Influence of pH and Ionic Strength on the Steric Mass-Action Model Parameters around the Isoelectric Point of Protein

Abstract

The ion-exchange equilibrium and the dependence of the parameters in the steric mass-action (SMA) model on salt concentration and buffer pH around the isoelectric point of protein were studied. Bovine serum albumin (BSA, isoelectric point = 5.4) was used as a model protein and DEAE Sepharose FF as an ion exchanger. Finite batch adsorption experiments and isocratic elution chromatography were performed for the determination of the model parameters (i.e., characteristic charge, equilibrium constant, and steric factor). The results showed that pH had significant effects on the parameters. With an increase of pH from 4.5 to 6.5, the characteristic charge increased from 0.9 to 3.0 and leveled off as a plateau at pH above 5.5. The charge groups in the contact region of protein surface were considered to play a crucial role on the characteristic charge. The decrease of pH and increase of salt concentration lowered the absolute value of the zeta potential of the protein surface and led to a decrease of the equilibrium constant. The steric factor remained unchanged at about 31 at pH 5.5 and 6.0 and increased to 44.5 at pH 5.0 and 96.8 at pH 4.5, mainly as a result of the lower adsorption capacity of BSA at pH <5.5. Furthermore, the increase of the molecular volume of BSA at pH 4.5 would be an additional reason for the increase of the steric factor. Taking into account the effect of the pH and salt concentration on these parameters, the SMA model described the ion exchange equilibrium of protein more accurately.

Adsorption of Proteins onto Ion-Exchange Chromatographic Media: A Molecular Dynamics Study

“…From the adsorption of bHb it is also indicated that, though the net charge of the total bHb molecule (charge of +2) is akin to that on Q Sepharose FF (positively charged), the bHb surface still has negative charges to interact with the adsorbent. Similar results for other proteins were obtained by experiments. ,, …”

Adsorption of Proteins onto Ion-Exchange Chromatographic Media: A Molecular Dynamics Study

“…From the adsorption of bHb it is also indicated that, though the net charge of the total bHb molecule (charge of +2) is akin to that on Q Sepharose FF (positively charged), the bHb surface still has negative charges to interact with the adsorbent. Similar results for other proteins were obtained by experiments. ,, …”

Improved purification of immunoglobulin G from plasma by mixed‐mode chromatography

“…This trend is consistent with the behavior described in our previous publication . The selectivity ( S d ) of the AN SepFF adsorbents for IgG over BSA was calculated using the following equation : where is the capacity factor, which can be expressed as where βis the phase ratio . When , .…”

Fluorescence Turn-on Responses of Anionic and Cationic Conjugated Polymers toward Proteins: Effect of Electrostatic and Hydrophobic Interactions

“…These data show that the fluorescence of PFEBT/protein complexes can be effectively manipulated by pH, due to the pH-dependent change in protein net charges . As reported previously, the net charges of BSA at pH 7.4 and 4.5 are around −17 and +29, respectively, and the net charges of lysozyme at pH 7.4 and 12 are around +8 and −3, respectively . Accordingly, decreasing the pH from 7.4 to 2 gradually reverses the net charge of BSA from negative to positive, resulting in reinforced electrostatic attractions between PFEBT − and BSA at low pH; likewise, increasing the pH from 7.4 to 14 induces and in turn strengthens electrostatic attractions between PFEBT + and lysozyme.…”

Adsorption of Proteins onto Ion-Exchange Chromatographic Media: A Molecular Dynamics Study

“…From the adsorption of bHb it is also indicated that, though the net charge of the total bHb molecule (charge of +2) is akin to that on Q Sepharose FF (positively charged), the bHb surface still has negative charges to interact with the adsorbent. Similar results for other proteins were obtained by experiments. ,, …”

Improved purification of immunoglobulin G from plasma by mixed‐mode chromatography

“…This trend is consistent with the behavior described in our previous publication . The selectivity ( S d ) of the AN SepFF adsorbents for IgG over BSA was calculated using the following equation : where is the capacity factor, which can be expressed as where βis the phase ratio . When , .…”

Fluorescence Turn-on Responses of Anionic and Cationic Conjugated Polymers toward Proteins: Effect of Electrostatic and Hydrophobic Interactions

“…These data show that the fluorescence of PFEBT/protein complexes can be effectively manipulated by pH, due to the pH-dependent change in protein net charges . As reported previously, the net charges of BSA at pH 7.4 and 4.5 are around −17 and +29, respectively, and the net charges of lysozyme at pH 7.4 and 12 are around +8 and −3, respectively . Accordingly, decreasing the pH from 7.4 to 2 gradually reverses the net charge of BSA from negative to positive, resulting in reinforced electrostatic attractions between PFEBT − and BSA at low pH; likewise, increasing the pH from 7.4 to 14 induces and in turn strengthens electrostatic attractions between PFEBT + and lysozyme.…”

Adsorption of Proteins onto Ion-Exchange Chromatographic Media: A Molecular Dynamics Study

“…From the adsorption of bHb it is also indicated that, though the net charge of the total bHb molecule (charge of +2) is akin to that on Q Sepharose FF (positively charged), the bHb surface still has negative charges to interact with the adsorbent. Similar results for other proteins were obtained by experiments. ,, …”

Improved purification of immunoglobulin G from plasma by mixed‐mode chromatography

“…This trend is consistent with the behavior described in our previous publication . The selectivity ( S d ) of the AN SepFF adsorbents for IgG over BSA was calculated using the following equation : where is the capacity factor, which can be expressed as where βis the phase ratio . When , .…”

Fluorescence Turn-on Responses of Anionic and Cationic Conjugated Polymers toward Proteins: Effect of Electrostatic and Hydrophobic Interactions

“…These data show that the fluorescence of PFEBT/protein complexes can be effectively manipulated by pH, due to the pH-dependent change in protein net charges . As reported previously, the net charges of BSA at pH 7.4 and 4.5 are around −17 and +29, respectively, and the net charges of lysozyme at pH 7.4 and 12 are around +8 and −3, respectively . Accordingly, decreasing the pH from 7.4 to 2 gradually reverses the net charge of BSA from negative to positive, resulting in reinforced electrostatic attractions between PFEBT − and BSA at low pH; likewise, increasing the pH from 7.4 to 14 induces and in turn strengthens electrostatic attractions between PFEBT + and lysozyme.…”