Reductive unfolding of serum albumins uncovered by Raman spectroscopy.

The reductive unfolding of bovine serum albumin (BSA) and human serum albumin (HSA) induced by dithiothreitol (DTT) is investigated using Raman spectroscopy. The resolution of the S-S Raman band into both protein and oxidized DTT contributions provides a reliable basis for directly monitoring the S-S bridge exchange reaction. The related changes in the protein secondary structure are identified by analyzing the protein amide I Raman band. For the reduction of one S-S bridge of BSA, a mean Gibbs free energy of -7 kJ mol(-1) is derived by studying the reaction equilibrium. The corresponding value for the HSA S-S bridge reduction is -2 kJ mol(-1). The reaction kinetics observed via the S-S or amide I Raman bands are identical giving a reaction rate constant of (1.02 +/- 0.11) M(-1) s(-1) for BSA. The contribution of the conformational Gibbs free energy to the overall Gibbs free energy of reaction is further estimated by combining experimental data with ab initio calculations.

[1]  G. Marino,et al.  Early intermediates in the PDI‐assisted folding of ribonuclease A , 2008, Protein science : a publication of the Protein Society.

[2]  S. Foley,et al.  A Raman spectroscopy and theoretical study of zinc–cysteine complexation , 2007 .

[3]  L. Poole,et al.  Cysteine reactivity and thiol-disulfide interchange pathways in AhpF and AhpC of the bacterial alkyl hydroperoxide reductase system. , 2007, Biochemistry.

[4]  R. Glockshuber,et al.  Kinetics of the intramolecular disulfide exchange between the periplasmic domains of DsbD. , 2007, Journal of molecular biology.

[5]  P. Kahn,et al.  Unfolding and refolding of bovine serum albumin at acid pH: ultrasound and structural studies. , 2006, Biophysical journal.

[6]  R. Schweitzer‐Stenner.  Advances in vibrational spectroscopy as a sensitive probe of peptide and protein structure A critical review , 2006 .

[7]  H. Scheraga,et al.  A localized specific interaction alters the unfolding pathways of structural homologues. , 2006, Journal of the American Chemical Society.

[8]  C. Dobson,et al.  Enthalpic and entropic contributions mediate the role of disulfide bonds on the conformational stability of Interleukin‐4 , 2006, Protein science : a publication of the Protein Society.

[9]  J. Winther,et al.  Increasing the reactivity of an artificial dithiol-disulfide pair through modification of the electrostatic milieu. , 2005, Biochemistry.

[10]  M. Karlsson,et al.  Denaturant-assisted formation of a stabilizing disulfide bridge from engineered cysteines in nonideal conformations. , 2005, Biochemistry.

[11]  K. Murayama,et al.  Heat-induced secondary structure and conformation change of bovine serum albumin investigated by Fourier transform infrared spectroscopy. , 2004, Biochemistry.

[12]  H. Scheraga,et al.  Dissimilarity in the reductive unfolding pathways of two ribonuclease homologues. , 2004, Journal of molecular biology.

[13]  S. Franzen,et al.  Single-pass attenuated total reflection Fourier transform infrared spectroscopy for the analysis of proteins in H2O solution. , 2002, Analytical chemistry.

[14]  Giovanni Scalmani,et al.  New developments in the polarizable continuum model for quantum mechanical and classical calculations on molecules in solution , 2002 .

[15]  H. Gilbert,et al.  Discrimination between Native and Non-native Disulfides by Protein-disulfide Isomerase* , 2001, The Journal of Biological Chemistry.

[16]  H. Scheraga,et al.  Disulfide bonds and protein folding. , 2000, Biochemistry.

[17]  K. Kobayashi,et al.  Crystal structure of human serum albumin at 2.5 A resolution. , 1999, Protein engineering.

[18]  S. Cramer,et al.  A holistic approach to protein secondary structure characterization using amide I band Raman spectroscopy. , 1999, Analytical Biochemistry.

[19]  C. T. Reimann,et al.  Proteins in vacuo. Denaturing of disulfide-intact and disulfide-broken lysozyme probed by molecular dynamics simulations , 1998 .

[20]  Y. Ozaki,et al.  Conformational changes in seventeen cystine disulfide bridges of bovine serum albumin proved by Raman spectroscopy , 1997, FEBS letters.

[21]  S. Anderson,et al.  Correlation between disulfide reduction and conformational unfolding in bovine pancreatic trypsin inhibitor. , 1997, Biochemistry.

[22]  A. Hinck,et al.  Engineered disulfide bonds in staphylococcal nuclease: effects on the stability and conformation of the folded protein. , 1996, Biochemistry.

[23]  Harold A. Scheraga,et al.  Mechanism of reductive protein unfolding , 1995, Nature Structural Biology.

[24]  J. Fontecha,et al.  Infrared and Raman Spectroscopic Study of Casein in Cheese: Effect of Freezing and Frozen Storage , 1993 .

[25]  D. Goldenberg,et al.  Probing the determinants of disulfide stability in native pancreatic trypsin inhibitor. , 1993, Biochemistry.

[26]  H. Scheraga,et al.  Equilibrium and kinetic constants for the thiol-disulfide interchange reaction between glutathione and dithiothreitol. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[27]  S. Krimm,et al.  Vibrational studies of the disulfide group in proteins. Part V. Correlation of SS stretch frequencies with the CCSS dihedral angle in known protein disulfide bridges , 1992, Biopolymers.

[28]  K. Kuwajima,et al.  Kinetics of disulfide bond reduction in alpha-lactalbumin by dithiothreitol and molecular basis of superreactivity of the Cys6-Cys120 disulfide bond. , 1990, Biochemistry.

[29]  M Levitt,et al.  Stabilization of phage T4 lysozyme by engineered disulfide bonds. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[30]  S. J. Burton,et al.  Refolding human serum albumin at relatively high protein concentration. , 1989, European journal of biochemistry.

[31]  H. Mantsch,et al.  Secondary structure of the hydrophobic myelin protein in a lipid environment as determined by Fourier-transform infrared spectrometry. , 1987, The Journal of biological chemistry.

[32]  A. Kossiakoff,et al.  The crystallographically determined structures of atypical strained disulfides engineered into subtilisin. , 1986, The Journal of biological chemistry.

[33]  R. Lord,et al.  Laser-excited Raman spectroscopy of biomolecules. VIII. Conformational study of bovine serum albumin. , 1976, Journal of the American Chemical Society.