On the Adsorption of Proteins on Solid Surfaces, a Common but very complicated phenomenon

On the Adsorption of Proteins on Solid Surfaces, a Common but very complicated...

(Parte 1 de 4)

JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 91, No. 3, 233-244. 2001

On the Adsorption of Proteins on Solid Surfaces, a Common but Very Complicated Phenomenon

Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, 3-1-1 Tsushima-Naka, Okayama 700--8530, Japan

Received 25 December 2000/Accepted 9 January 2001

Adsorption of proteins on solid surfaces and their interaction are major concerns in a number of fields such as biology, medicine, biotechnology and food processing, and play an important role from various points of view. Based on practical viewpoints, information on the conformation of the adsorbed protein as well as adsorption characteristics is essential for a system's performance. Although there are still many problems to be solved, extensive studies in recent years, owing to the development in instrumentation and instrumental tech- niques, reveal the adsorption behavior of proteins in detail. Here, we stress the importance and interesting aspect of protein adsorption on solid surfaces by reviewing findings that have been obtained in recent years.

\[Key words: adsorption, protein, enzyme, peptide, amino acid, conformational change\]

An interface that is formed between two different phases usually has a higher standard free energy than the bulk phase. As a result, the interface is apt to be thermo- dynamically stabilized by adsorbing any substances that are different from the solvent molecules. The substances are more or less varied in their structures upon adsorp- tion on the surface and sometimes change their func- tions. In particular, adsorption of proteins on a solid sur- face is a generally observed phenomenon in various fields and the changes in their structures and functions upon adsorption as well as the adsorbed amounts some- times have a very important consequence. In this paper, we intended to review the findings obtained thus far in- cluding those of our recent studies on the adsorption behavior of proteins at the solid/liquid interface and the mechanisms of interaction, in connection with industrial- related fields.

Adsorption of proteins on a surface and its interaction are major concerns in a number of fields, such as biol- ogy, medicine, biotechnology, and food processing and sometimes play an important role in a system's perfor- mance. Table 1 lists various fields concerned with the adsorption of proteins or enzymes with respect to im- portant related factors. In preparing immobilized enzymes by the adsorption method either with or without crosslinking, their activity is greatly influenced by their adsorption behavior; not only the adsorbed amount of enzyme but also its orienta- tion upon adsorption affects the activity and stability of the immobilized enzyme (1, 2). An enzyme-linked im- munoassay is often used to measure trace amounts of protein by the antigen-antibody interaction, in which the antigen or antibody is first allowed to adsorb onto a plas-

* Corresponding author, e-mail: kazuhiro@biotech.okayama-u.ac.jp. phone: +81-86-251-8200 fax: +81-86-251-8264 tic surface. When the antigen is adsorbed on the surface, its epitope should be oriented in such a way that it can properly recognize the antibody (3, 4). Thus, the orienta- tion of the protein molecule on the surface would affect analytical sensitivity.

In the design of biocompatible materials for surgical implants, particular adsorption of fibrinogen should be suppressed as much as possible to avoid blood agglutina- tion. From this viewpoint, the adsorption behavior of fibrinogen on the surface of materials such as hydroxy- apatite in the presence and absence of other proteins has been extensively investigated (5-7).

The adsorption behavior of proteins onto chromato- graphic columns affects the separation efficiency in chro- matographies, such as hydrophobic, ion-exchange, and hydroxyapatite chromatographies, since hydrophobic and electrostatic forces affect the proteins/surface interac- tions. In ion-exchange chromatography, an overall elec- trical charge usually determines the elution profiles of the proteins. However, the electrical property of a limit- ed region of the surface of the protein molecule would sometimes critically affect the elution profiles (8).

Protein solutions or liquid foods containing proteins or microbial suspensions are sometimes treated by mem- brane separation methods, such as ultrafiltration and reverse-osmosis using a porous membrane, for concentra- tion or dialysis. The main problem in these techniques is the adhesion of proteins or proteinaceous soils onto the membrane surface (9). Similarly, in food manufacturing processes and bioprocesses including those that use biore- actors, proteinaceous soils adhere to the wall surface of the equipment or pipes. The adherence behavior of soil particles on the solid surfaces affects the efficiency of detergents during the cleaning process (10, 1).

In biosensors for monitoring cell culture or blood glu- cose level in diabetic patients in situ, their specificity, sen- sitivity, and durability depend on protein adsorption on the sensor surface (12). Similarly, the proper transport and delivery of drugs from polymeric microcarriers are intimately linked with protein adsorption (13). In these

234 NAKANISHI ET AL. J. BIOSCL BtOENG., TABLE 1. Various fields or events in which adsorption of proteins onto solid surfaces plays an important role

Various fields or events Important factors

Adsorbed amount Selectivity for adsorption Orientation/conformation of the adsorbed protein

Design of immobilized enzyme Enzyme-linked immunoassay Design of biocompatible materials Adsorption chromatography Membrane separation Foods/bioproducts manufacturing processes Biosensors Cultivation of animal cells Drug delivery systems Reaction using soluble enzyme

For the factor(s) that is primarily important in the function or efficiency, the "circle" symbol is given.

cases, the extent of protein adsorption should be re- duced as much as possible.

In the cultivation of anchorage-dependent cells, it seems likely that cell adhesion in the presence of serum is governed by a competition between adsorption of extracellular matrix (ECM) proteins secreted by the cells or proteins that are already present in the serum, mainly fibronectin, vitronectin, and collagen, and adsorption of the serum proteins (14): the adsorption of serum pro- teins prior to that of ECM proteins inhibit or delay cell adhesion. In the formation of dental plaque and adher- ence of microorganisms on surfaces in a marine environ- ment, preformation of a protein layer is requisite. Thus, with a view to their prevention, the adsorption of pro- teins has been studied (15, 16).

In enzymatic reactions in glass or plastic vessels, en- zymes are apt to be inactivated on adsorption onto the wall. Thus, when the enzyme concentration is sufficiently low, this effect of inactivation of enzyme adsorbed on the wall could not be ignored.

Although in liquid foods composed of emulsion, emul- sification properties such as stability and dispersibility of micelles are influenced by the adsorption of the peptide moiety onto the micelle surface, these subjects will not be discussed in this article.

Adsorption of proteins on the solid surfaces has been elucidated from both points of view, i.e., adsorbed amount and conformational changes upon adsorption. Here, experimental methods for determining conforma- tional changes as well as the adsorbed amount are briefly shown.

Methods for measuring amount of adsorbed proteins

Measurement of the amount of adsorbed proteins re- quires high accuracy since the adsorbed amount per unit area is extremely low. At room temperature, the amount of proteins adsorbed is on the order of several milligrams per unit square meter as will be shown later. Various techniques have been used to measure the amount of adsorbed proteins on solid surfaces yielding several other information. In Table 2, the experimental techniques are summarized including a brief description of their principles and obtainable information other than the amount of adsorbed proteins. In a conventional adsorption experiment, hereafter referred to as the depletion method, the amount of ad- sorbed proteins is determined based on the decrease in protein concentration in the solution after the solid sur- face have come in contact with the solution. In general, this method requires a large surface area to be sufficient- ly accurate. Therefore small-particle substances or latex are often used as substrates (17-21).

The concentration of the adsorbate in the solution is measured using an appropriate assay. For example, pro- teins may be quantified by the Lowry method or labeled with a radioisotope, 125I for example (2, 23). In the latter case, the decrease in radioactivity in the solution is a measure of the adsorption. The radioactive tracer tech- nique can be used to study not only the adsorption from a single-component solution but also the competitive adsorption of various components of a mixture (24).

Direct measurement of the amount of proteins adsorb- ed on the surface can be conducted using several tech- niques. For example, an enzyme-linked immunosorbent assay (28) and radiolabeling (25) are used for this pur- pose, although these two methods can be used only with residual proteins after the surfaces are rinsed. The quartz crystal microbalance (QCM) technique is based on the change in the oscillating frequency of piezoelec- tric devices upon mass loading. The most common device used in this technique is an AT-cut quartz crystal with electrodes positioned on opposite sides. The QCM technique can provide information about the adsorption and desorption processes, and has been used to monitor the adsorption of proteins on gold and chromium sur- faces employed as electrodes (3, 26, 27).

Some optical techniques have also been used for direct measurements of the amount of proteins adsorbed on solid surfaces. One example is ellipsometry, which is based on changes in the state of polarized light upon reflection. The changes measured are interpreted in terms of the refractive index and thickness of a thin adsorbed film. The refractive index gives the average adsorbed protein concentration. Thus, on the basis of the thickness and concentration of the protein layer, the adsorbed state of the protein can be predicted. In addition to the static measurement of the amount of protein on the sur- face (29, 30), in situ measurements of adsorption and desorption of proteins have been conducted for various surfaces using ellipsometry (31, 32).

Total internal reflection fluorescence (TIRF) is another optical technique. When a beam of light is incident on an interface between two transparent media from the side of the medium with the higher refractive index so that it is completely reflected at the interface, an evanes- cent wave penetrates into the medium with a lower re-

Vot. 91, 2001 ADSORPTION OF PROTEIN 235

TABLE 2. Techniques used to investigate protein adsorption behavior

Technique Principle Information to be obtained Ref.

Depletion Decrease in solute concentration after incubation Amount of adsorbed molecules 17-21 with solid surface

Radiotracer Decrease in concentration of radioisotope-labeled Amount of molecules adsorbed from single- 21-25 molecules in solution and multicomponent solutions Radioactivity on surface due to Amount of irreversibly adsorbed molecules radioisotope-labeled molecules adsorbed

Quartz crystal Change in oscillating frequency of Courses of adsorption and desorption 3, 26, 27 microbalance (QCM) piezoelectric devices upon mass loading

Enzyme-linked Epitope recognition by primary antibodies Amount of irreversibly adsorbed molecules 28 immunosorbent assay (ELISA)

Ellipsometry Change in the state of polarized light upon Amount and thickness of adsorbed protein and 29-32 reflection their changes

Total internal reflection Fluorescence due to surface-adsorbed Amount of fluorophores adsorbed on surface 3-35 fluorescence (TIRF) molecules excited by evanescent field

Neutron reflection Reflectivity of neutrons at solid-water interface Amount and layer thickness of protein 36, 37 adsorbed on surface

Fourier transform Change in infrared absorption spectrum of Conformation of protein adsorbed on surface 29, 38 infrared spectroscopy protein on adsorption (FTIR)

Fluorescence spectroscopy Change in fluorescence spectrum of protein Conformation of protein molecules on surface 39 on adsorption

Atomic force microscopy Atomic interaction between surface and Three-dimensional image of surface 40 (AFM) scanning probe

fractive index. TIRF instruments are designed to mea- sure fluorescence intensities due to surface-adsorbed macromolecules excited by the evanescent field. The TIRF technique was used to study the adsorption of BSA, labeled with such fluorescent molecules as fluorescein isothiocyanate (FITC) and eosin isothiocyanate (EITC), from flowing solutions onto a thin polymer film coated on a glass plate (3, 34). The TIRF technique has a high potential for providing information on competitive ad- sorption, interfacial conformational changes, and sur- face mobility of the adsorbed protein (35). Recently, neutron reflection has also been applied to the study of proteins adsorbed at a solid-liquid interface (36, 37).

Methods for measuring conformational changes

The structure or conformation of adsorbed proteins has also been studied extensively using some of the tech- niques shown in Table 2. Ellipsometry provides the aver- age thickness and density of the adsorbed protein layer and thus some information could be obtained on the protein structure as described above.

Using Fourier transform infrared reflection (FTIR), more detailed information on the structure of proteins upon adsorption can be obtained. Fourier transform in- frared reflection adsorption spectroscopy (FTIR-RAS) was combined with ellipsometry to study the conforma- tion of ~-lactoglobulin (hereafter abbreviated as /%Lg) adsorbed on gold (29). FTIR spectroscopy coupled with attenuated total reflectance optics (FTIR-ATR) was also used to study conformational changes of adsorbed pro- teins (38).

Measurement of fluorescence due to tryptophan residues, in addition to circular dichroism (CD) spec- troscopy, was used to obtain information about the structure of protein molecules on such small particles as Teflon latex (diameter: d~215 nm) and silica suspension

(d~ 15-18 nm) (19, 39). Deposits of proteins on graphite and gold surfaces were observed using atomic force microscopy (AFM) (40). In the study on the adsorption behavior of enzymes, additional information on the structural change could be obtained by monitoring the activity change.

Thus far, a number of data on adsorption behavior of proteins onto solid surfaces have been reported particu- larly at room temperature where their denaturation is usually negligible.

Amounts of adsorbed proteins Table 3 shows some typical experimental data on the amounts of pro- teins adsorbed on surfaces of solid materials measured at room temperature. The experimental or estimated plateau (saturated) values in the adsorption isotherm were taken from the literature. The amount of proteins adsorbed at room temperature is on the order of several milligrams per square meters, varying with the kind of protein, type of surface, and adsorption conditions.

(Parte 1 de 4)

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