Investigations into the Thermodynamics of Polypeptide Interaction with Nonpolar Ligands

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• 作者：Milton T. W. Hearn and Guoling Zhao
• 刊名：Analytical Chemistry
• 出版年：1999
• 出版时间：November 1, 1999
• 年：1999
• 卷：71
• 期：21
• 页码：4874 - 4885
• 全文大小：189K
• 年卷期：v.71,no.21(November 1, 1999)
• ISSN：1520-6882

文摘

In this paper, we describe a general procedure to evaluatethe thermodynamics of the interaction between polypeptides and hydrophobic ligands in the presence of aquo-organic solvent mixtures. These studies address experimental requirements for the determination of the linearfree energy relationships, derivation of partition coefficients or other extrathermodynamic parameters such ascontact areas, or assessment of the conformational changesthat may occur when polypeptides or proteins interactwith immobilized nonpolar ligands. Not unexpectedlyfrom thermodynamic arguments, the trends and magnitudes of free energy parameters, such as the enthalpy ofassociation, as previously derived in many studies fromgradient elution reversed-phase high-performance liquidchromatographic (RP-HPLC) measurements are oftendifferent from the data for the same parameters derivedfrom equilibrium binding or microcalorimetric determinations. To reconcile these divergencies and to more closelyexamine the thermodynamic basis of the interaction ofpolypeptides with nonpolar ligands, the dependency of thelogarithmic capacity factor, ln k', on temperature, T, forseveral polypeptides (bombesin, mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-endorphin, glucagon)have been investigated using a n-butylsilica and acetonitrile-water or methanol-water mixtures of definedsolvent compositions. With low-pH, acetonitrile-watermixtures, the van't Hoff plots, i.e., the plots of ln k' versus1/T, were nonlinear over the range of T = 278-358 K,although within a narrow temperature range, e.g., fromT = 278-308 K, the experimental data for these polypeptides could be approximated by a linear relationship. Thisnonclassical van't Hoff behavior was associated withinteractive processes that involved temperature-dependent enthalpic, entropic, and heat capacity changes. Incontrast, with low-pH, methanol-water mixtures, the van'tHoff plots showed dependencies that were essentiallylinear over the range of T = 278-358 K. The slopes ofthe van't Hoff plots with acetonitrile-water and methanol-water mixtures at a defined T value and solvent composition were significantly larger than those found for thecorresponding experiments carried out under gradientelution RP-HPLC conditions. From these plots of ln k'versus 1/T, the changes in the apparent enthalpy ofassociation (mages/gifchars/Delta.gif" BORDER=0 >) and the apparent entropy of association (mages/gifchars/Delta.gif" BORDER=0 >) for the interaction of these polypeptides with the solvated n-butyl ligands at different T andsolvent compositions have been determined. For thesepolypeptides, both mages/gifchars/Delta.gif" BORDER=0 > and mages/gifchars/Delta.gif" BORDER=0 > exhibited lineardependencies on the volume fraction, mages/entities/phiv.gif">, of the organicsolvent over a narrow range of T, but the slopes of theseplots were dependent on the T range examined. Thedependencies of the slope term, S, and the intercept term,ln k_o, derived from the plots of ln k' versus mages/entities/phiv.gif"> as a functionof T, have also been investigated. A new relationshiplinking the S values with mages/gifchars/Delta.gif" BORDER=0 > and mages/gifchars/Delta.gif" BORDER=0 > as a functionof T and mages/entities/phiv.gif"> has been derived and validated. In addition,the relationship between S, mages/gifchars/Delta.gif" BORDER=0 >, mages/gifchars/Delta.gif" BORDER=0 >, the apparentchange in heat capacity, mages/gifchars/Delta.gif" BORDER=0 >, and the accessiblesurface area, mages/gifchars/Delta.gif" BORDER=0 >A_tot, of these polypeptides has been examined, thus providing a linkage of these thermodynamicand extrathermodynamic parameters to the partitioncoefficient, P, and the molecular properties of thesepolypeptides. The results confirm that entropy-enthalpycompensation effects participate in the interaction ofpolypeptides with hydrophobic ligands. This investigationhas confirmed that the use of solvent-water mixtures ofdefined composition, rather than the more convenientpractice of using gradient elution methods, is essential ifthermodynamically consistent values of the binding affinities and partition coefficients are to be quantitativelyderived. Similar considerations apply to the derivation ofextrathermodynamic parameters associated with the conformational transitions of polypeptides (or proteins) whenthey interact with nonpolar n-alkyl ligands. This study thusprovides a general approach to evaluate the interactionthermodynamics of polypeptides or proteins in these andsimilar lipophilic systems.