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.

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.

Flow microcalorimetric measurements for bovine serum albumin on reversed-phase and anion-exchange supports under overloaded conditions.

Heat of adsorption data using flow microcalorimetry is reported for the adsorption of bovine serum albumin (BSA) on C18 and C4 chromatographic supports. Exothermic heats were obtained in all cases. Data for the effect of salt indicate that conformational changes in adsorbed protein appear to be greatest in the absence of salt. Also, the specific surface area of the support was found to influence behavior more strongly than the length of the carbon ligand. Heats of adsorption of BSA on an ion-exchange support were also measured. Endothermic heats were obtained in all cases. The data indicate that the observed heat effects may be strongly influenced by the release of water from the surface.