Isothermal Titration Microcalorimetric Studies of the Effect of Temperature on Hydrophobic Interaction between Proteins and Hydrophobic Adsorbents

This study attempted to comprehend how temperature affects hydrophobic interaction between proteins and hydrophobic adsorbents. By equilibrium batch analysis, we measured the adsorption isotherm to evaluate the protein-adsorbent affinity, while isothermal titration calorimetry was used to measure the adsorption enthalpy. In addition, the affinity and enthalpy differences between two proteins, alpha-chymotrypsinogen A and trypsinogen, with two adsorbents, butyl-Sepharose and octyl-Sepharose gel, under varying temperatures were studied with respect to the exposed hydrophobic segments of the protein and ligand hydrophobicity. The enthalpies obtained in this investigation can be used to more thoroughly understand the hydrophobic interaction between proteins and adsorbents. First, the adsorption isotherm experiments reveal that the adsorption quantity of the proteins with the Sepharose gels increases with temperature. For a microcalorimetric measurement, as temperature is increased from 298 to 310 K, the DeltaH value of alpha-chymotrypsinogen A with butyl-Sepharose increases, while the DeltaH value of trypsinogen is reduced. This is likely due to the fact that alpha-chymotrypsinogen A has a higher area of exposed hydrophobic segments than trypsinogen does. This observation also implies that as temperature increases, the interaction mechanism of alpha-chymotrypsinogen A with butyl-Sepharose changes from an adsorption-dominated process to a partitioning process. In addition, for octyl-Sepharose, the DeltaH value of alpha-chymotrypsinogen A is positive and decreases with temperature increment. However, the DeltaH value of trypsinogen was positive and increased with temperature. Therefore, we conclude that as temperature increases, the interaction mechanism of the proteins for octyl-Sepharose is a partitioning-dominated process. Copyright 2000 Academic Press.     

Hydrophobic contribution of amino acids in peptides measured by hydrophobic interaction chromatography

The adsorption behaviors of amino acids in short chain peptides were examined. Each amino acid, aliphatic or charged, was inserted between the two tryptophans of a peptide, GWWG. The capacity factors of these peptides on an Ocytl-Sepharose column were measured. The adsorption enthalpies, entropies, and the number of repelled water molecules after adsorption were estimated to analyze the contribution of each different amino acid to its hydrophobic adsorption. The peptides inserted with aliphatic amino acids owned the highest capacity factors but released the least amount of adsorption heat among all the peptides under examination. It was found that the hydrophobic contribution of aliphatic amino acids was derived from the entropy gain by repelling the ordered water surrounding them. The insertion of negatively charged amino acids greatly reduced the capacity factors but still repelled a significant number of water molecules after adsorption. This indicated that the water molecules surrounding ionic amino acids were not orderly aligned. The dehydration cost energy but the water repelling did not offer enough entropy to drive the adsorption. Subsequently, lower retention was obtained from the peptides inserted with negatively charged ionic amino acids. The insertion of lysine increased the adsorption enthalpy but repelled no water molecules after adsorption. It was speculated that the inserted lysine still interacted with hydrophobic ligands but disturbed the interaction between ligands and adjacent tryptophans. Therefore, the adsorption enthalpy increased and the capacity factors decreased. Different amino acids contributed to hydrophobic interaction in different ways. The simultaneous analysis of capacity factor, adsorption enthalpy, adsorption entropy, and the number of repelled water molecules facilitated the understanding of the adsorption processes.     

Microcalorimetric studies of the mechanism of interaction between designed peptides and hydrophobic adsorbents

To understand the mechanism of interaction between peptides and peptides with hydrophobic ligands, the oligomers (GWG, GWWG, GWWWG) were designed and synthesized to study adsorption behavior with octyl sepharose and CM-octyl sepharose. By batch equilibrium binding analysis and dilution heat of peptide solution measurement, the binding isotherm and adsorption enthalpy were obtained and the binding thermodynamics parameters were calculated and analyzed. In the isotherm analysis, we reveled that the affinity of GWG for both adsorbents is stronger than that of GWWG and GWWWG. The results demonstrate that the cation-pi interaction between the peptides and the buffer molecules is significant for solutions of peptides with tryptophan residues, and the solvation is competitive with the hydrophobic interaction between the peptides and the hydrophobic ligands. From the dilution heat measurements, we observed an endothermic dilution heat for GWG and exothermic for GWWG and GWWWG. All these results indicate that the increased tryptophan chain length can promote the solvation behavior of the peptides by the peptide-buffer interaction in this buffer system. Comparing the types of ligands reveals that the binding affinities of each peptide for the two adsorbents are similar. However, the mechanism of adsorption for peptides with hydrophobic ligands might be quite different with respect to the binding enthalpy between peptides and adsorbents. The adsorption of the peptides on octyl sepharose is an entropy-driven process for all the peptides. In contrast, the adsorption of CM-octyl sepharose with GWG and GWWG is an enthalpy-driven process, whereas that with GWWWG is entropy-driven. These findings indicate that the amount of tryptophan controls the characteristics of the peptides and the interaction mechanism in the binding procedure. This study of the adsorption mechanism of the designed peptide could provide fundamental information for peptide purification and amino acid residue behavior in peptide drug design.     

Fractions of thermodynamic functions for native lysozyme adsorption onto moderately hydrophobic surface

Calorimetric measurement of adsorption enthalpies of native lysozyme(Lyz) on a moderately hydrophobic surface at 25°C, pH 7.0 and various salt concentrations was performed. Based on the thermodynamics of stoichiometric displacement theory (SDT), we calculated the fractions of thermodynamic functions involving four subprocesses during a displacement adsorption process from the directly determined enthalpies in combination with adsorption isotherm measurements. The thermodynamic fractions reveal the relative degree of the four subprocesses for contributions to enthalpy, entropy and free energy. The results show that native Lyz adsorption on a moderately hydrophobic surface is an entropy driven process contributed mainly by conformational loss of adsorbed Lyz.     

Performance of hydrophobic interaction ligands for human membrane‐bound catechol‐O‐methyltransferase purification

Despite of membrane catechol‐O‐methyltransferase (MBCOMT, EC 2.1.1.6) physiological importance on catecholamines’ O‐methylation, no studies allowed their total isolation. Therefore, for the first time, we compare the performance of three hydrophobic adsorbents (butyl‐, epoxy‐, and octyl‐Sepharose) in purification of recombinant human COMT (hMBCOMT) from crude Brevibacillus choshinensis cell lysates to develop a sustainable chromatographic process. Hydrophobic matrices were evaluated in terms of selectivity and hMBCOMT's binding and elution conditions. Results show that hMBCOMT's adsorption was promoted on octyl and butyl at ≤375 mM NaH₂PO_4, while on epoxy higher concentrations (>850 mM) were required. Additionally, hMBCOMT's elution was promoted on epoxy, butyl, and octyl using respectively 0.1–0.5, 0.25–1, and 1% of Triton X‐100. On butyl media, a stepwise strategy using 375 and 0 mM NaH₂PO₄, followed by three elution steps at 0.25, 0.7 and 1% Triton X‐100, allowed selective hMBCOMT isolation. In conclusion, significant amounts of MBCOMT were purified with high selectivity on a single chromatography procedure, despite its elution occurs on multiple peaks. Although successful applications of hydrophobic interaction chromatography in purification of membrane proteins are uncommon, we proved that traditional hydrophobic matrices can open a promising unexplored field to fulfill specific requirements for kinetic and pharmacological trials.     

Displacement processes on hydrophilic/hydrophobic surfaces in 1-propanol-water mixtures

Selective adsorption of 1-propanol-water mixtures was investigated on adsorbents of various surface character. The enthalpy of displacement of 1-propanol by water was studied on graphitized PRINTEX-80, K-60 silicagel, and n-alkylammonium vermiculites. The free enthalpy and entropy isotherms of displacement were derived. The adsorption between the silicate layers of hydrophobic vermiculites was followed by x-ray measurements, and thermodynamic functions were calculated for the structural changes in the adsorption layers. Formation of alcohol-water clusters on the surfaces plays an important role.     

Adsorption behaviour of lipase from Staphylococcus carnosus on a hydrophobic adsorbent.

The adsorption of proteins on a solid surface with a subsequent desorption is a well known final purification step in downstream processing. Here the adsorption behaviour of a microbial lipase on the hydrophobic Fractogel TSK butyl 650 in a crude fermentation supernatant is investigated. The measured equilibrium curves differ from fermentation to fermentation by up to +/- 65%. The adsorption capacity increases with decreasing particle diameter of the adsorbent and is influenced by the method of contacting the supernatant with the adsorbent. The rate of desorption depends largely on the adsorption conditions, which is an indication of different orientations of the adsorbed enzyme.     

Synthesis of new hydrophobic adsorbents based on homologous series of uncharged alkyl sulphide agarose derivatives

A homologous series of uncharged thioalkyl derivatives of agarose were prepared by a simplified synthetic route and their adsorption behaviour towards human serum proteins was evaluated and compared with that of a commercially available alkyl ether derivative of agarose. The influence of the spacer arm length on the adsorption efficiency was also investigated. The degree of substitution of the derivatives can be estimated conveniently by sulphur analysis. The four different types of thiolkyl derivatives (C6, C8, C12 and C14) investigated here behave in all respects like hydrophobic adsorbents. The coupling yield obtained is high (75% or more) and is better than that obtained by alternative synthetic routes reported so far. The adsorption capacity towards serum proteins of the various derivatives increases with increasing alkyl chain length and degree of substitution. Desorption is achieved by a progressive decrease in the polarity of the eluent and the recovery of the applied material is in the range 80-90%. The role played by the thioether as a possible modulator of the observed hydrophobic adsorption is discussed. For the group separation of serum proteins the optimum adsorbent, as regards capacity combined with ease of elution of adsorbed material, should be substituted with chains of six or eight carbon atoms and have a ligand concentration in the range 80-120 mumole g-1 dry gel.     

Fluoroalkyl and alkyl chains have similar hydrophobicities in binding to the "hydrophobic wall" of carbonic anhydrase

The hydrophobic effect, the free-energetically favorable association of nonpolar solutes in water, makes a dominant contribution to binding of many systems of ligands and proteins. The objective of this study was to examine the hydrophobic effect in biomolecular recognition using two chemically different but structurally similar hydrophobic groups, aliphatic hydrocarbons and aliphatic fluorocarbons, and to determine whether the hydrophobicity of the two groups could be distinguished by thermodynamic and biostructural analysis. This paper uses isothermal titration calorimetry (ITC) to examine the thermodynamics of binding of benzenesulfonamides substituted in the para position with alkyl and fluoroalkyl chains (H(2)NSO(2)C(6)H(4)-CONHCH(2)(CX(2))(n)CX(3), n = 0-4, X = H, F) to human carbonic anhydrase II (HCA II). Both alkyl and fluoroalkyl substituents contribute favorably to the enthalpy and the entropy of binding; these contributions increase as the length of chain of the hydrophobic substituent increases. Crystallography of the protein-ligand complexes indicates that the benzenesulfonamide groups of all ligands examined bind with similar geometry, that the tail groups associate with the hydrophobic wall of HCA II (which is made up of the side chains of residues Phe131, Val135, Pro202, and Leu204), and that the structure of the protein is indistinguishable for all but one of the complexes (the longest member of the fluoroalkyl series). Analysis of the thermodynamics of binding as a function of structure is compatible with the hypothesis that hydrophobic binding of both alkyl and fluoroalkyl chains to hydrophobic surface of carbonic anhydrase is due primarily to the release of nonoptimally hydrogen-bonded water molecules that hydrate the binding cavity (including the hydrophobic wall) of HCA II and to the release of water molecules that surround the hydrophobic chain of the ligands. This study defines the balance of enthalpic and entropic contributions to the hydrophobic effect in this representative system of protein and ligand: hydrophobic interactions, here, seem to comprise approximately equal contributions from enthalpy (plausibly from strengthening networks of hydrogen bonds among molecules of water) and entropy (from release of water from configurationally restricted positions).     

Comparison of the protein adsorption selectivity of salt-promoted agarose-based adsorbents: Hydrophobic, thiophilic and electron donor–acceptor adsorbents

Protein adsorption of human serum onto six different agarose-based chromatographic gels that were representative of the salt-promoted adsorbent family [octyl- and phenyl-Sepharose, mercaptoethanol–divinyl sulfone agarose (T gel), mercaptomethylene pyridine-derivatized agarose gel (MP gel), tricyanoaminopropene–divinyl sulfone agarose (DVS–TCP gel), tricyanoamino-propene–bisoxirane agarose (bisoxirane–TCP gel)] was studied in the presence of moderate or high concentrations of the water structuring salt, sodium sulfate. Study of the protein adsorption selectivity by two-dimensional gel electrophoresis revealed an opposed selectivity for hydrophobic interaction adsorbents and electron donor–acceptor adsorbents. The T gel, MP gel and TCP gels belonged to the electron donor–acceptor adsorbents, displaying a main selectivity for immunoglobulins, whereas octyl-Sepharose belonged to the hydrophobic adsorbents, displaying a main selectivity for ‘hydrophobic' proteins. Phenyl-Sepharose for its part was described as an example of a composite selectivity of both families. The conclusion of this work is two-fold: (1) hydrophobic interaction chromatography (HIC) and electron donor–acceptor chromatography (EDAC) have opposed protein selectivities and are both salt-promoted. As a main consequence, it means that high concentrations of a water-structuring salt can promote different types of weak molecular interactions, resulting in different protein adsorption selectivities: (2) thiophilic adsorption chromatography (TAC) should be renamed EDAC as similar protein selectivity is demonstrated for electron donor–acceptor ligand devoid of sulfur atoms.     

Rapid and efficient screening of adsorbent for oligopeptide using molecular docking and isothermal titration calorimetry

A rapid and efficient method based on molecular docking and isothermal titration calorimetry (ITC) was developed to identify effective adsorbents for the target peptide Ser‐Glu‐Ala‐Asp‐His (SEADH). Preliminary screening of five candidate adsorbents using molecular docking revealed that three suitable structures (A1, A2, and A3) either with or without coordination of Zn²⁺ should be effective. The three selected structures were then prepared and further screened by evaluating their affinities for the peptide SEADH using ITC. The screening results revealed that the adsorbent A2 coordinated with Zn²⁺ was the best adsorbent, and subsequent static adsorption experiments confirmed the reliability of the screening method. Further ITC analysis, combined with molecular docking, was performed to provide the possible adsorption mechanism.     

Microcalorimetric determination of displacement adsorption enthalpies of protein refolding on a moderately hydrophobic surface at 308 K

Both microcalorimetric determination of displacement adsorption enthalpies ΔH and measurement of adsorbed amounts of guanidine – denatured lysozyme (Lys) refolding on the surface of hydrophobic interaction chromatography (HIC) packings at 308 K were carried out and compared with that at 298 K. Study shows that both temperature and concentration of guanidine hydrochloride (GuHCl) affect the molecular mechanism of hydrophobic interaction of protein with adsorbent based on the analysis of dividing ΔH values into three kinds of enthalpy fractions. The adsorption in higher concentrations of GuHCl (>1.3 mol L^–1) at 308 K is an enthalpy-driving process, and the adsorption under other GuHCl concentrations is an entropy-driving process. The fact that the Lys denatured by 1.8 mol L^–1 GuHCl forms a relatively stable intermediate state under the studied conditions will not be changed by temperature.     

Distribution coefficients in the water-dodecane system and heats of adsorption of ethylene glycol monoalkyl ethers on hydrophilic and hydrophobic carriers

The distribution coefficients in the water-dodecane system and the heats of adsorption of ethylene glycol monoalkyl ethers on hydrophilic (Silochrom S-80) and hydrophobic (Apiezon L on Chromaton) carriers were determined by gas chromatography. At low concentrations and 25°C, ethers with C₁-C₄ alkyl radials predominantly occurred in the aqueous phase, whereas the amyl ether of ethylene glycol was better soluble in the organic phase. Ethers adsorbed formed monomolecular and polymolecular coatings on the hydrophobic and hydrophilic adsorbents, respectively. The heats of adsorption of ethers on the hydrophilic adsorbent were higher than the heats of adsorption on the hydrophobic adsorbent by factors of from 1.93 to 2.20.     

Single-step purification and immobilization of penicillin acylase using hydrophobic ligands

Five differenthydrophobic ligands immobilized on 4% (4XL) and 6% (6XL) crosslinked agarose were used to study the single-step purification of penicillin acylase from cell lysate. The 4XL gels showed relatively higher specific activity and recovery than the 6XL gels. In single-step purification, highly active enzyme (42 U/mg) was obtained using moderately hydrophobic ligand (octyl). The crude enzyme immobilized on octyl gel by adsorption showed significant operational stability over a period of 30 d at room temperature. Reactor studies demonstrated the feasibility of hydrophobic ligands as a medium for immobilization.     

Quantitative description of the hydrophobic effect: the enthalpic contribution

A new method of experimental determination of the hydrophobic effect enthalpy is proposed. The method is based on regarding the hydration enthalpy as the sum of the nonspecific hydration enthalpy, specific hydration enthalpy, and the hydrophobic effect enthalpy. The hydrophobic effect enthalpies of noble and simple substance gases, alkanes, arenes, and normal aliphatic alcohols are determined. For the noble gases and alkanes, the hydrophobic effect enthalpy is found to be negative and independent of the size of molecule. For aromatic hydrocarbons, it is positive and grows up with the size of the hydrocarbon. The hydrophobic effect enthalpies of normal aliphatic alcohols are determined by assuming that the specific interaction enthalpies of alcohols in water and in methanol are equal. The hydrophobic effect enthalpy values for the aliphatic alcohols (-10.0 +/- 0.9 kJ.mol(-1)) were found to be close to the alkanes hydrophobic effect enthalpies (-10.7 +/- 1.5 kJ.mol(-1)).     

Calorimetric investigation of the adsorption of nitrogen bases and nucleosides on a hydrophobic interaction sorbent

Heats of adsorption for nitrogen bases and nucleosides on Sepharose CL-6B, a hydrophobic interaction adsorbent, were collected through flow microcalorimetry in order to ascertain the thermodynamic driving force for adsorption in each case. It was determined that enthalpy changes associated with base stacking self-interactions can contribute significantly to the observed heats of adsorption. Accordingly, the observed heats were the net effect of the adsorbate/adsorbent interactions and the adsorbate stacking self-interactions. Since base stacking proceeds beyond the dimer stage, multi-layer adsorption of these compounds is possible, even at low solution concentrations.     

Isotherm analysis of phenol adsorption on polymeric adsorbents from nonaqueous solution

Macroporous poly(methyl methacrylate-co-divinylbenzene) (PMMA), interpenetrating polymer adsorbent based on poly(styrene-co-divinylbenzene) (PS) and poly(methyl methacrylate-co-divinylbenzene) (PMMA/PS), and macroporous cross-linked poly(N-p-vinylbenzyl acetylamide) (PVBA) were prepared for the adsorption of phenol from cyclohexane. The sorption isotherms of phenol on the three polymeric adsorbents were measured and fitted to Langmuir and Freundlich isotherms. It is shown that the Langmuir isotherm, which is based on a homogeneous surface model, is unsuitable to describe the sorption of phenol on the adsorbents from nonaqueous solution and the Freundlich equation fits the tested three adsorption systems well. The isosteric enthalpy was quantitatively correlated with the fractional loading for the sorption of phenol onto the three polymeric adsorbents. The surface energetic heterogeneity patterns of the adsorbents were described with functions of isosteric enthalpy. The results showed that the tested three polymeric adsorbents exhibited different surface energetic heterogeneity patterns. The initial isosteric enthalpy of phenol sorption on polymeric adsorbent has to do with the surface chemical composition and is free from the pore structure of the polymeric adsorbent matrix. Forming hydrogen bonds between phenol molecules and adsorbent is the main driving force of phenol sorption onto PVBA and PMMA adsorbent from nonaqueous solution. When phenol is adsorbed on PMMA/PS, pi-pi interaction resulting from the stacking of the benzene rings of the adsorbed phenol molecules and the pendant benzene ring of adsorbent is involved.     

A review on the ligand binding studies by isothermal titration calorimetry

Thermodynamics of biomacromolecule ligand interaction is very important to understand the structure function relationship in proteins. One of the most powerful techniques useful to obtain additional information about the structure of proteins in biophysical chemistry field is isothermal titration calorimetry (ITC). An ITC experiment is a titration of a biomacromolecule solution by a solution containing a reactant (ligand) at constant temperature to obtain the exchanged heat of the reaction. The total concentration of ligand is the independent variable under experimental control. There are many reports on data analysis for ITC to find the number of binding sites (g), the equilibrium constant (K), the Gibbs free energy of binding process (ΔG), the enthalpy of binding (ΔH) and the entropy of binding (ΔS). Moreover, ITC gives information about the type of reaction, electrostatic and hydrophobic interactions, including determination of cooperativity characterization in binding process by calculating the Hill coefficient (n). A double reciprocal plot and a graphical fitting method are two simple methods used in the enzyme inhibition and metal binding to a protein. Determination of a binding isotherm needs more ITC experiments and more complex data analysis. Protein denaturation by ligand includes two processes of binding and denaturation so that ITC data analysis are more complex. However, the enthalpy of denaturation process obtained by ITC help to understand the fine structure of a protein.     

Synthesis, characterization and adsorption performance of a novel post-crosslinked adsorbent

In this paper a post-crosslinked polymeric adsorbent PDHT-2 with high specific surface area was prepared by Friedel-Crafts reaction of the pendant vinyl groups without an externally added crosslinking agent. It was obvious that both the specific surface area and the pore volume of starting copolymer PDHT-1 increased significantly after post-crosslinking. Batch adsorption runs of phenol from aqueous solution onto adsorbent PDHT-1 and PDHT-2 were researched, and commercial macroporous resin XAD-4 was chosen for comparison purpose. Experimental results showed that the adsorption isotherms could be fitted by Langmuir model and Freundlich model and the adsorption capacity onto PDHT-2 was much larger than that onto PDHT-1 and XAD-4 with respect to phenol and phenolic compound, which possibly resulted from its larger specific surface area. The adsorption process for phenol onto the three adsorbents was proved to be exothermic and spontaneous in nature. The thermodynamic parameters such as Gibb's free energy (ΔG), change in enthalpy (ΔH) and change in entropy (ΔS) had been calculated. The adsorption kinetic curves obeyed the pseudo-second order model and the intraparticle diffusion process was the rate-controlling step.     

Microcalorimetric studies of the interaction mechanisms between proteins and Q-sepharose at pH near the isoelectric point (pI) effects of NaCl concentration, pH value, and temperature

This study examined the interaction mechanisms of beta-lactoglobulins A and B (Lg A, Lg B) with an anion exchanger, Q-Sepharose at pH near the isoelectric point at which the proteins are expected to be electrically neutralized under various NaCl concentrations and temperatures by the equilibrium binding analysis and the adsorption enthalpy directly measured by isothermal titration calorimetry. The data evaluated from isotherms fitted by the Langmuirean model reveal that the addition of NaCl considerably reduced the binding affinities and capacities of both the proteins with Q-Sepharose at pH 5.2, indicating that electrostatic forces are dominant during the adsorption. However, the hydrophobic interaction seems to be involved in adsorption as well at a higher NaCl concentration, and the adsorption enthalpies confirm this suggestion. In addition, the effects of temperature on the equilibrium binding behaviors for Lg A or Lg B with Q-Sepharose were found to be salt concentration-dependent, probably due to their different binding mechanisms at 0.03 M and 0.3 M NaCl. Where, at 0.3 M NaCl, the hydrophobic interaction plays a more pronounced role. This implication was again supported by the adsorption enthalpies. The presented data provide further insight to the interaction mechanisms between proteins and ion exchangers, facilitating the optimization of protein separations.