Studies On Solution Thermodynamics Of Protein And Its Model Molecules

Protein is an indispensable material in living body,which plays an important role in life process.Serum albumin(SA)is a kind of protein which can bind with intrinsic and extrinsic materials,and transfers them to every parts of the body.Study on interaction between small organic molecules and serum albumin is helpful to understand the importance of such weak interaction as hydrophilic-hydrophobic interaction in biomacromolecule solution more deeply,thereby disclose the mystery of relevant life process.Amino acids,small peptides,acylamides and their derivatives have been used extensively as the most important biological model compounds.Sugars and polyols help in stabilizing the native conformation of globular proteins.The principle reasons for studying the thermodynamics of amino acids or acylamides with polyhydric compounds and the influence of pH value on such systems are to obtain(â…°)the information that contributes to the growing body of knowledge about solute solvation and solute -solute interactions in aqueous media,and(â…±)a better understanding of their role played in the conformational stability and unfolding behavior of proteins.Thermometry is an important method in the modern thermodynamic and thermochemical research,which has incomparable advantages than other methods in the thermodynamic research of life system containing many components.21^th Century is the century of life science.Study on interaction between biology macromolecule and small molecule in life body solutions by utilizing microcalorimetry combined with spectroscopy methods is of great significance for human being to explore the life essences and to disclose the life mystery.As a part of the project supported by National Natural Science Foundation (No.20273061),the present work consists of five parts.The first part:interactions of human and bovine serum albumin(HSA and BSA) with bis-quatemary ammonium surfactants. In this part,the interactions of serum albumin with two bis-quatemary ammonium surfactants,(C_nN)₂Cl₂(n = 12,14),in buffer solutions(pH=7.0)has been investigated by isothermal titration calorimetry at 298.15 K.The binding site number, binding constant and thermodynamic function change were obtained by fitting the experimental data.In addition,the influence of the two surfactants on the secondary structure of protein has been studied by Circular dichroism(CD)spectra.The results show that:(1)There are two classes of binding sites on HSA and BSA molecules for the two surfactants.One is high affinity binding(corresponding to larger K value)caused by electrostatic interaction of surfactant head groups with ionic sites on the protein surfaces,the other is low affinity binding(corresponding to smaller K value)due to the binding of alkyl chains of the surfactant to hydrophobic cavities of the protein molecules.The two classes of binding processes respectfvely cause positive and negative thermal effect.(2)The entropy effects for the two surfactants binding sites are all positive. Elongation of the alkyl chains in the surfactant molecules can strengthen their binding to HSA or BSA,and the binding force of the both surfactants to HSA is stronger than that to BSA.For high affinity sites,the difference of the binding site number between the two surfactants is small.However,for low affinity sites,the binding site number for(C₁₄N)₂Cl₂ is much smaller than that for(C₁₂N)₂Cl₂,this is because the alkyl chains of(C₁₄N)₂Cl₂ molecules are too long to be completely enclosed in the molecular cavity of a single binding site,which would lead to the reduction of evolved heat and the increasing of entropy.(3)The binding of the two surfactants to HSA or BSA can change the secondary molecular structure of the two kinds of proteins.The change of the secondary structure is mainly the conversion fromα-helix toβ- sheet.This is because the adsorption of surfactant cations on the protein macromolecule surface would lead to a swelling of the macromolecule and exposing of the hydrophobic residues.Thus the originalα-helices are partly broken,which gives a more open disordered structure.The second part:interactions of serum albumin with paeonol and two of its isomers.In this part,the interaction of serum albumin with paeonol (2'-hydroxyl-4'-methoxyacetophone,Pae)as well as two of its isomers (2'-hydroxyl-5'-methoxyacetophone,Hma and 4'-hydroxyl-3'-methoxyacetophone, Ace)in buffer solutions(pH=7.0)has been determined by isothermal titration calorimetry at 298.15 K.The binding constants,changes of enthalpy,entropy and Gibbs free energy are obtained by fitting experimental data.Moreover,the influence of the three drug molecules on the secondary structure of protein has been studied by CD spectra.The results show that:(1)There are two classes of binding sites on SA molecules for the three drugs. The first-class binding is caused by electrostatic interaction and hydrogen bonding of substitute groups on benzene ring of drug molecules with amino acid residues on SA surface,and the second one is due to the binding of benzene ring of drug to hydrophobic cavities of SA.(2)The binding process is predominantly driven by enthalpy according to the characteristics of standard Gibbs energy change.For the binding of the same drug molecule,the evolved heat and binding constant to HSA are smaller than those to BSA.On the same class of binding site,the negative value ofΔH°decreases in the order of Pae,Hma and Ace.The difference of thermodynamic data is caused by the different protein structure and locations of substitute groups on aromatic benzene ring of guest molecules.(3)The binding of the three isomers to HSA or BSA can change the secondary structure of the two protein molecules.These results indicate that the interaction includes contributions of the binding and the partial change of molecular structure of SA induced by the three isomers.(4)The second binding site number of Pae- SA system is much larger than that of other systems.So the chance for the binding of Pae to SA is largest under the same conditions,which may be helpful to understand that Pae has more therapeutic efficacy than its two isomers. The third part:interactions of serum albumin with three imidazole-based ionic liquids.In this part,the interactions of serum albumin with three imidazole-based ionic liquids([bmim]BF₄,[bmim]PF₆,[omim]BF₄)in buffer solutions(pH=7.0)have been determined by isothermal titration calorimetry at 298.15 K.The binding constants, changes of enthalpy,entropy and Gibbs free energy are obtained by the non-linear fitting of experimental data.In addition,the influence of the three ionic liquids on the secondary structure of protein has been studied by CD spectra.The results show that:(1)There are two classes of binding sites on SA molecules for the three ionic liquids.The first-class binding is caused by electrostatic interaction and hydrogen bonding of anions of ionic liquids with amino acid residues on SA surface,and the second one is due to the binding of imidazole ring of ionic liquid and the alkyl chains thereon to hydrophobic cavities of SA.(2)The binding processes are both entropy driven according to the characteristics of standard Gibbs energy change.For the binding of the same ionic liquid,the evolved heat and binding constant to HSA are larger than those to BSA.The binding constant for the hydrophobic interaction of[omim]BF₄ with SA is much larger than that for[bmim]BF₄,which shows that the match degree between alkyl chains and hydrophobic cavity of protein for[omim]BF₄ is better than that for[bmim]BF₄.In addition,the first-class binding site number for the binding of[bmim]PF₆ to SA is much larger than that for the binding of[bmim]BF₄,which may be caused by the increasing of binding chance of anion PF₆^- to the surface of SA molecule.(3)The binding of the three inoic liquids to HSA or BSA can change the secondary structure of the two protein molecules,mainly the decrease ofα-helix content.These results proved the interactions of SA with the three ionic liquids.The fourth part:Enthalpic interactions of formamide and N, N-dimethylformamide with myo-inositol in aqueous sodium chloride solutions.In this part,the enthalpies of mixing of formamide and N,N-dimethylformamide with myo-inositol along with those of their dilution in aqueous sodium chloride solution of different concentration have been determined by using flow-mix-isothermal microcalorimetry at 298.15 K.These results were used to determine the heterotactic enthalpic interaction coefficients(h_xy,h_xxyand h_xyy) according to the McMillan-Mayer theory.The pairwise interactions between the two acylamide and myo-inositol have been discussed by solute-solute interactions and solute-solvent interactions.The results show that:(1)The heterotactic enthalpic pairwise interaction coefficients h_xybetween formamide and myo-inositol in water and aqueous sodium chloride solution of different concentration are all negative,which shows that the value of h_xyis mainly dependent on the dipole-dipole interaction between solute molecules.(2)The h_xyvalues for interaction between DMF and myo-inositol are all positive over the whole concentration of the salt aqueous solutions investigated.This indicates that the cooperative effects of the hydrophobic-hydrophilic interaction between the methyl group of DMF molecule and the hydroxyl group of myo-inositol molecule and the partial dehydration of the two solutes can surpass the dipole-dipole interaction between the polar group of DMF molecule and the hydroxyl group of myo-inositol molecule.(3)In aqueous sodium chloride solution of different concentration,the heterotactic enthalpic pairwise interaction coefficients(h_xy)between formamide and myo-inositol become less negative with increase of the concentration of sodium chloride.In contrast,the values of h_xybetween N,N-dimethylformamide and myo-inositol become more positive with elevated concentration of sodium chloride solutions.This mainly due to the increase of dehydration effects of solute molecules caused by the increase of the concentration of sodium chloride.The fifth part:Enthalpies of dilution of D-p-hydroxyphenylglycine in buffer solutions at different pH.In this part,enthalpies of dilution of D-p-hydroxyphenylglycine in phosphate buffer solutions at different pH have been determined by isothermal titration microcalorimetry at 298.15 K.The corresponding homogeneous enthalpic interaction coefficients have been calculated according to the McMillan-Mayer.The results are discussed according to solute-solute interactions and solute-solvent interactions.The results shows:(1)The enthalpies of dilution of D-pHPG in phosphate buffer solutions at different pH are all positive,while the enthalpic pair interaction coefficients h₂ are all negative.This suggests that the hydrogen bond,ion - dipole interaction and electrostatic interaction dominate the pairwise interactions.(2)There is a minimum for the enthalpies of dilution at pH 7.0,which leads to the enthalpic pair interaction coefficient of D-pHPG pass through a maximum at pH 7.0.This is because the pH value is near to the isoelectric point(pI)of D-pHPG (6.6),at which D-pHPG becomes electro-neutralized,the electrostatic interactions between the cation and anion of electrolyte with D-pHPG are minimized.When pH>pI,the higher the pH value is,the more electric charges D-pHPG molecule will possess,so the electrostatic interactions reinforces,which results in the increase of dilution enthalpies.(3)At pH = 6,7,8,negative value of partial differential of molar enthalpy of dilution versus final molality of the diluted component(m_f)when m_f→0 (i.e.-((?)Δ_dilH_m/(?)m_f)_mf→0)is approximately zero,while such values are much larger when pH = 9,10.But when pH = 11,-((?)Δ_dilH_m/(?)m_f)_mf→0value decreases again. This kind of difference can be attributed to their different interaction between solutes.