Mechanism and Kinetics of Protein Adsorption onto Particles

There were some inconsistencies in the results on protein adsorption reported by different researchers. This is attributed to the different conditions for adsorption, i.e., different surface properties, different concentration of particles, different dispersion media, etc. However, some general facts have been observed as presented below.

1. Mechanism of Protein Adsorption

The factors controlling protein adsorption were comprehensively studied by Haynes, Norde, et al. [1]. They concluded that protein adsorption is controlled by the following factors: (1) electrostatic force, (2) hydrophobic interaction force, (3) hydrogen bonding force, (4) conformational change of a protein molecule, and (5) change of dissociation upon adsorption. These are illustrated in Fig. 1 and explained in the following sections.

(a) Electrostatic Force. The electrostatic force plays the most significant role in adsorption. Proteins are amphoteric compounds. Therefore, the value and sign of their electrical charge and so the amount of adsorption depends on the

Substrate

Mult i-point contact

FIG. 1 Events following protein adsorption onto a substrate.

Change in the distribution of ionic groups

Deformation

Substrate

Mult i-point contact

FIG. 1 Events following protein adsorption onto a substrate.

pH of the medium. There are two conditions under which the maximal adsorption is realized. In one case, the maximal adsorption was observed at the pH where the particle surface and the proteins have opposite charges so conferring strong electroattractive force. In the other case, proteins adsorb the maximum at the isoelectric point (IEP) of the protein. This happens because the cross-section area per protein molecule becomes least when the protein molecules adsorbed adjacently have no charge, so that they have the least repulsive force between them. Immunoglobulin molecules were supposed to close their antibody-binding fragments (Fab) tightly and occupy the minimal surface area of carrier at the IEP [2].

Which of the former or the latter of the above-mentioned two cases takes place depends on the ionic strength of particles. It must be mentioned that in such a system the adsorption of proteins is accompanied by redistribution of charged groups.

In the adsorption system in which electrostatic interaction is dominant, the increase in ionic strength or compression of the electric double layer leads to a decrease in adsorption. Therefore, the proteins once adsorbed in the medium of low ionic strength can be desorbed from the particles by the addition of salts.

(b) Hydrophobicity. Generally speaking, a protein is better adsorbed on a hydrophobic surface than a hydrophilic one. This is due to hydrophobic interaction of the protein and the surface, i.e., both prefer hydrophobic atmosphere in an aqueous phase and touch each other.

It is believed that excellent bioinertness of hydrophilic materials results from less adsorption of proteins on the hydrophilic surface. Coating of particle surface with polyethylene glycol has been one of the most common methods to make the surface bioinert [3]. However, an extremely hydrophilic surface is rather susceptible to protein adsorption, although the reason for this is unknown. Ta-naka et al. argued that a moderately hydrophilic polymer surface adsorbs the lowest proteins [4]. This was attributed to a water phase with specific structure located on the polymer surface. Wavering of hydrated chains of the surface is also believed to be effective for the restriction of nonspecific protein adsorption [5].

(c) Hydrogen Bond. Protein adsorption is sometimes affected by hydrogen bonding. Proteins in water are hydrogen-bonded to the water molecules. Hydrogen bonding also works on some substrates, i.e., between the substrate and water or between the substrate molecules. When protein approaches the substrate surface, rearrangement of hydrogen bonds can take place among the protein, water, and the substrate.

(d) Conformation. Every protein shelters its hydrophobic part in the core and possesses its stable conformation in the solution. As the protein meets with a hydrophobic substrate, it may not need to keep the native conformation any longer and change it if the protein is soft. In other words, a protein having low native state stability is apt to suffer the breakdown of the native conformation when adsorbed. From the perspective of thermodynamics, such a conformational change can occur when the increase in entropy is compensated by the increase in enthalpy.

As easily expected, a larger molecule can have more contact points with the substrate and therefore receives more benefit from the conformational change upon adsorption.

(e) Degree of Dissociation. The change of dissociation of ionizable groups in protein molecules upon adsorption was determined by Haynes et al. [1]. As described in Ref. 1, electrostatic interaction has the most significant role in protein adsorption, but the interaction can be adjusted by self-regulation of protein dissociation. This means that the degree of dissociation is different between dissolved and adsorbed proteins, depending on the nature of substrate as well as the ionic strength.

2. Kinetics of Protein Adsorption

Protein adsorption behavior is generally described by the Langmuir isotherm if the protein concentration is not very high. Despite this, the adsorption is seldom reversible. It is attributed to the nature of proteins, i.e., the great size and flexibility of protein molecules. A protein molecule first contacts at one point with the substrate, and the number of contacting points increases with time. When the protein molecules have numerous contacts with the substrate, they become difficult to leave from the substrate and thus the adsorption becomes irreversible.

As the protein concentration increases, adsorption becomes saturated and multilayered adsorption of protein takes place. Therefore, the adsorption isotherm of such systems has a plateau range before the second ascent.

The adsorption kinetics of multiprotein systems is worth discussing. In competitive adsorption of two or more proteins, replacement of proteins proceeds obeying a certain role, i.e., the Vroman effect, in which the protein of low binding constant but high concentration is preferentially adsorbed but is gradually replaced by the protein of low concentration but high binding constant. This is the main reason for a large molecule to occupy most adsorption sites finally [6].

3. Balance of Contribution of Different Factors to Protein Adsorption

Yoon et al. prepared two series of functionalized particles. One series consisted of carboxylated particles having different carboxyl group content, prepared by controlling carboxylation reaction [7]. The other consisted of sulfonated particles. In both systems, adsorption minimum, corresponding to the smallest plateau of Langmuir-Freundlich isotherm, was observed at a certain density of functional groups. The transition point was regarded as the point at which hydro-

phobic interaction and hydrogen bonding (in the carboxylated system) or electrostatic interaction (in the sulfonated system) was balanced. Comparison of the plateau values of adsorption revealed that the adsorption was most affected by electrostatic force and least by hydrophobic interaction among three. In contrast, a kinetic factor in the Langmuir-Freundlich equation, the K value in the equation, was maximal at the same transition point. The results showed that adsorption by electrostatic force was slowest.

4. Application of Protein Adsorption

The above-mentioned protein adsorption behavior can be utilized for bioengineering and biotechnology applications such as protein separation. For example, cationic particles can selectively collect acidic proteins (proteins having low isoelectric points) from the mixture of proteins at a low pH because only the acidic protein has no electrorepulsive force with the particle under this condition [8]. If the adsorption of proteins onto cationic particles is carried out at a high pH, various kinds of proteins are adsorbed including acidic protein. But, with adding salts, other proteins are gradually desorbed and the acidic protein remains on the particle surface to the last. Such protein separation has so far been done with gel column chromatography equipped with a diethylami-noethyl Sepharose gel column. Cationic particle dispersion was superior to gel column chromatography in terms of the selectivity and yield of the desired protein.

Continuous separation of proteins having different isoelectric points was carried out with the use of a stirred cell charged with carboxylated and sulfonated particles [9].

Was this article helpful?

0 0

Post a comment