Calorimetric approach to understand pH and salt influence on the adsorption mechanism of lysozyme to a traditional cation exchanger.

Built upon our interest in illustrating the complexity of protein adsorption onto chromatographic supports and to understand the rule of nonspecific interactions in the ion exchange adsorption process, a traditional model system (lysozyme - carboxymethyl cellulose) was used to determine the charge influence during biomolecule adsorption. Flow microcalorimetry (FMC) was exploit as a dynamic technique that provides adsorption and desorption heat signals for a specific system, permitting an improved understanding of the driving forces and mechanisms involved during the interaction. For this purpose, measurements were made at pH 8 at both absence and presence of salt (NaCl 50 mM) and compared with previous studies performed at pH 5. Distinct FMC profiles were observed regarding pH. For most of the experiments, two exothermic heat signals are observed at pH 8 while at pH 5 one endothermic and one exothermic peak are shown. This difference was justified with a less energy demanding for desolvation at pH 8. Lysozyme adsorption was shown to be a multi-step process involving desolvation, primary protein adsorption and secondary adsorption after reorientation with distinct contributions to the overall energy. At pH 8, the exothermic contribution to the adsorption process is lower compared to pH 5, which is justified by the lower charge density that lysozyme presents at pH 8 compared to pH 5.

Inhibited enzymatic reaction of crosslinked lactate oxidase through a pH-dependent mechanism.

Lactate oxidase (LOx), recognized to selectively catalyze the lactate oxidation in complex matrices, has been highlighted as preferable biorecognition element for the development of lactate biosensors. In a previous work, we have demonstrated that LOx crosslinking on a modified screen-printed electrode results in a dual range lactate biosensor, with one of the analysis linear range (4 to 50 mM) compatible with lactate sweat levels. It was advanced that such behavior results from an atypical substrate inhibition process. To understand such inhibition phenomena, this work relies in the study of LOx structure when submitted to increased substrate concentrations. The results found by fluorescence spectroscopy and dynamic light scattering of LOx solutions, evidenced conformational changes of the enzyme, occurring in presence of inhibitory substrate concentrations. Therefore, the inhibition behavior found at the biosensor, is an outcome of LOx structural alterations as result of a pH-dependent mechanism promoted at high substrate concentrations.

Thermodynamics of the adsorption of monoclonal antibodies in phenylboronate chromatography: Affinity versus multimodal interactions.

The aim of this work was to investigate the complex phenomena underlying the adsorption of an anti-human IL-8 (anti-IL8) monoclonal antibody (mAb) to m-aminophenylboronate (m-APBA) by Flow Microcalorimetry (FMC) and to understand the role of non-specific interactions in the adsorption process. FMC was exploited as a dynamic on-line method to measure instantaneous heat energy transfers in order to understand the surface phenomena underlying mAb's adsorption towards the synthetic ligand m-APBA under different pH (7.5, 8.5, 9.0, 9.5 and 10.0) and salt concentrations (0 and 150 mM NaCl). Results showed that the adsorption of anti-IL8 mAb to m-APBA is enthalpically driven (ΔHads<0), as expected for the predominant reversible esterification reaction between boronates and cis-diols-containing molecules. For all the pH conditions studied, thermograms presented a first exothermic peak, characteristic of the reversible esterification reaction between mAb (pI≥9.3) and m-APBA (pKa = 8.8), except at pH 9.0 in the presence of 150 mM NaCl, for which the thermogram presented a first endothermic peak. The heat of adsorption (ΔHads) obtained at conditions where cis-diol interactions were predominant was approximately -243 ± 38 kJ/mol against -82 ± 14 kJ/mol (p-value < 0.05) obtained at pH 9.0 with 150 mM NaCl. The observed shift in the thermogram profile at pH 9.0, 150 mM NaCl, and the consequent decrease of 60-70% in ΔHads were indicative of the promotion of electrostatic interactions between the protein and the ligand. Overall, and whereas the binding of the PBA ligand to mAb molecules has been described for decades as being affinity-based, our study demonstrates the multimodal behaviour of this interaction and contributes towards the understanding of the adsorption thermodynamics.

Acylated-naproxen as the surface-active template in the preparation of micro- and nanospherical imprinted xerogels by emulsion techniques.

A strategy based on water-in-oil emulsion for the dispersion of a sol-gel mixture into small droplets was employed with the view of the production of naproxen-imprinted micro- and nanospheres. The procedure, aiming at a surface imprinting process, comprised the synthesis of a naproxen-derived surfactant. The imprinting process occurred at the interface of the emulsions or microemulsions, by the migration of the NAP-surfactant head into the sol-gel drops to leave surficial imprints due mainly to ion-pair interaction with a cationic group contained within the growing sol-gel network. The surface-imprinted microspheric particles exhibited a log-normal size distribution with geometric mean diameter of 3.1µm. A mesoporous texture was found from measurements of the specific surface area (206m(2)/g) and pore diameter (Dp 2nm). Evaluation of the microspheres as packed HPLC stationary phases resulted in the determination of the selectivity factor against ibuprofen (α=2.1), demonstrating the successful imprinting. Chromatographic efficiency, evaluated by the number of theoretical plates (222platescm(-3)), emerged as an outstanding feature among the set of all relatable formats produced before, an advantage intrinsic to the location of the imprinted sites on the surface. The material presented a capacity of 3.2µmolg(-1). Additionally, exploratory work conducted on their nanoscale counterparts resulted in the production of nanospheres in the size order of 10nm providing good indications of a successful imprinting process.

Modeling Langmuir isotherms with the Gillespie stochastic algorithm.

The overall goal of this work is to develop a robust modeling approach that is capable of simulating single and multicomponent isotherms for biological molecules interacting with a variety of adsorbents. Provided the ratio between the forward and reverse adsorption/desorption constants is known, the Gillespie stochastic algorithm has been shown to be effective in modeling isotherms consistent with the Langmuir theory and uptake curves that fall outside this traditional approach. We have used this method to model protein adsorption on ion-exchange adsorbents, hydrophobic interactive adsorbents and ice crystals. In our latest efforts we have applied the Gillespie approach to simulate binary and ternary isotherms from the literature involving gas-solid adsorption applications. In each case the model is consistent with the experimental results presented.

Disposable immunosensor for human cytomegalovirus glycoprotein B detection.

Combining the advantages of the biosensor field with the problem of detecting Human Cytomegalovirus (HCMV) in human samples, an inexpensive, simple and disposable electrochemical immunosensor for glycoprotein B detection in urine is proposed. Glycoprotein B has been chosen once is the dominant antigen of HCMV. The approach is based on a sandwich-type immunoassay, with the HCMV glycoprotein B sandwiched between the Anti-HCMV antibody adsorbed onto the working electrode, and the Anti-HCMV labeled with gold nanoparticles. Glycoprotein B detection was carried out through electrochemical stripping analysis of silver nanoparticles quantitatively deposited on the immunosensor through catalysis by the nanogold labels. The influence of different steps in the immunosensor construction, namely the silver deposition time, silver enhancer concentration, antibody concentration, BSA concentration and glycoprotein B incubation time, were examined and optimized. The method showed a linear dependence between glycoprotein B concentration and the corresponding anodic stripping peak current, resulting in detection limits of 3.3±1.7ng/mL for samples prepared in buffer and 3.2±0.2ng/mL for urine samples, suggesting that the biological matrix does not interfere with the immunosensor detection capability. Given its mode of preparation, by physical adsorption of the capture antibody in the working electrode, the immunosensor also exhibited an acceptable reproducibility, with a residual standard deviation of 13.5% for samples prepared in buffer and 11.2% for urine samples, thereby presenting a promising development potential for clinical applications.

Effect of salts and temperature on the adsorption of bovine serum albumin on polypropylene glycol-Sepharose under linear and overloaded chromatographic conditions.

The interaction thermodynamics associated with bovine serum albumin (BSA) adsorption on polypropylene glycol (PPG)-Sepharose CL-6B gel, using ammonium and sodium sulfate was studied. Analysis of data under linear conditions was accomplished with the stoichiometric displacement retention model and preferential interaction approach. Preferential interaction analysis indicated a strong entropic driving force due to the release of a large amount of solvent on adsorption. Flow microcalorimetry provided direct heat of adsorption measurements under overloaded conditions and confirmed that the adsorption of BSA on PPG-Sepharose was entropically driven within the range of conditions studied. Using these data in combination with isotherm measurements, it is shown that protein surface coverage, salt concentration, salt type and temperature affect the enthalpic and entropic behavior in hydrophobic interaction chromatography (HIC). This study shows that protein-sorbent interactions can be strongly influenced by the degree of water release, protein-protein interactions on the surface, and the re-orientation and/or reconfiguration of the adsorbed protein.

Study of hydrophobic interaction adsorption of bovine serum albumin under overloaded conditions using flow microcalorimetry.

The adsorption behavior of bovine serum albumin (BSA) on a Sepharose based hydrophobic interaction support has been studied. Flow microcalorimetry has been used to determine the heat of adsorption under overloaded chromatographic conditions. These data have been complemented with capacity factor and isotherm measurements to provide insight on the mechanisms of adsorption. The heat of adsorption data have confirmed that the hydrophobic interaction adsorption of BSA under linear isotherm conditions is driven by entropy changes. Under overloaded (non-linear) conditions, however, it has been shown that the changes in enthalpy can drive adsorption; this behavior is not evident from analyses of capacity factor data. It is postulated that for BSA adsorption on the Sepharose derivative of interest, attractive force interactions between adsorbed protein molecules drive the adsorption process under overloaded conditions in a high (NH4)2SO4 environment. It is further postulated that these interactions are due to a change in confirmation of the adsorbed protein under these conditions.

Thermodynamic study of the interaction between linear plasmid DNA and an anion exchange support under linear and overloaded conditions.

Anion-exchange chromatography has been successfully used in plasmid DNA (pDNA) purification. However, pDNA adsorption mechanism using this method is still not completely understood, and the prediction of the separation behavior is generally unreliable. Flow microcalorimetry (FMC) has proven its ability to provide an improved understanding of the driving forces and mechanisms involved in the adsorption process of biomolecules onto several chromatographic systems. Thus, using FMC, this study aims to understand the adsorption mechanism of linear pDNA (pVAX1-LacZ) onto the anion-exchange support Fast Flow (FF) Q-Sepharose. Static binding capacity studies have shown that the mechanism of pDNA adsorption onto Q-Sepharose follows a Langmuir isotherm. FMC experiments resulted in thermograms that comprised endothermic and exothermic heats. Endothermic heat major contributor was suggested to be the desolvation process. Exothermic heats were related to the interaction between pDNA and Q-Sepharose primary and secondary adsorption. Furthermore, FMC revealed that the overall adsorption process is exothermic, as expected for an anion-exchange interaction. Nevertheless, there are evidences of the presence of nonspecific effects, such as reorientation and electrostatic repulsive forces.

Lysozyme adsorption onto a cation-exchanger: mechanism of interaction study based on the analysis of retention chromatographic data.

In this study, based on the analysis of retention chromatographic data, we examined the adsorption of lysozyme onto carboxymethyl cellulose. Lysozyme retention data was collected at pH 5 and pH 8. The sodium chloride (NaCl) concentration in the mobile phase ranged from 300mM to 500mM and the temperature for this study varied from 288K to 308K. The retention measurements generated from these experimental conditions were analyzed with the Van't Hoff method, the preferential interaction model and the stoichiometric displacement model. Endothermic heats-of-adsorption and increases in entropy were observed under certain experimental conditions. These data suggest the presence of entropic driving forces such as the release of water and/or possibly structural changes in lysozyme molecules adsorbed to the surface of carboxymethyl cellulose. The modest observed exergonic adsorption ΔG° and the preferential interaction analysis corroborate the presence of water-release for this study. Additional analysis with the stoichiometric displacement model method revealed negligible changes in the structure of lysozyme molecules in contact with the surface of carboxymethyl cellulose.

Enthalpy contributions to adsorption of highly charged lysozyme onto a cation-exchanger under linear and overloaded conditions.

An investigation of the adsorption mechanism of lysozyme onto carboxymethyl cellulose (CMC) was conducted using flow calorimetry and adsorption isotherm measurements. This study was undertaken to provide additional insight into the underlying mechanisms involved in protein adsorption that traditional approaches such isotherm measurements or van't Hoff analysis can't always provide, particularly when protein adsorption occurs under overloaded conditions. Lysozyme and CMC were selected for this study because the characteristics of the protein and the adsorbent are well known, hence, allowing the focus of this work to be on the driving forces influencing adsorption. Calorimetry results have showed that lysozyme adsorption onto CMC produced both exothermic and endothermic heats of adsorption. More specifically flow calorimetry data coupled with peak deconvolution methods illustrated a series of chronological events that included dilution, primary protein adsorption, rearrangement of surface proteins and a secondary adsorption of lysozyme molecules. The observations and conclusions derived from the experimental work presented in our figures and tables were developed within the mechanistic framework proposed by Lin et al., J. Chromatogr. A. 912 (2001) 281.

The effects of ligand chain length, salt concentration and temperature on the adsorption of bovine serum albumin onto polypropyleneglycol-Sepharose.

The interaction thermodynamics associated with bovine serum albumin adsorption on polypropylene glycol (n=3)-Sepharose CL-6B and polypropylene glycol (n=7)-Sepharose CL-6B, using ammonium sulfate as the modulator was studied. Analysis of data under linear conditions was accomplished with the stoichiometric displacement retention model, preferential interaction approach and van't Hoff plots applied to HIC systems. Preferential interaction analysis indicated a strong entropic driving force under linear conditions, due to the release of a large amount of solvent on adsorption. In contrast, flow microcalorimetry under overloaded conditions showed that the adsorption of bovine serum albumin may be entropically or enthalpically driven. It is postulated that adsorption in the nonlinear region is influenced by the degree of water release, protein-protein interactions on the surface, reorientation of ligand, and conformational changes in the protein.