Advances in Colloid and Interface Science, 35 (1991) 139-172 Elsevier Science Publishers B.V., Amsterdam
OF PLASMA PROTEINS ONTO POLYMER
TOSHIRO SUZAWA and HIROYUKI SHIRAHAMA
Department ofApplied Chemistry, Faculty OfEngineering, Hiroshima Shitami Saijo-cho, Hlgashi-Hiroshima, 724 (Japan)
CONTENTS Abstract ...................... ................... 1. Introduction 2. Surface Characterization of Latices ............ 2.1. Conductometric and potentiometric titrations ....... 2.2. Zeta-potentials ................. 2.3. Water-soluble polymer layers ............ 3. Protein Adsorption ................. 3.1. Adsorption isotherms ............... 3.2. Effects of pH and ionic strength ........... 3.3. Contribution of latex surfaces (particularly, effects of water-soluble polymer layers) ................. 3.4. Denatured-proteins adsorption ............ 3.4.1. Heat-denatured albumin ............ 3.4.2. Urea-denatured albumin ............ 3.5. Participation of coexistent electrolyte ions ........ 4. Concluding Remarks ................. References .....................
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ABSTRACT In this article we review the adsorption of plasma proteins onto polymer latices on the basis of our experimental data. First, the surface characteristics of the latices were examined. Hydrophilic polymer layers (water-soluble polymer layers) were found to exist on the surfaces of copolymer latices, e.g., a polyacrylamide (polyAAm) layer existed on the surface of the styrene/acrylamide copolymer [P(St/AAm)] latex. These diffuse layers strongly affected the protein adsorption, that is, the amount of plasma proteins adsorbed onto copolymer latices (viz. P(StJAAm) and styrene/2-hydroxyethyl methacrylate copolymer [P(St/HEMA)l latices), particularly in the alkaline pH region, was much smaller than that onto a hydrophobic polystyrene (PSI latex. The protein adsorption was also studied as a function of pH, ionic strength and electrolyte concentration. Further, the adsorbability of heat- and urea-denatured albumins was investigated. A higher affinity of denatured components for polymer latices was observed compared with that of the native components. oool-8686/w/$11.90
0 1991 Elsevier Science Publishers B.V.
140 1. INTRODUCTION
Proteins are highly surface-active due to their amphiphilic properties. Hence, proteins can interact with any interface they encounter, generally leading to adsorption of the proteins. In many practical fields, such as in the cosmetic, biomedical, pharmaceutical and foodstuff industries, the adsorption behavior of proteins at interfaces is of great importance. Recently, the adsorption of plasma proteins onto polymer surfaces has been investigated (e.g., see Refs [l-31) for developing and improving biomaterials such as the antithrombogenic artificial heart and kidney. The primary occurrence when these biomaterials are in contact with blood is the rapid adsorption of plasma proteins. This initial rapid adsorption of proteins, which can greatly affect the platelet adhesion, is strongly dependent on the chemical and physical properties of the polymer material surface. On the other hand, polymer latices have hitherto mainly been used in such industrial fields as paint, adhesive, paper and textile manufacture. In recent years their application to medical diagnostics 141,enzyme-immobilized latices 151 and materials for evaluating the affinity of plasma proteins 16-91 is being actively examined. The most advantageous point in the application of polymer latices as adsorbents for proteins is that the surface area of the latices is extremely large. Moreover, the surface properties of latex particles, e.g., the hydrophobicity (or hydrophilicity) of the latex surface, can be varied relatively easily. Therefore, one can investigate protein adsorption onto polymer latices as a function of these surface properties etc. From the above point of view, in this article we will first discuss the surface characterization of polymer latices. Subsequently, we would like to describe the adsorbability of plasma proteins onto the latices; that is, the effects of pH, ionic strength and the surface properties of the latices on the adsorption of plasma proteins will be investigated, which may provide researchers engaged in related subjects with some useful information. 2. SURFACE CHARACTERIZATION OF LATICES
As described later, protein adsorption is strongly dependent on the surface characteristics of the polymer latices. In our laboratory many different types of soap-free polymer latices have been prepared in a highly monodisperse, stable state. We describe here the surface characterization of polymer latices, which were examined mainly by using surface-chemical procedures.
In a soap-free system, the latex surfaces are stabilized only by the ionic end groups originating from initiator fragments and/or comonomers. To confirm the presence of end groups on the latex surfaces, conductometric and potentiometric titrations were carried out at 25°C under a nitrogen atmosphere. Figure 1 shows the conductometric titration curve of polystyrene (PS) latex [lO,lll. Two distinct inflection points (viz., end points of titration) are shown in the curve. The first end point (a) corresponds to the equivalence point of strong acid (-OSOg, derived from the initiator fragments (potassium persulfate). The second end point (b) probably corresponds to that of weak acid (-COO_). This weak acid group appears to originate from the oxidation of -OH formed by the Kolthoff reaction I121of -0SOj. On the basis of the results of potentiometric titrations, the surface charge densities (a) of polymer latices as.a function of pH can be determined. The results obtained are shown in Fig. 2 [lO,lll. The latices used
of 5x10e3N NaOH (ml )
Fig. 1. Conductometric titration
curveof PS latex (25’C).
were hydrophobj~PS hom~pol~er and hydro~~~~~c coworkers, i.e., styre~e~St?~-hydroxyethyl m~~hac~la~ ~~~MA~ copolymer ~~S~~M~~ and S~ac~~ic acid @AI ~~o~ymer ~~~S~/~~~~atices.The su~scr~~~ 2 and 5 of the copolymer Iatices in Fig, 2 represent the mol% of AA or HEmA used in the co~ol~eri2a~ion* As can be seen from &is ~gure~ tfre pH de~~A~ latkes is hardly distinpendence of the ~-values for FS and PfS guishable, because the quantities of carboxyl groups (-COOH, which diss~~a~ greatly in the &~ka~ino pH region) on these la&es are far fewer than those on P(S~~} latiees. On the other band, o-values for ~~S~~~ fatices increase remarkably with increasing pW. However, in the acidic region up to about pW 5, the a-value for carboxy~a~d latices is almost equal to that of other la&es, since,carboxy~groups hardly dissociate in this pH region. The o-values (except in the acidic region) for ~rbo~~a~d latices are proportioned to the amount of AA used in their copo~~arizatians*
2.2. Zeta-potentials In referring to the surface properties of materials, the zeta-potential has an important significance, for the interaction between adsorbates (proteins) and adsorbents (materials) is strongly affected by the sign and magnitude of the zeta-potentials. Figure 3 shows the pH dependence of zetapotentials for PS and P(StkIEMA) latices 111,13,141. Although little difference in the a-values between these two latices was observed (see Fig. 21, the zeta-potential of PCWHEMA) particles was considerably smaller than that of PS particles over the whole pH range measured, and decreased with increasing the amount of copolymerized HEMA. This discrepancy could probably be attributed to the structures of the electrical double layer for these latices; namely, the surface charge density (a) represents the amount of all ionized groups existing on the latex surface, while the zetapotential is the potential at the slipping plane of the electrical double layer. To clarify this, we attempted to estimate the position (t) of the slipping plane by using the following Eversole-Boardman equation [151. In tanh CzeZj4hT) = In tanh Cze$d4kZ’l-
where z is the valence of ions, e is the charge of an electron, h is the Boltzmann constant, T is the absolute temperature, & is the Stern poten-
50 40 I 30-
-Q- P(St/HEMAs) -.-
P( StlHEMAlo) 9
PH Fig. 3. Zeta-potentials of PS and PW/HEMA) latex particles as a function of pH (25X, ionic strength 0.01).
tial, and x is the Debye-Huckel parameter. The results obtained are given in Table 1 of the next section [11,121.As shown in this table, the values of t for P(StJHEMA)latices are about two times greater than that for PS latex, and t increases with increasing the content of HEMA copolymerized. From the above results, a schematic representation of the electrical double layer for PS and PWHEMA) latices can be given, see Figure 4. That is to say, a hydrated polyHEMA layer (water-soluble polymer layer) exists on the latex surface, and this layer shifts the position of the slipping plane away from the latex surface. Thus, in spite of having much the same surface charge density, the potential at the slipping plane (i.e., zeta-potential) for the P(St/HEMA) latex could be lower compared with that for the PS latex. Also the smaller Stern potential ”
aSurface charge density obtained by conductometric bPosition of slipping plane cSt.ern potential dThickness of water-soluble
Fig. 6. Transmission electron micrographs of OsOestained thin sections for copolymer latex particles.
tetroxide (OsO&stained thin sections of these latex particles. In the case of P(St/HEMA) latex, both the larger (the interior parts of latex particles) and the smaller particles (the parts near the surface of the latex particles) have microdomain structures consisting of polyHEMA domains (stained black with OsOd and PS domains (not stained with 0~0~). This means that polyHEMA domains exist not only on the surface but also in the interior of the latex particles. Whereas, for P(St/AAm) latex, only the smaller particles are stained black with Os04, indicating that polyUm can mostly exist near the latex surface but that little polyAAm exists in the latex interior. In other words, the surface of P(St/AAm) latex is covered uniformly with a polyAAm layer. Thus, there is a difference in the surface distribution of water-soluble polymer layers on the latex particles, depending on the hydrophilicity of comonomers (HEMA or AAm) used. 3. PROTEIN ADSORPTION Although more than one hundred proteins exist in plasma, we describe here mainly the adsorption of serum albumin (which is the most abundant protein in plasma and whose physicochemical properties have been studied extensively) onto polymer latices in the following ways.
3.1. Adsorption isotherms By way of example, the adsorption isotherms of bovine serum albumin (BSA) onto PS 1a t ex are given in Fig. 7 1221.The measurements were carried out at pH values of 4.2, 5.0 and 7.4. These pH values correspond to
of BSA ( mgldl
Fig. 7. Adsorption isotherms of BSA onto PS latex (25’C, ionic strength 0.01).
the pH lower than the isoelectric point (i.e.p.1of BSA, the pH in the vicinity of the i.e.p., and the pH higher than the i.e.p., respectively. The initial parts of the isotherms directly reflect the interaction between the protein and the latex, because little interactiaodn between sorbed protein molecules occurs at lower concentrations of the protein. Thus, the initial slope of the isotherm at pH 7.4 (here the electrostatic repulsion acts between the latex and the protein) is relatively lower than those at pH 4.2 and 5.0 (where the electrostatic attraction or little repulsion occurs between them). The isotherms, except that at pH 7.4, show steps which probably reflect a conformational rearrangement of adsorbed protein molecules into more compact and ordered structures [6,7,9,13,22-241 rather than a multilayer adsorption. Taking the BSA molecule to be an ellipsoid with major and minor axes, 2a (14nm) and 2b (4nm), respectively, the cross-sectional area 0 of the protein molecule is given by aab (or xbb). And, by using the following equation 1251,a closely packed monolayer coverage (A) of unperturbed ‘side-on’ adsorbed molecules yields a surface concentration of 2.5 mg/m2, but ‘endon’ adsorption requires 8.6 mg/m2.
where 3c/2& is the ‘packing factor’, M is the molecular weight of the protein (6.7 lo4 for BSA), and NA is the Avogadro number. l
From these theoretical values and the plateau values of the amount adsorbed in Fig. 7, thus, at least near the i.e.p. region, BSA molecules appear to adsorb onto hydrophobic PS latex in its ‘side-on’ form. This ‘side-on’ form of adsorption of BSA onto PS latex is also supported by photon correlation spectroscopy experiments [71. 3.2. Effects ofpH and ionic strength It is essential to examine protein adsorption under physiological conditions (i.e., at pH 7.4, ionic strength 0.15, and temperature 37°C) with regard to using polymer materials in Co. However, considering the application of the materials to protein separators, bioreactors, etc., the protein adsorption must be investigated in a wider range of pH and ionic strength. Nevertheless, there is little reported in the literature covering a wide range of conditions. Figure 8 shows the pH dependence of BSA adsorption onto PS latex at different ionic strengths 113,221.The amount adsorbed shows a maximum near the i.e.p. (ca pH 5) of BSA. This is probably because BSA molecules form the most compact structures near the i.e.p. region, and hence, more molecules can adsorb on a given surface area. Moreover, the pH at maximum adsorption shifts to a more acidic pH with increasing ionic strength. This phenomenon probably correlates with the shift of the i.e.p. of BSA toward an acidic pH with increasing ionic strength 1261,further discussion is given in Section 3.5. 4
PH Fig.8.pH dependence of BSA adsorption onto PS latex at different ionic strengths (25’C).
In the acidic pH region lower than the i.e.p. of BSA, the amount adsorbed increases with increasing ionic strength. The electrostatic repulsion in the interior of protein molecules and the lateral repulsion between adsorbed protein molecules decrease with increasing ionic strength. These cause more protein molecules to adsorb on the given surface area. The amount adsorbed in this pH region decreases with decreasing pH, despite the greater electrostatic attraction between the protein and the latex with decreasing pH. This decrease in the amount adsorbed is probably due to the greater size of the protein molecule in the more acidic pH region. Thus, the adsorbability of the protein (BSA) depends not only on the electrostatic interaction but also on the conformational alteration of the protein molecule. In the i.e.p. region of BSA it seems that the conformational alteration of BSA is not affected very much by ionic strength, and if so, the amount adsorbed probably remains constant, regardless of ionic strength. However, as is seen in Fig. 8, the amount adsorbed in this pH region decreases with increasing ionic strength. This result suggests that BSA adsorption is affected by the electrostatic interaction to some extent, even in the i.e.p. region. In the alkaline pH region (pH > 7), the amount adsorbed increases with increasing ionic strength similar to the case in the acidic region. At a low ionic strength (O.OOl),little adsorption in this pH region is observed because of the greater electrostatic repulsion between the protein and the latex. At a higher ionic strength (0.1) BSA adsorption is influenced little by pH. In this case hydrophobic interaction between BSA molecules and the PS latex is probably a dominant factor in the adsorption, because electrostatic interactions decrease with increasing ionic strength. Also, the greater conformational stability of a protein molecule at high ionic strength [271 seems to affect this small change in the amount adsorbed. Judging from the amount adsorbed, at high ionic strength, it seems that BSA molecules adsorb onto hydrophobic PS latex in a ‘side-on’ form over the whole pH range. Bovine y-globulin (ByG) adsorption onto PS latex as a function of pH and ionic strength is shown in Fig. 9 [281.The amount adsorbed shows a maximum near the i.e.p. (ca pH 7) of ByG similar to BSA adsorption. From the amount of ByG adsorbed at an ionic strength of 0.154, we can obtain the area Gs) for an adsorbed ByG molecule as a function of pH by using Eqn (4). The result obtained is given in Fig. 10. As is known, the By G (or immunoglobulin G, IgG) molecule has a Y-shaped structure with hinge-like flexible arms (Fabfraction, see Fig. 10; the other WC>fraction is more hydrophobic). Judging from the S-values obtained, it is likely that a
Ionic strength 0.154
J?ig.9. B$ adsorption onto PS latex as a function of pH and ionic strength (25X).
3000 8401 Calculated area/ molecule ( 8’ 1 Conformation
Fig. 10. Conformation of ByG adsorbed at the PS latex surface.
Be molecule adsorbs onto hydrophobic PS latex via its F, fraction as shown in Fig. 10. In the neighborhood of the i.e.p. of ByG, since the protein molecule has the most compact structure (viz., the collapsed form), the area for an adsorbed molecule becomes minimum. As the pH is moved away from the i.e.p., the molecule extends its arms gradually, and hence,
the amount adsorbed decreases as shown in Fig. 9. The conformation of ByG (or IgG) adsorbed or immobilized on polymer latices plays an important role in the antigen-antibody reaction (viz., medical diagnostics). 3.3. Contribution
of latex surfaces (particularly, polymer layers)
effects of water-soluble
As described in Section 2, it was found that the surface characteristics (or properties) of the latices were very differentfrom one another. Accordingly, it seems to be of interest to discuss the effects of latex surface properties (particularly the effects of water-soluble polymer layers) on protein adsorption. Figures 11 and 12 show the pH dependence of BSA adsorption onto PWHEMA) and P(St/fUm) latices, respectively, at a high ionic strength 0.1 [13,181. At a low ionic strength, such as 0.001, the predominant driving force for adsorption is mainly the electrostatic interactions between the proteins and the latices. Therefore, the surface properties of polymer latices probably little affect protein adsorption at a low ionic strength. Thus, it is preferable to compare protein adsorption at a high ionic strength. As described in Section 3.2., the amount of BSA adsorbed onto hydrophobic PS latex is little affected by pH-change, and is quite large. This is because the hydrophobic interaction between the latex and the protein acts
-m- P(StlHEMA5) +
Fig. 11. pH dependence of BSA adsorption onto PS and P(St/HEMA) latices (25’C, ionic strength 0.1).
153 + PS
F’ig. 12. pH dependence of BSA adsorption onto PS and P(St/AAm) latices (25’C, ionic strength 0.1).
over a wide pH range at a high ionic strength. On the other hand, BSA adsorption onto hydrophilic P(St/I-IEMA) and P(St/AAm) latices is much smaller than that onto PS latex, particularly in the pH region higher than the i.e.p. of the protein. Further, as is shown in Figs 11 and 12, this tendency is proportional to the content of HEMA or AAm copolymerized. In particular, the amount of BSA adsorbed onto P(St/AAm) latex decreases with increasing the content of AAm copolymerized, even in the acidic pH region. As described in Section 2, there exist water-soluble polymer layers on these two copolymer latices, i.e., polyHEMA and polyAAm layers exist on the surfaces of P(St/HEMA) and P(St/AAm) latices, respectively. And the thickness of these polymer layers (particularly that of polyAAm layer) increases with increasing the content of AAm (or HEMA) copolymerized (see Table 1). Protein molecules also have similar hydrated polymer layers on their surfaces. When two surfaces which have such water-soluble polymer layers approach each other, the steric repulsion (arising from the decrease in a conformational entropy due to the overlap of these hydrated polymer layers on the surfaces) acts between the two surfaces. This steric repulsion is referred to as ‘volume restriction effect’ (or ‘diffuse layer effect’), and its magnitude increases with the thickness of hydrated polymer layers 1291.Therefore, the small BSA adsorption onto these copolymer latices could be mainly attributed to steric repulsion, in addition to the decrease in the hydrophobic interaction between the latices and the protein [13,14, 18,30,311. This adsorption behavior, due to steric repulsion, is observed
most remarkably in the case of P(St/AAm) latex (see Fig. 12 and compare Table 1). Figure 13 shows the comparison between BSA adsorption onto various polymer latices at an ionic strength of 0.1[14,321. As mentioned above, the amounts adsorbed onto PWHEMA) and P(St/AAm) latices in the pH region higher than the i.e.p. (ca pH 5) of BSA are much smaller compared with that adsorbed onto PS latex. Since electrostatic attraction acts between BSA and each latex, the amount adsorbed is relatively large in the acidic pH region (pH < 4.5). In this pH region, however, the amount adsorbed onto P(St/HEMA) latex is comparable to that adsorbed onto PS latex, whereas BSA adsorption onto PCWAAm) latex remains small. This result is probably attributed to the difference in the surface structures of these two copolymer latices as shown in Fig. 6. That is to say, because there exist large domains of PS on the surface of the PWHEMA) latex, the adsorbability of BSA onto this latex shows much the same tendency as that onto PS latex; while the amount adsorbed onto the latex is much smaller than that onto PS latex, since the surface of the PCWAAm) latex is covered uniformly with a polyAAm layer. On the other hand, BSA adsorption onto (hydrophilic) P(St/AA) latex (which has a large amount of surface carboxyl groups) shows rather complicated features. This is probably because the hydrogen bonding and the electrostatic interactions between the latex and the protein affect BSA adsorption greatly 1221. 6
Fig. 13. pH dependence of BSA adsorption onto various polymer latices (%‘C, ionic strength 0.1).
Figure 14 shows bovine hemoglobin (BHb) adsorption onto various polymer latices [331. Although hemoglobin (Hb) is the most abundant protein in whole blood, very little is ordinarily present free in the plasma. However, since hemolysis accompanies surgical treatment and blood contact with foreign surfaces, Hb may be present in sufficient concentrations in the plasma at local regions where sublethal hemolysis occurs 1341.
PH Fig. 14. BHb adsorption onto various polymer latices as a function of pH (25’C, ionic strength 0.01).
As can be seen from Fig. 14, the maximum adsorption of BHb onto each latex is obtained near the i.e.p. (ca pH 6.8) of the protein as in the case for BSA adsorption (see Fig. 13). Onto hydrophilic copolymer latices [viz. P(St/HEMA) and P(St/AAm) laticesl, the amount of BHb adsorbed in the alkaline pH region is hardly distinguishable because of steric repulsion (caused by water-soluble polymer layers existing on the copolymer latex surface) and the weaker hydrophobic interaction between BHb and these latices. In the regions pH < 4 and pH > 11, an increase in the amount of BHb adsorbed onto hydrophobic PS latex is observed, but it does not occur in the case of copolymer latices. Typical hemoglobins are tetrameric proteins consisting of two a-subunits and two (3-subunits (viz., c~&). In the neutral pH region, the Hb tetramer molecule is stable, while in the regions pH
10, it dissociates into subunits (dimer and/or monomer, i.e., CX& + 2@ (and/or +2a + 26), 1351.This change in the dissociated state of the Hb molecule is irreversible in the regions pH < 4 and pH > 12 , and was confirmed by gel chromatography 1331.Hydrophilic (amino acid) groups of the protein molecule are generally located on its surface, and hydrophobic groups are buried in its interior. However, in view of the dynamics of protein structures, hydrophobic clefts exposed to the aqueous environment can instantaneously produce hydrophobic surface patches. These hydrophobic patches and the hydrophobic contacts [341between the Hb subunit chains (particularly between dissimilar chains of a-6) are likely to be exposed to the aqueous phase by dissociation of the Hb molecule. Thus, the hydrophobic interactions between these hydrophobic sites and PS latex will increase. This increased hydrophobic interaction and more efficient packing of the subunits in the adsorbed layer will result in greater adsorption of BHb onto PS latex in the regions pH < 4 and pH > 11. On the other hand, such an increase in the amount of BHb adsorbed is not observed with copolymer latices in these pH regions, for the hydrophobic interaction between copolymer latices and the (dissociated BHb) subunits is much weaker than that between PS latex and the subunits. Moreover, this lower adsorbability onto these copolymer latices was observed with bovine pancreas ribonuclease (RNase, which is not a plasma protein) 1371. As described above, protein adsorption onto surfaces having water-soluble polymer layers [viz.,onto PWAAm) and P(St/HEMA)laticesl is much smaller compared with that onto hydrophobic PS latex. The surfaces onto which little adsorption of plasma proteins occurs are expected for biocompatible (particularly antithrombogenic) materials, for the interaction between these surfaces and blood components (or living cells) is very weak [13,18,301.Prom this point ofview, the surfaces grafted with water-soluble monomers such as AAm and HEMA seem to be preferable as blood compatible materials. 3.4. Denatured-proteins
Originally, denaturation of protein means a variation of its conformation from the native state to another state; more exactly, it signifies that noncovalent bonds (viz., hydrogen bonds, hydrophobic interaction, etc., which keep the conformation of a protein molecule stable) are broken by some denaturing factors, and that leads to a change such as unfolding in the protein conformation. Generally it does not involve the breaking of covalent bonds (peptide bond). Denaturation factors such as heat (temperature), pH, chemical (urea, etc.), pressure, y-ray are considered, but we refer here to heat- and urea-denatured proteins particularly.
Before describing the adsorbability of heat-denatured proteins, we first mention the difference in the nature between native and denatured proteins. Figure 15 shows the (polyacrylamide) gel electrophoresis patterns of native and heat-denatured BSA. It can be seen from this that native BSA contains not only component 1 (albumin monomer) but also other small amounts of components 2’ (dimer) and a very small amount of 3 (probably trimer). It has been reported that a commercial native BSA contains about 10% of component 2’ (Refs f38-401; native BSA used in this work contained 8% of 2’). On the other hand, in the case of heat-denatured BSA (denatured under the conditions mentioned in Fig. 15), new components 1’ (modified monomer) and 2 (dimer of 1’) were formed accompanying a drastic decrease in the content of component 1. The analysis by densitometry indicated that the contents of components 1,1’, 2 and 3 were 11, 66, 20 and 3%, respectively. Component 1’ is formed by an intramolecular SH/S-S exchange reaction as a result of some unfolding of component 1 141,421 and is different from component 1 in the helix content and affinity for dyes. Component 2 is mainly formed by an intermolecular SH/S-S exchange reaction 141-431, i.e., by l’(SH) + l’(S S) + 2.
Migration 0 I
Fig. 15. Polyacrylamide gel electrophoresis patterns of native and heat-denatured BSA, native BSA aqueous solution (pH 19.5, ionic strength 0.01) was incubated at 65°C for 90 min.
Aoki et al. [4421analyzed the heat-denatured BSA using gel isoelectric focusing, by fractionating it into components, (defatted BSA was used but the denaturation condition was much the same as ours) and found that the values of the i.e.p. of the denatured components (1’ and 2) were pH 5.9. At an elevated temperature, BSA molecules unfold and their hydrophobic amino acid residues, such as tryptophan etc. (which are masked in the interior of the native protein), are exposed to the aqueous phase. This exposure of hydrophobic amino acid residues was confirmed by observing the greater absorbance of heat-denatured BSA than that of native BSA in the ultraviolet region near 230 nm (see Fig. 16; the absorption of light near this region is due to these hydrophobic amino acid residues). Much information on the heat-denaturation of BSA has been obtained [38,41,44-461, but little on the adsorption of the denatured BSA has been reported. Figure 17 shows the pH dependence of native and heat-denatured BSA adsorption onto PS latex by densitometry and spectrophoto2.0
aJ g 2ki 1.c Lo 2
( nm )
Fig. 16. UV-absorption spectra of native and heat-denatured 0.25 wt%, pH 6.0, ionic strength 0.01) at 25°C.
BSA @SA concentration
Fig. 17. Native and heat-denatured BSA adsorption onto PS latex as measured by densitometq and spectrophotometry (25”C, ionic strength 0.01).
metry. By using densitometry, we can determine the adsorbability of each component of heat-denatured BSA, while spectrophotometry gives the total amount of (heat-denatured) BSA adsorbed. As illustrated in Fig. 17, the amount of native BSA adsorbed determined by densitometry has almost the same value as that determined by spectrophotometry. However, the amount of heat-denatured BSA adsorbed is much greater than that of native BSA. The amount of native BSA adsorbed shows a maximum near the i.e.p. (ca pH 5) of this protein, as described previously, whereas that of heat-denatured BSA shows a maximum at a more alkaline pH (at ca pH 5.7). These results will be discussed in the next Section. Figure 18 shows the pH dependence of heat-denatured BSA adsorption onto various polymer latices. The results obtained show much the same tendency as native BSA (cf., Figs 13 and 18). That is to say, in the alkaline pH region (in the whole pH region for PCZUArn) latex), BSA adsorption onto hydrophilic copolymer latices is much smaller than that onto PS latex, and in the acidic pH region, its adsorbability onto PS and P(StfHEMA) latices shows a similar tendency. This high adsorption affinity of heatdenatured BSA compared with native BSA is probably due to increases in the hydrophobic interaction and London-van der Waals force between the latices and heat-denatured BSA. The increases in these interactions probably arise from the unfolding of BSA molecules caused by heat denaturation. As mentioned above, the pH at maximum adsorption for
PH Fig. 18. Heat-denatured BSA adsorption onto various polymer latices as a function of pH by densitometry (25”C, ionic strength 0.01).
heat-denatured BSA shifts to a more alkaline region than that for native BSA (see Fig. 17, cf. Figs 13 and 18). Assuming that maximum adsorption is obtained near the i.e.p. of BSA (the i.e.p. pH for heat-denatured components is higher than that of native BSA as mentioned before), and the denatured components (1’ and 2) adsorb preferentially onto polymer latices, the pH at maximum adsorption would shift to a more alkaline pH region. These will be discussed in more detail in the following section. In recent years the high-performance liquid chromatography (HPLC) technique has been used for analyzing the competitive adsorption of plasma proteins or albumin (containing oligomers) onto latices 140,471. However, it seems that components 1 and 1’ (contained in heat-denatured BSA) cannot be separated by HPLC, though the components (1, or l’, and 2) whose molecular weights are different from one another can be separated. Therefore, in this study both polyacrylamide gel electrophoresis and densitometry techniques were simultaneously used for analyzing the adsorbability of heat-denatured BSA components onto the latices. Figure 19 shows each component adsorption of heat-denatured BSA onto hydrophobic PS latex. The solid lines (reference) and the dashed lines indicate the absorption curves drawn by the densitometer before and after BSA adsorption onto the latex, respectively, and pH values denoted are those of adsorption experiments. The difference in area between the solid
and dashed lines corresponds to the adsorbed amount of the heat-denatured components. This value for each component is shown as the “adsorption ratio (%)” in this figure, though the adsorption ratio of component 3 is not shown because this component scarcely exists in the native and heatdenatured BSA. Reference -----
pH 9.70 1’
Fig. 19. Adsorption of each component of heat-denatured BSA onto PS latex by densitometry; solid (reference) and dashed lines are absorption curves by densitometer before and after adsorption, respectively. pH values denoted are for each adsorption experiment.
As can be seen from Fig. 19, at pH 3.95 there is not much difference in the adsorbability between the native (1) and heat-denatured (1’ and 2) components, because the electrostaticattractionacts between the components and the latex at this pH. However, at pH 6.10 (near the i.e.p. (PH 5.9) of the heatdenatured components), the denatured components (1’ and 2) adsorb preferentially onto PS latex as compared with the native component (1). This preferential adsorption of the denatured components is probably attributed to the greater hydrophobic interaction and London-van der Waals force between these denatured components and PS latex than those between the native component and the latex. As described previously, (native) BSA molecules are unfolded and made more hydrophobic (as a result of the exposure of hydrophobic amino acid residues to the aqueous phase) by heat treatment. Thus, the denatured components will be able to adsorb preferentially onto PS latex. Since the electrostaticrepulsion acts between the protein and the latex, the adsorption of each component onto PS latex at pH 9.70 is considerably lower than that at pH 6.10. Nevertheless, component 1’ (modified monomer) can adsorb to some extent, even at pH 9.70.
The result for P(St/AAm) latex is shown in Fig. 20. The adsorbability of each component onto this latex is much weaker than that onto PS latex (cf’. Figs 19 and 20). As is described in the section of surface characteristics of polymer latices, the surface of P(St/AAm) latex is covered uniformly with the hydrophilic polyacrylamide (polyAAm) layer, and, the steric repulsion referred to as “the diffuse layer effect” (viz., the volume restriction effect) acts between the hydrated layer of P(St/AAm) latex and BSA molecules. Therefore, the lower affinity of heat-denatured components for P(St/AAm) latex is probably due to this steric repulsion and to the smaller hydrophobic interaction between this latex and BSA molecules [13,181. The steric repulsion appears to be more effective in the alkaline pH region (see the result at pH 8.90 in Fig. 20 where the electrostatic repulsion acts between the protein and the latex). However, in general, the adsorbability of heat-denatured components is greater than that of the native component. There is probably a range of nonelectrostatic forces involved (London-van der Waals force being one among them) which can drive the protein adsorption. On the other hand, in the case of P(St/HEMA) latex having a microdomain structure on its surface, results similar to that for PS latex in the acidic and the i.e.p. region, and to that for P(St/AAm) latex at the alkaline pH were obtained (the data are not shown); that is, heatdenatured components adsorbed preferentially onto P(St,!HEMA) latex in the acidic and i.e.p. region, while at the alkaline pH each component hardly adsorbed onto this latex.
pH 8.90 1’
Fig. 20. Adsorption of each component of heat-denatured BSA onto P(St,&Am) latex by densitometry; solid and dashed lines are the same as in Fig. 19.
3.4.2. Urea-denatured albumin LIZ41 Urea is known as a denaturant of proteins. The denaturation mechanism of BSA in aqueous urea solution has been studied by many authors 139,~541. Their results can be summarized as follows: urea breaks hydrogen bonds and hydrophobic interactions which contribute to the stability of the tertiary structure of the protein, and unfolds the protein molecules 1511. In concentrated urea solution (above 5 iV), unfolded BSA molecules show a tendency to aggregate 1541. This is much more pronounced in the neutral and the alkaline pH region [39,501. The aggregated forms of BSA seem to be induced by an intermolecular SlWS-S exchange reaction 139,50,531. The i.e.p. of BSA in urea solution shifts to a more alkaline pH region than in the absence of urea 149,531. Thus, also in the case of urea-denatured BSA, almost the same components as heat-denatured BSA are probably formed in urea solution. Figure 21 shows the adsorption isotherms of BSA onto (hydrophobic) polymethyl methacrylate (PMMA) latex in the presence of 8 M urea. The isotherms are considerably different from those in the absence of urea (cf., Figs 7 and 21). The initial slopes of the isotherms in urea solution are very sharp, regardless of pH; that is, high affinity types of isotherms are obtained. In the presence of urea, hydrophobic amino acid residues masked in the interior of native BSA molecules are exposed to the aqueous phase [51,55,561. Therefore, this high affinity of urea-denatured BSA for PMMA latex could be due to the increment of hydrophobic interactions between unfolded BSA molecules and the latex.
60 40 EEA concentration
Fig. 21. Adsorption isotherms of BSA onto PMMA latex in 8Murea (25X, ionic strength 0.01).
Figure 22 shows the effect of urea on BSA adsorption onto PMMA latex as a function of pH. As can be seen from this figure, the pH at maximfum adsorption in the presence of urea shifts to a more alkaline pH compared with that in the absence of urea (i.e., ca pH 5 + 6). Further, in the pH region higher than ca pH 5.2, the amount adsorbed in urea solution is considerably greater than that in the absence of urea. As described in Section 3.4.1., these results are probably due to the preferential adsorption of ureadenatured components of BSA compared with the native component. On the contrary, in the acidic pH region (pH < 51, the amount of urea-denatured BSA adsorbed is smaller than that of native BSA. The degree of denaturation of BSA by urea depends on pH, and it is higher at alkaline pH than at acidic pH (in the case of heat-denatured BSA, this had already been confirmed 1571). Probably this is one reason for the above result. Moreover, the presence of urea in the solution makes a BSA molecule expand regardless of denaturation. This expanded form of the protein molecule will lead to a decrease in the amount adsorbed.
0 PH Fig. 22. Effect of urea on BSA adsorption onto PMMA latex (25’C, ionic strength 0.01).
As described above, the amount of urea-denatured BSA adsorbed shows a maximum at more alkaline pH compared with native BSA, similarly to heat-denatured BSA. Further, as mentioned before, also in the cases of other (plasma) proteins, the maximum adsorption was obtained near the
Fig. 23. Effect of urea on electrophoretic mobilities of BSA-covered PMMA latex particles (25-C, ionic strength 0.01).
i.e.p. of each protein. Figure 23 shows the electrophoretic mobilities of BSA-covered PMMA particles in the presence and absence of urea. It can be seen that the i.e.p. of BSA (adsorbed onto the latex particle) shifts to a more alkaline region with increasing urea concentration, and at 5 M urea the i.e.p. is pH 6 which agrees with the pH at maximum adsorption in Fig. 22. Thus, in general, it may be concluded that the maximum adsorption of the protein is obtained near its i.e.p. pH (more accurately, the i.e.p. of the protein adsorbed onto polymer materials). 3.5. Participation of coexistent electrolyte ions There are many electrolyte ions in blood and living cells. Serum albumin is well known as a carrier protein, because numerous inorganic and organic ions, nutrients, and drugs are easily bound to albumin molecules. This high binding affinity of ions, etc., to albumin is probably due to the configurational adaptability of this protein (which can vary its steric conformation according to the circumstances) and the hydrophobic patches existing on the protein surface . First, the effects of electrolyte anions on BSA adsorption was studied [261.
Figure 24 shows the effects of three electrolyte anions (Cl-, CH,COO-, and SCN_) on BSA adsorption onto PS latex at ionic strength 0.1 1261. These electrolyte anions were used as the sodium salt (i.e., NaCl, CH&OONa and NaSCN) to avoid the effects of (electrolyte) cations on the adsorption. In the system, however, a small amount of HCl or NaOH was present for adjusting the pH. As can be seen from this figure, the adsorbability of BSA in both Cl- and CH&OO- media shows almost the same tendency. While the adsorption behavior in SCN medium is very different from that in the other anions (Cl-and CH$003 media. That is, in SCN medium, the pH at maximum adsorption shifts to a more acidic pH and the maximum adsorption is greater compared with those in other media. The binding affinity of SCN- to BSA molecule is much greater than that of Cl- (C!H&OO_), especially in the i.e.p. and acidic pH regions f59-621. Hence, the i.e.p. of BSA in the SCN- medium shifts to a more acidic pH than that in the Cl- medium 163,641,and more compact structures of BSA molecules form in the SCN- medium because of the decrease in the electrostatic repulsion 1651.These probably contribute to the above-mentioned BSA adsorbability in the SCN- medium (in the pH region lower than the i.e.p.). On the other hand, in the pH region higher than the i.e.p. of BSA, the amount adsorbed in the SCN- medium is somewhat smaller than that in other anionic media. It was reported that SCN- reacted mainly with tryptophan (i.e., hydrophobic amino acid residues) of BSA in the neutral
2 ’ m t ‘2 1
Fig. 24. Effects of electrolyte anions on BSA adsorption onto PS latex (25’C, ionic strength 0.1).
pH region, in addition to the electrostatic interactions with positively charged groups surrounding the hydrophobic area 1661.And, the binding entropy of SCN to BSA molecule, AS (positive value), is larger than that of Cl- 1671.Moreover, the electrolyte anions used here are in the lyotropic series (or Hofmeister series) in the order, SCN- < Cl- < CHsCOO- 163,681. All these facts indicate that the hydrophobic interaction (between BSAand the latex) and the dehydration power of the SCN- medium are smaller than those in other media. Thus, in this pH region, the amount of BSA adsorbed in the SCN- medium will decrease. Figure 25 shows the electrophoretic mobilities of BSA-covered PS particles as a function of pH in three anionic electrolyte media 1261.As can be seen from this figure, in all anionic media, the i.e.p. of (the adsorbed) BSA shifts to a more acidic pH with increasing ionic strength. This result is probably related to the pH shift of the maximum adsorption of BSA to the acidic side with an increase of ionic strength (see Fig. 8). Further, the i.e.p. of (the adsorbed) BSA in the SCN- medium shifts to a more acidic pH compared with that in other anionic media, which agrees with the results obtained in Fig. 24.
Fig. 25. Effects of electrolyte anions on electrophoretic mobilities of BSA-covered PS latex particles (25%).
Next, we describe the participation of electrolyte cations in BSA adsorption 1691.In particular, to investigate the effects of divalent cations on protein adsorption is interesting and important for developing and im roving biomaterials, for example, considering that the calcium ion (Ca8+) is the only blood coagulation factor which is not a protein (other factors are
proteases). Three different kinds of electrolyte cations (Na+, Ca2+, and Mg2’) were used as the chloride. Figure 26 shows the effects of these electrolyte cations on BSA adsorption onto PS latex 1691.In the acidic pH region lower than the i.e.p. of BSA (PH < 51,the differences in the amounts adsorbed in these media are hardly distinguishable because of the electrostatic attractions between the latex and the protein. Since a BSA molecule is positively charged as a whole in this pH region, there seems to be little cation binding to the protein molecule. It is generally known that monovalent cations such as Na+ hardly bind to serum albumin [701. In fact, ion chromatography analysis revealed that no binding of electrolyte cations was observed at a pH < 5 1691.Moreover, the cation binding to PS latex in the acidic region (particularly in the case of Na+) was negligibly small [691.Thus, these facts lead to small differences in BSA adsorption in all cationic media. On the other hand, in the pH region higher than the i.e.p. (pH > 5), the amounts adsorbed in divalent cationic media are greater than those in a monovalent medium. In this pH region, the electrostatic repulsion acts between PS latex and BSA. Norde and van Dulm pointed out that electrolyte cations were incorporated into the contact interface between the latex and albumin molecule upon adsorption, and divalent cations were incorporated to a greater extent compared with monovalent cations [71,721. We also
“E z 2.0-0 _?I ;; z ld l.O;: m 4
PH F'ig. 26.Effects ofelectrolyte cations on BSA adsorption onto PS latex (25’C, ionic strength
measured the amounts of electrolyte cations incorporated into the BSAlatex interface [691. As a result, the divalent cation (Ca2’) has a much stronger tendency toward incorporation compared with the monovalent cation (Na+). Owing to the negatively charged BSA adsorbed onto the (negatively charged) latex, anion charges may be accumulated in the BSAlatex interface at pH > 5. Accordingly, it can be considered that the incorporation of divalent electrolyte cations into the contact interface prevents such an accumulation of anionic charge. Further, zeta-potentials of PS particles in divalent cationic media were smaller than that in a monovalent medium [691. These facts lead to the decrease in the electrostatic repulsion between the latex and BSA in divalent media. Furthermore, the dehydration power of a divalent cation dominates that of a monovalent cation. In the region pH > 5, consequently, BSA adsorption in divalent media will become greater than that in a monovalent medium. From the above results, a schematic representation for the participation of the divalent cation (Ca2’) in the protein (BSA) adsorption onto the (PS) latex is given in Fig. 27. The divalent cation seems to play the role of bridging between the surface acid groups of the latex and carboxyl groups of the BSA molecule upon adsorption 1721. Probably, the incorporation of divalent cations prevents the accumulation of anion charges and facilitates the protein adsorption. On the other hand, there is a little incorporation of a monovalent cation (Nat) to prevent the anion accumulation, but the amount incorporated is considerably smaller 1691because of its inability to bridge between both anionic groups of the latex and the BSA molecule.
BSA molecule l --+
Fig. 27. Schematic representation for a divalent cation (Ca2’) participation in BSA adsorption onto the (PSI latex (pH > 5); other small ions such as H’ and Cl-are not depicted to avoid complexity.
170 4. CONCLUDING REMARKS
We described in this article plasma protein adsorption onto polymer latices. From the measurements of surface characteristics of polymer latices, it was found that watersoluble polymer layers (i.e., polyAAm and polyHEMA layers) existed on the surfaces of P(St/AAm) and P(SljHEMA) latices. Plasma protein adsorption onto these copolymer latices was hardly discernible particularly in the pH region higher than the i.e.p. of each protein. This suggests that such latices as P(St/AAm) and P(St/HEMA) having water-soluble polymer layers on their surfaces can be expected to act as antithrombogenic materials. Heat- and urea-denatured BSA adsorption onto polymer latices was also studied. As a result, the phenomena ofgreater adsorption and the shift of pH for maximum adsorption to a more alkaline region were observed for denatured BSA. These results are due to the preferential adsorption of denatured components of BSA compared with the native component. Lately, composites prepared from heat-denatured albumin and hydrophilic vinyl polymers as carriers for controlled slow release of anticancer drugs have been used [731.Hence, it has become of great interest to investigate the adsorption of denatured proteins, not only from a basic aspect, but also for practical applications. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
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