C. N. Pace, D. V. Laurents and R. E. Erickson, “Urea Denaturation of Barnase: Ph Dependence and Characterization of the Unfolded State,” Biochemistry, Vol. 31, No. 10, 1992, pp. 2728-2734. doi:10.1021/bi00125a013
[43] M. H. Brumano and M. G. Oliveira, “Urea-Induced Denaturation of b-trypsin: An Evidence for a Molten Globule State,” Protein and Peptide Letters, Vol. 11, No. 2, 2004, pp. 133-140. doi:10.2174/0929866043478257
[44] T. A. Dar, L. R. Singh, A. Islam, F. Anjum, A. A. Moo- savi-Movahedi and F. Ahmad, “Guanidinium Chloride and Urea Denaturations of b-Lactoglobulin at pH 2.0 and 25?C: The Equilibrium Intermediate Contains Non-Native Structures (Helix, Tryptophan and Hydrophobic Patches),” Biophysical Chemistry, Vol. 127, No. 3, 2007, pp. 140-148. doi:10.1016/j.bpc.2007.01.006
[45] Y. J. Shiu, U. S. Jeng, Y. S. Huang, Y. H. Lai, H. F. Lu, C. T. Liang, I. J. Hsu, C. H. Su, C. Su, I. Chao, A. C. Su and S. H. Lin, “Global and Local Structural Changes of Cytochrome C and Lysozyme Characterized by a Multigroup Unfolding Process,” Biophysical Journal, Vol. 94, No. 12, 2008, pp. 4828-4836. doi:10.1529/biophysj.107.124214
[46] A. R. H. Babbes, E. T. Powers and J. W. Kelly, “Quanti?cation of the Thermodynamically Linked Quaternary and Tertiary Structural Stabilities of Transthyretin And Its Disease-Associated Variants: The Relationship between Stability and Amyloidosi,” Biochemistry, Vol. 7, 2008, pp. 6969-6984. doi:10.1021/bi800636q
[47] A. C. Ferreon and D. W. Bolen, “Thermodynamics of Denaturant-Induced Unfolding of a Protein That Exhibits Variable Two-State Denaturation,” Biochemistry, Vol. 43, No. 42, 2004, pp. 13357-13369. doi:10.1021/bi048666j
[48] H. Kokubo, J. R?sgen, D. W. Bolen and B. M. Pettitt, “Molecular Basis of the Apparent Near Ideality of Urea Solutions,” Biophysical Journal, Vol. 93, No. 10, 2007, pp. 3392-3407. doi:10.1529/biophysj.107.114181
[49] M. C. Stumpe and H. Grubmüller, “Interaction of Urea with Amino Acids: Implications for Urea-Induced Protein Denaturation,” Journal of the American Chemical Society, Vol. 129, No. 51, 2007, pp. 16126-16131. doi:10.1021/ja076216j
[50] T. E. Creighton, “Toward a Better Understanding of Protein Folding Pathways,” Proceedings of the National Academy of Sciences, USA, Vol. 85, No. 14, 1988, pp. 5082-5086.

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