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Water Interface
JOURNAL OF COLLOID AND INTERFACE SCIENCE ARTICLE NO.
178, 426–435 (1996)
0137
The Role of Electrostatic Forces in Anomalous Adsorption Behavior of Phosvitin at the Air/Water Interface SRINIVASAN DAMODARAN 1
AND
SHIQUIAN XU
Department of Food Science, University of Wisconsin—Madison, 1605 Linden Drive, Madison, Wisconsin 53706 Received February 16, 1995; accepted August 22, 1995
The adsorption of phosvitin at the air–water interface has been studied to elucidate the influence of electrostatic forces on protein adsorption at liquid interfaces. Because of its polyanionic character, adsorption of phosvitin at the air–water interface takes place only at low pH, but not near neutral pH. Phosvitin adsorbed at an initial pH of 2.0 is completely desorbed from the interface when the pH is increased to neutral pH. The saturated monolayer coverage for phosvitin is about 1.25 mg/m 2 at pH 2.0. However, in spite this significant amount of adsorption, no decrease in surface tension occurs. Instead, a consistent increase in surface tension of the solution occurs, which apparently violates the Gibbs adsorption equation. A model based on the configuration of phosvitin at the interface has been proposed to explain the thermodynamic reasons for this apparent violation of the Gibbs equation. It is shown that phosvitin is anchored to the interface only via a short C-terminus hydrophobic segment and the rest of the highly hydrophilic molecule is suspended in the subsurface. These suspended loops exert an electrostatic pull on surface water molecules, causing an increase in surface tension. However, the reduction in free energy resulting from removal of the hydrophobic segment from water to the interface is much greater than the increase in surface tension caused by charge–dipole interactions, so that there is actually a net reduction in free energy of the system. Thus, although adsorption of phosvitin apparently violates the Gibbs adsorption equation, it does not violate the basic thermodynamic principle. The results also show that proteins can adsorb to an interface against seemingly excessive electrostatic repulsive forces through attachment of only a small hydrophobic peptide segment. q 1996 Academic Press, Inc.
Key Words: phosvitin; adsorption; air–water interface; electrostatic forces; kinetics of adsorption.
(1, 2). Although it is recognized that the tendency of proteins to adsorb at phase boundaries, especially at air/water and oil/water interfaces, arises because of their amphiphilic nature, the exact role of electrostatic forces in the adsorption process is not well understood. Several studies relating to adsorbed and spread proteins films at various interfaces have been reported (3–14). However, only a few of those studies have dealt with the role of electrostatic forces in the kinetics of protein adsorption at the air–water interface (6, 8). MacRitchie and Alexander (6) reported on the effects of spread monolayers of various negatively and positively charged molecules on adsorption of lysozyme from a dilute solution to the air–water interface. Although this approach is useful for elucidating the effects of charge–charge interactions on protein adsorption to a charged monolayer, information regarding interaction of the force field of a clean interface with the electrostatic free energy of a protein on the latter’s rate of adsorption at the interface cannot be obtained from such an approach. The influence of electrostatic free energy of a protein on its adsorption at liquid interfaces can be best understood by studying the kinetics of adsorption of phosvitin. Phosvitin is a phosphoglycoprotein found in egg yolk of all avian species. At pH 7.0 it has a net charge of about 0179. The high net charge and the sensitivity of its conformation to changes in its electrostatic free energy make phosvitin an ideal candidate for studying the influence of electrochemical potential on adsorption of proteins at interfaces. MATERIALS AND METHODS
INTRODUCTION
Adsorption and film formation of proteins at interfaces is a complex phenomenon, influenced by the interaction of the interfacial force field with various molecular chemical potentials, such as hydrophobic, electrostatic, hydration, and conformational (entropic) chemical potentials, of proteins 1
To whom correspondence should be addressed.
0021-9797/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Phosvitin has a molecular weight of about 35,000 and contains 217 amino acid residues, of which 123 residues are serine residues (15, 16). Of the 123 serine residues, 118 are phosphorylated and 5 are ‘‘free’’ (17). The 4 threonine residues in phosvitin also are phosphorylated. The amino acid sequence of phosvitin (Fig. 1) shows that it contains only about 10% nonpolar amino acid residues, 6.5% acidic residues (Asp / Glu), and 18% basic residues (Lys / Arg / His). The region between residues 55 and 155 contains long stretches of phosphoserine residues interspersed with
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FIG. 1. Amino acid sequence of hen egg phosvitin (from Ref. (7)).
lysine, arginine, and asparagine residues. The only significant grouping of nonpolar amino acid residues is found at positions 205–216. The remainder of the molecule is extremely hydrophilic. Phosvitin carries a net charge of about 0179 at pH 7.0. Because of this high net negative charge, the physicochemical properties of phosvitin resemble that of a typical polyanionic polymer. At neutral pH, phosvitin predominantly exists in a random coil conformation, whereas at pH 2.0 its conformation is about 67% b-sheet and 11.5% a-helix (18–20). Hen egg phosvitin was obtained from Sigma Chemical Co. (St. Louis, MO). All salts used in this study were of ultrapure (Gold Label) grade from Aldrich Chemical Co. (Milwaukee, WI). The surface tensions of salt solutions were comparable to reported values and did not change during aging, which indicated that the salts were free of lowmolecular-weight organic impurities. [ 14C]Formaldehyde was from New England Nuclear Co. (Boston, MA). All other reagents were of analytical grade. Extreme care was taken in purifying water for adsorption studies. A Milli-Q ultrapure water purification system (Millipore Corp., Bedford, MA) with a Qpak1 cartridge package (composed of activated charcoal, reverse osmosis, ion exchange, and ultrafiltration cartridges) capable of removing inorganic and organic impurities was used to purify the water. The resistivity of water was usually 18.2 mV cm. To check the water quality, the surface tension of water was measured at 207C. If the surface tension of water was not 72.9 { 0.1 mN/m and did not remain constant during 24 h of aging, it was discarded. Phosvitin was radiolabeled by reductive methylation of amino groups with [ 14C]formaldehyde at pH 7.0 as described in detail elsewhere (1). Phosvitin concentration was determined using an E 1% value of 5.09 at 275 nm (21). The specific radioactivity of the labeled protein was about 0.608 mCi/mg. The kinetics of adsorption of phosvitin at the air–water interface was studied as described in detail elsewhere (1, 2,
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22). Briefly, protein solutions for adsorption studies were prepared by mixing an aliquot of a stock solution ( Ç0.55 mg/ml) of 14C-labeled phosvitin with 125 ml of water preadjusted to the required pH. The final pH of the dilute solution was noted. An aliquot of the final solution (120 ml) was poured into a Teflon trough (21 1 5.56 1 1.27 cm) and the surface was cleaned by sweeping with a fine capillary attached to an aspirator. The rate of change of surface concentration of the radiolabeled protein was monitored by measuring surface radioactivity using a rectangular gas proportional counter (8 1 4 cm) (Ludlum Measurements, Inc., Sweetwater, TX) as described elsewhere (1). The entire experimental setup was housed in a refrigerated incubator maintained at 24 { 0.27C and 90–95% relative humidity. A calibration curve relating cpm versus surface radioactivity ( mCi/m 2 ), constructed by spreading 14C-labeled b-casein on the air–water interface, was used to convert cpm to mCi/ m 2 . The methods of spreading, construction of the standard curve, and the rationale for using radiolabeled b-casein as a calibration standard have been described in detail elsewhere (22). The surface concentration (mg/m 2 ) was calculated by multiplying surface radioactivity with specific radioactivity ( mCi/mg) of the protein. The contribution of bulk radioactivity to cpm was corrected using a standard curve relating cpm versus specific radioactivity ( mCi/ml) of CH3 14COONa solutions. The rate of change of surface pressure was monitored by the Wilhelmy plate method using a thin sand-blasted platinum plate (1-cm width) hanging from an electrobalance (Cahn Instruments, Co., CA) as a probe. Both surface concentration and surface pressure were monitored simultaneously. The adsorption isotherm of phosvitin at the air–water interface was studied by incubating protein solutions for about 24 h at 247C, and measuring equilibrium surface concentration at various bulk concentrations. Circular dichroism (CD) spectra of phosvitin were taken using a computerized Olis Circular Dichroism spectrometer (On-Line Instrument Systems, Jefferson, GA). The instrument was calibrated with (1S)-( / )-10-camphorsulfonic acid (23). A quartz cell with a light path of 0.1 cm was used. Ten scans of each sample were averaged, and the mean residue ellipticity, [ u], values, expressed in deg cm2 dmol 01 , were calculated using a value of 115 for the mean residue molecular weight. The secondary structure parameters were estimated by the CDESTIMA software developed by Chang et al. (24). RESULTS
The CD spectra of phosvitin in water at various pH are presented in Fig. 2. At pH 7.0 the spectrum shows a strong negative band at 196 nm, which is indicative of a highly disordered conformation. The intensity of this negative band decreases and a broad negative trough appears in the 210-
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FIG. 2. Circular dichroic spectra of phosvitin at various solution pH. — , pH 7.0; – – – , pH 2.8; – r – r, pH 2.1; rrr, pH 1.5.
to 218-nm region as the pH is decreased from 7.0 to 2.1. At pH 1.5, the negative band at 196 nm disappears and a strong negative trough appears at 215 nm. The negative trough at 215 nm is characteristic of proteins with b-sheet structure. The secondary structure contents of phosvitin, estimated from the CD spectra using an algorithm developed by Chang et al. (24), at various pH are presented in Table 1. At pH 7, the conformation of phosvitin is predominantly random, with 64.5% random coil, 31% b-turns, and 4.5% b-sheet. Lowering of pH from 7.0 to 2.1 results in a dramatic increase in b-sheet content at the cost of reductions in random coil and b-turns. At pH 2.1, the conformation of phosvitin is 64.5% b-sheet, 1% b-turn, and 34.5% random coil. At pH 1.5, which is below the pK1 of phosphoserine residues, the b-sheet content increases to 71% and about 10% a-helix forms at the expense of a reduction in the random coil content to about 19%. These CD analyses indicate that a decrease in the electrostatic energy of phosvitin at acidic pH promotes conversion of random coil to b-sheet, and phosvitin predominantly exists as a b-type protein at pH 2. The effect of pH on adsorption of phosvitin from a dilute solution (1.5 mg/ml) to the air–water interface is shown in Fig. 3A. The rate and extent of adsorption increase progressively with decreasing pH. No significant adsorption occurs at pH 7.1. Although the rate and extent of adsorption increase significantly below pH 3.2, adsorption occurs only after a long lag period. At pH 3.2 and 2.5 the lag time is about 110 min, whereas at pH 2.0 it is only about 25 min. The effect of pH on adsorption of phosvitin is attributable to changes in its net charge as a function of pH. The net charge of phosvitin (calculated from its amino acid composition and assuming a value of 2.1 and 6.8 for pK1 and pK2 , respec-
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tively, of phosphoserine residues) at pH 2.0, 2.5, 3.2, and 7.1 is 015, 048, 071, and 0184, respectively. The inhibition of adsorption at pH 7.1 and an initiation of adsorption at pH 3.2 suggest that a net elimination of about 113 negative charges in phosvitin is required to initiate its adsorption at the interface. Further reduction of the net charge from 071 to 015, caused by decreasing the pH from 3.2 to 2.0, dramatically increases both the rate and the extent of adsorption at the interface. Although adsorption of phosvitin occurs at low pH, no decrease in surface tension of phosvitin solutions occurs as a result of adsorption (Fig. 3B). In fact, the surface tension actually increases slightly with adsorption time. The net increase of surface tension at pH 2.0 is about 0.6 mN/m, whereas at pH 7.1 it is only about 0.25 mN/m. This behavior, which is contrary to what one would expect for an adsorbed protein film at the air–water interface, was highly reproducible and was observed only in the case of phosvitin and not with other proteins, such as b-casein and bovine serum albumin. It must be pointed out that the initial surface tension (at zero time) of the solution was about 72 mN/m, which indicated absence of organic impurities at the surface. Thus, the net increase in surface tension caused by adsorption of phosvitin cannot be due to anything other than the affect of the adsorbed protein on surface water molecules. It appears that when phosvitin adsorbs to the air–water interface, attractive interaction between surface water molecules and charged (hydrophilic) segments of the protein in the subsurface causes a net increase of surface tension. This situation might be analogous to salt solutions, where ion–dipole interaction between surface water molecules and ionic solutes at the subsurface results in an increase in surface tension of salt solutions compared to that of pure water. Generally, protein adsorption at the air–water interface leads to a decrease in free energies of both the sorbate and the sorbent. This, however, need not always be the case. More often than not, only a part of the polypeptide chain lies in the interface, and the remainder is suspended into the subsurface. The thermodynamic state of the interface would be affected by the nature of its interaction with the adsorbed and suspended segments. Phosvitin is an atypical protein. The amino acid sequence clearly indicates that only the Cterminus stretch of about 17 amino acid residues (8% of the total amino acid residues) has the possibility of attaching itself to the interface. The rest of the molecule (92%) is highly charged, which requires it to be suspended into the subsurface. In this orientation, it is quite possible that the charge–dipole interaction between the suspended segments and surface water molecules may actually cause a slight increase in surface tension. As for all equilibrium processes, the only thermodynamic requirement for adsorption of a solute to an interface is a reduction in the free energy of the system. In this case, the system is composed of a sorbate and a sorbent. Thermodynamically, so long as the sum of
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FIG. 3. (A) Influence of bulk-phase pH on the kinetics of adsorption of phosvitin at the air–water interface. h, pH 2.0; L, pH 2.5; s, pH 3.2; n, pH 7.1. The bulk-phase protein concentration was 1.5 mg/ml. (B) Variation of surface pressure during adsorption of phosvitin at the air–water interface. Symbols are the same as above.
the free energy changes of the sorbate and the sorbent is negative, adsorption should proceed. Conversely, if the net reduction in free energy of the sorbate is greater than the net increase in free energy of the sorbent, then adsorption should occur. This appears to be the case for adsorption of phosvitin. The fact that positive adsorption takes place despite an apparent increase in surface tension indicates that the net decrease in the free energy of adsorbed phosvitin molecules is greater than the net increase in the free energy of the surface, such that the overall change in the free energy of the system is negative. In other words, it is probably the decrease in the free energy of the sorbate, and not that of the sorbent, that drives the adsorption process. The lack of surface pressure development also might indicate that even at 1.25 mg/m 2 surface concentration, the adsorbed phosvitin molecules do not form a continuous film, and probably might exist in a gaseous state because of lack of cohesive interaction between them. In other words, in the adsorbed state, only a small hydrophobic segment of each phosvitin molecule might be actually in the interface and TABLE 1 Secondary Structure Content of Phosvitin in Water at Various pH pH
% a-helix
% b-sheet
% b-turns
% aperiodic
7.0 2.8 2.1 1.5
0 0 0 10
4.5 32.5 64.5 71.0
31.0 18.5 1.0 0
64.5 49.0 34.5 19.0
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the bulk of the charged segments of the molecule might be suspended into the subsurface. The rate of adsorption of phosvitin was further analyzed using a diffusion-controlled adsorption model (7, 25) according to the relationship Gt Å 2C0 (D/3.1416) 1 / 2t 1 / 2 ,
[1]
where Gt is surface concentration at time t, D is the diffusion coefficient, C0 is the bulk protein concentration, and t is the time. It should be noted that Eq. [1] is valid only under irreversible adsorption conditions where there is no back diffusion from the interface. However, whether or not back diffusion of phosvitin occurs or an energy barrier to adsorption exists can be elucidated by analyzing the extent of deviation of the apparent diffusion coefficient (estimated from Eq. [1]) from that of the solution diffusion coefficient. The apparent diffusion coefficients of phosvitin at various pH (and net charge) were calculated from the slopes of the initial linear regions of G –t 1 / 2 plots (Fig. 3A). Figure 4 shows that the relationship between the apparent diffusion coefficient and the net charge of phosvitin follows an exponential model, with a correlation coefficient (r 2 ) of 0.995. The apparent diffusion coefficient of phosvitin at pH 7.1 is about 4 1 10 09 cm2 /s. This value is at least two orders of magnitude lower than the solution diffusion coefficient of a protein with a comparable molecular weight. This low apparent diffusion coefficient may be attributable to a high electrostatic energy barrier for adsorption of polyanionic phosvitin at the hydrophobic air–water interface. Decreasing the pH
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FIG. 4. Effect of bulk pH ( s ) and net charge ( h ) on the apparent diffusion coefficient of phosvitin. The apparent diffusion coefficient values were calculated from the slopes of linear regions of curves in Fig. 3A. The solid line is the fitting curve showing the exponential relationship (y Å 2.56 1 10 0.01 x , r 2 Å 0.996) between the net charge and the apparent diffusion coefficient.
from 7.1 to 2.0 increases the apparent diffusion coefficient to 2.06 1 10 07 cm2 /s. This value is still at least sixfold lower than that for a protein with comparable molecular weight and net charge (26). These lower-than-expected apparent diffusion coefficient values of phosvitin may be attributable to two possible phenomena. First, the hydrodynamic size of phosvitin at pH 7.1 is probably much larger than that of a protein with comparable molecular weight. Grizzuti and Perlmann (27) reported that at constant ionic strength the intrinsic viscosity of phosvitin increased dramatically in the pH range 5–7. This is due to ionization of the phosphoserine residues, resulting in extensive hydration and electrostatic repulsion-induced expansion of the molecule at pH 7.0. This is supported by the fact that phosvitin essentially exists in a random coil state at pH 7.0 (Table 1). Even though phosvitin assumes a b-sheet conformation at pH 2.0, it may still have a large hydrodynamic size as a result of extensive hydration of phosphoserine residues. Thus, the lower-than-expected apparent diffusion coefficient of phosvitin might be the result of its unusually large hydrodynamic size. Second, because of its high electrostatic free energy at pH 7.1, interaction of phosvitin with the low dielectric air–water interface would be thermodynamically unfavorable, resulting in a slowerthan-diffusion-controlled rate of adsorption (1). Incidentally, the hydrophilic protein lysozyme, but not by the hydrophobic protein b-casein, also has been shown to exhibit
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this behavior (2). Although the electrostatic free energy of the molecule is considerably lower at pH 2.0 than that at pH 7.1, the probability of its adsorption at the interface might be low because of its extensively hydrated state and high energy input required to dehydrate the molecule prior to its adsorption at the interface. In reality, both phenomena are likely to be responsible for the slow rate of adsorption of phosvitin to the air–water interface. The adsorption isotherm of phosvitin at pH 2.0 is presented in Fig. 5. The plot of Geq against equilibrium bulk concentration shows a plateau at 1.5–3.0 mg/ml, indicating formation of a saturated monolayer. Above 3.0 mg/ml bulk concentration, the surface concentration increases progressively, indicating formation of multilayers. The Ccrit value, that is, the minimum concentration above which formation of a saturated monolayer begins, is about 1.5 mg/ml. Since all adsorption studies were performed at 1.5 mg/ml bulk concentration, the rates of adsorption essentially reflect the kinetics at saturated monolayer coverage conditions. The saturated monolayer coverage (plateau region) for phosvitin at pH 2.0 is about 1.25 mg/m 2 . This value corresponds to ˚ 2 per molecule. an interfacial area of about 4516 A Adsorption of proteins at interfaces follows Langmuirian kinetics when the bulk concentration is below Ccrit (28), and under these conditions Geq is given by the relationship ln K 0 lG n Å ln
G , C(1 0 aG )
[2]
FIG. 5. Adsorption isotherm of phosvitin at pH 2.0. The bulk phase was water adjusted to pH 2.0 with 1 N HCl at 247C. Cb is equilibrium bulk concentration of phosvitin.
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FIG. 6. Plot of low bulk concentration adsorption isotherm data of phosvitin (Fig. 5) according to Eq. [2].
where K is the equilibrium constant, a is the average area occupied per protein molecule at monolayer coverage (i.e., 1/ G at saturated monolayer coverage), C is equilibrium bulk concentration of protein, G is the surface concentration at equilibrium, n is an exponent related to cooperativity among adsorbing protein molecules, and l is related to an activation energy barrier for adsorption. The adsorption isotherm of phosvitin was analyzed according to Eq. [2] in order to determine the equilibrium constant K. The right-hand side of Eq. [2] was plotted against G n for n Å 1, 2, and 3. The lowest value of n that gave a straight line with the highest correlation coefficient was selected as its value. Figure 6 shows that a best fit is obtained with n Å 1, indicating that there is no cooperativity among phosvitin molecules adsorbing at the interface. However, a negative value of l indicates that the activation energy barrier for adsorption decreases with increasing surface coverage. The equilibrium binding constant, determined from the intercept, is 293 mg/ m 2 wt%. The effects of NaCl and CaCl2 on adsorption of phosvitin at the air–water interface are shown in Fig. 7. At pH 7, neither NaCl nor CaCl2 facilitates adsorption of phosvitin, indicating that charge neutralization by counterions alone is not sufficient to promote adsorption of phosvitin. At pH 2.0, addition of 0.1 M NaCl has no significant effect on the rate and extent of adsorption of phosvitin compared to those of the pH 2.0 control. However, at pH 2.0, addition of increasing amounts of CaCl2 increases both the rate and the extent of adsorption of phosvitin at the interface. The apparent diffusion coefficient of phosvitin, calculated from the initial slopes of the curves in Fig. 7, increases with increasing concentrations of CaCl2 in the bulk phase (Table 2). In 0.1 M CaCl2 at pH 2.0, the apparent diffusion coefficient is about an order of magnitude greater than that in the absence of
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FIG. 7. Effects of CaCl2 and NaCl on the kinetics of adsorption of phosvitin at the air–water interface at pH 2.0 and 7.0: s, no salt, pH 2.0; l, 0.1 M CaCl2 , pH 7; /, 0.5 M CaCl2 , pH 7; , 0.1 M NaCl, pH 2.0; h, 0.001 M CaCl2 , pH 2.0; n, 0.01 M CaCl2 , pH 2.0; l, 0.1 M CaCl2 , pH 2.0. The bulk-phase protein concentration was 1.5 mg/ml.
CaCl2 , and is in the range expected for a protein of comparable molecular weight. In 0.1 M CaCl2 at pH 2.0, the surface concentration of phosvitin at equilibrium is about 2.5 mg/ m 2 , which is twice that of the value when no CaCl2 is present. However, even at this high surface concentration, the phosvitin film shows no positive surface pressure. Protein adsorption at interfaces is generally considered to be irreversible (7, 9, 27). However, the data in Fig. 8 show reversibility of adsorption of phosvitin at the air–water interface to changes in the pH of the bulk phase. In these experiments, phosvitin was first allowed to adsorb at the interface for 1000 min at pH 2.0. Aliquots (0.1–1.0 ml) of 1 M NaOH were then sequentially injected into the bulk phase. After each injection, the surface cpm of the adsorbed phosvitin film was monitored until it reached a new equilibrium value, TABLE 2 Effect of CaCl2 Concentration on the Apparent Diffusion Coefficient of Phosvitin Determined from the Rate of Adsorption at the Air–Water Interface CaCl2 concentration (M)
D 1 107 (cm7/s)
0 0.001 0.01 0.1
2.06 2.23 4.76 13.7
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DISCUSSION
The results presented here show that adsorption of phosvitin at the air–water interface exhibits several anomalous behaviors. First, adsorption occurs only at low pH, and complete and instantaneous desorption takes place when the pH of the bulk phase is increased to neutral pH. Second, adsorption causes a slight but reproducible increase, instead of a decrease, in surface tension. These anomalies must arise from the unusual charge and other physicochemical characteristics of phosvitin. Phenomenologically, the electrostatic charge of a protein molecule exerts two types of potential energy barriers for its adsorption at a hydrophobic interface, e.g., the air–water interface (8). In the initial stages of adsorption of a charged protein molecule at a clean air–water interface, if e is the net charge of the protein and e0 and e are dielectric constants of the aqueous phase and the gas (air) phase, respectively, then, according to the electrostatic theory, as the protein approaches the air–water interface an image charge equivalent to e * Å e( e0 0 e )/( e0 / e )
FIG. 8. pH-induced desorption of phosvitin from the air–water interface in the absence of CaCl2 in the bulk phase.
at which time a 0.5-ml aliquot of the bulk phase was withdrawn to check the final pH. Figure 8 shows that an increase in pH of the bulk phase causes a rapid decrease in surface cpm, indicating that an increase in the net charge of adsorbed phosvitin molecules causes instantaneous desorption of phosvitin from the interface. When the pH of the bulk phase is increased to 7.4, almost all adsorbed phosvitin is desorbed into the bulk phase. The effect of CaCl2 on pH-induced reversibility of adsorption of phosvitin at the air–water interface is shown in Fig. 9. In this case, phosvitin was first allowed to adsorb at the air–liquid interface from a solution containing 1.5 mg/ml phosvitin and 0.1 M CaCl2 , pH 2.0. After about 1400 min of adsorption, a predetermined amount of 1 M NaOH that would increase the pH of the bulk phase from 2 to about 8.2 was injected into the bulk phase using a syringe. The change in surface cpm was monitored with time. Figure 9 shows that an increase in pH of the bulk phase from an initial value of 2.0 to a final value of 8.2 causes desorption of phosvitin from the interface. However, the rate of desorption in the presence of 0.1 M CaCl2 is much slower than that in its absence (Figs. 8 and 9). This difference might arise from possible intermolecular crosslinking between adsorbed phosvitin molecules by divalent Ca 2/ ions. This Ca 2/ -induced cross bridging may occur between phosphoserine residues as well as between the sialic acid groups of the single carbohydrate chain in hen phosvitin.
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[3]
would appear in the low dielectric gas phase. As the protein approaches the interface, an electrostatic repulsive potential energy equivalent to
FIG. 9. pH-induced desorption of phosvitin from the air–water interface in the presence of 0.1 M CaCl2 in the bulk phase. s, surface cpm; h, surface pressure.
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Eele Å
ee * e 2 ( e0 0 e ) Å 2de0 2de0 ( e0 / e )
[4]
will develop, where d is the distance of the protein from the air–water interface. If the magnitude of this repulsive electrostatic potential is greater than any attractive hydrophobic potential, then no adsorption would take place. On the other hand, if the attractive hydrophobic potential is greater than the repulsive electrostatic potential, then adsorption would proceed, albeit at a slower rate. Once a certain amount of protein is adsorbed to the interface, the adsorbed protein would create an additional electrical barrier equivalent to Ep,ele Å
* e dc Å e c,
[5]
where c is the electrostatic potential in the two-dimensional plane of charged protein film at the interface. The kinetics of adsorption of phosvitin at the air–water interface might be affected by both these electrostatic energy barriers. Phosvitin essentially is a polyanionic polymer with a net charge of 0179 at neutral pH. The amino acid sequence of phosvitin shows that it has no discernible hydrophobic regions, except for a short stretch of nonpolar amino acid residues at positions 205–216 (Fig. 1). The lack of adsorption of phosvitin at pH 7.0 indicates that the increase in electrostatic repulsive potential energy of phosvitin as it approaches the interface is so large that it could not adsorb at the interface. For example, using Eq. [4] it can be shown that the electrostatic repulsive potential energy of phosvitin at pH 7 (with 0179 charges) would be equal to its thermal energy kT (where k is the Boltzman constant and T Å 297 K) when it is at a distance of 11.5 mm from the air–water interface. What this means is that, because of its high electrostatic free energy at pH 7, phosvitin would not be able to penetrate a subsurface layer 11.5 mm from the air–water interface by diffusion alone. Therefore, it is not surprising that it behaves purely as a macroion and lacks interfacial adsorptivity at pH 7. It is significant, however, that, even though phosvitin has a total of 93 charged residues (54 negatively charged and 39 positively charged residues) and a net charge of 015 at pH 2.0 and contains several uncharged hydrophilic residues, it is able to adsorb to a significant extent at this pH. Equation [4] predicts that the electrostatic potential energy of a molecule carrying a net charge of 015 will be equal to its thermal energy (at T Å 297 K) when it is about 0.158 mm from the air–water interface. This electrostatic potential energy barrier would not allow the molecule to adsorb at the interface. However, the fact that phosvitin is able to adsorb to the interface in spite of this electrostatic potential energy barrier indicates that a more favorable hydrophobic interaction that
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can more than offset the electrostatic barrier must exist. The C-terminus hydrophobic peptide segment 205–216 ought to be the segment that can hydrophobically interact with the air–water interface, since no other hydrophobic stretch can be found in the primary structure of phosvitin. The rest of the molecule might exist in the form of loops suspended into the subsurface. Previous studies have shown that initial anchoring of a protein to the air–water interface involves binding of only a small peptide segment of 4–6 amino acid residues (9–11). Recently, it has been shown that peptide segments with a repeating heptet sequence of –P–N–P–P– N–N–P– , where P and N are polar and nonpolar residues, respectively, readily form a-helices with amphiphilic surfaces in aqueous solutions (29). The binary code of peptide segment 204–216 resembles that of the above heptet with slight variations, suggesting that this segment might exist in the a-helix configuration at the interface. Construction of a helical wheel of the segment shows that one-half of the helix surface is hydrophobic with Val, Tyr, Ile, His, Leu, and Trp residues, and the other half of the helix surface is hydrophilic with Glu, Ser, Gly, Ser, Ile, Lys, and Arg residues. Based on these analyses, it appears that the hydrophobic surface of this short helical segment might be the site that anchors the protein to the air–water interface. The energetics of interaction of this surface with the interface appear to be favorable for overcoming the electrostatic repulsive interaction of the molecule with the interface. However, the fact that small changes in the pH of the subphase cause instantaneous desorption of phosvitin from the interface (Fig. 8) indicates that retention of the protein at the interface is highly sensitive to changes in the net charge of the molecule. Although phosvitin is able to adsorb to an extent of about 1.25 to 2.5 mg/m 2 at the air–water interface at pH 2, the surface tension of the solution apparently increases with increasing amounts of adsorption. It should be pointed out that at comparable surface concentrations, other proteins generally cause significant reductions in surface tension. For instance, at a monolayer coverage of 1.8 mg/m 2 , b-casein exerts a surface pressure of about 18 mN/m, and monolayers of lysozyme and bovine serum albumin display surface pressures of about 6.5 and 16 mN/m, respectively (9, 30). Although an increase in surface tension in spite of protein accumulation at the interface appears to be incongruous and seems to violate Gibbs’ adsorption equation, it can be explained thermodynamically. As pointed out earlier, the only thermodynamic requirement for adsorption is a reduction of free energy of the system. Whether this occurs from a loss of free energy of the interface or the adsorbed solute or both is irrelevant. In the case of phosvitin the overall decrease in free energy of the system seems to originate from the loss of free energy of adsorbed phosvitin and not from a reduction in the free energy of the interface. If we assume that the nonpolar residues of the C-terminus helix segment (Val, Tyr, Ile, Leu, and Trp) are involved in the attachment of
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negligible. It seems that even at this high surface coverage phosvitin does not exist as a film at the interface, but probably exists in a gaseous state because of a lack of cohesive interactions between the adsorbed segments. The contribution of electrostatic repulsive forces to the surface pressure of a protein film at the air–water interface is given by the Davies equation (5) Pele Å 6.1C 1 / 2[cosh sinh 01 (135/AeleC 1 / 2 ) 0 1], [7]
FIG. 10. Theoretical G versus Pele curves based on the Davies equation (Eq. [7]) for charged monolayers with z Å 015 at equivalent electrolyte concentrations of 0.001 ( s ) and 0.3 ( h ).
phosvitin to the interface, then, based on Tanford’s hydrophobicity scale (31), transfer of these nonpolar residues from the water phase to the interface would decrease the free energy of phosvitin by about 052 kJ/mol. For G Å 1.25 mg/m 2 (i.e., 3.6 1 10 012 moles/cm2 ), the reduction in free energy of the system, resulting from adsorbed phosvitin molecules, would be 01.89 erg/cm2 . In contrast, the increase in free energy of the system, resulting from an increase in the surface tension caused by charge–dipole interaction between suspended protein segments and surface water molecules, is only about 0.5 erg/cm2 . Therefore, in spite of an apparent increase in surface tension, the free energy of the system is decreased by about 01.4 erg/cm2 due to adsorption of phosvitin. The surface pressure of a protein film at any given surface concentration is given by (5) Pa / w Å Pkin / Pele / Pcoh ,
[6]
where Pkin is the surface pressure contribution from kinetic movement of molecules in the two-dimensional film, Pele is the contribution from electrostatic forces, and Pcoh is the contribution from cohesive interactions among molecules in the film. Since proteins are large, the contribution of Pkin to the surface pressure can be assumed to be negligible. The fact that the phosvitin film does not exhibit positive surface pressure even at a surface concentration of 1.25–2.5 mg/ m 2 indicates that the magnitudes of Pele and Pcoh also were
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where C is the concentration of equivalent electrolyte in the ˚ 2 ) per charge in bulk phase, Aele ( Å A/z) is the area (in A the surface of the protein film, and z is the net charge of the protein. Theoretical G versus Pele curves, calculated from Eq. [7], are shown in Fig. 10 for phosvitin at pH 2.0 (z Å 015) in water (C Å 0.001) and in 0.1 M CaCl2 (C Å 0.3). It should be noted that Eq. [7] predicts a value of Pele Å 2.5 mN/m for G Å 1.25 mg/m 2 (monolayer coverage when C Å 0.001), and a value of Pele Å 3 at G Å 2.5 mg/m 2 (monolayer coverage in the presence of 0.1 M CaCl2 , i.e., at C Å 0.3). The disagreement between experimental (negative surface pressure) and predicted surface pressure values indicates that, in the adsorbed state, the charged groups of phosvitin are not present in the interface. It is likely that all charged segments exist as loops suspended into the subphase. Hydration of charged segments and repulsive interactions between the loops via hydration-repulsion and electrostatic forces might prevent cohesive interactions between the adsorbed segments at the interface (shown schematically in Fig. 11). These might be the reasons for the lack of contribution of Pele and Pcoh to the surface pressure of the film. In addition, the highly charged loops in the subsurface might exert an electrostatic pull on surface water molecules, which might be the reason for the observed increase in surface tension. The results presented here show that, because of its high electrostatic free energy, phosvitin does not adsorb to the
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FIG. 11. Illustration of possible orientation of phosvitin at the air– water interface at pH 2.0.
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air–water interface at high pH. A decrease in electrostatic free energy at low pH increases the rate and extent of adsorption of phosvitin. A saturated monolayer of phosvitin formed in either the presence or the absence of CaCl2 at pH 2 exhibits a negative surface pressure, indicating that neither electrostatic repulsion nor cohesive interactions among the adsorbed segments occurs in the monolayer film, and the hydrophilic segments of phosvitin suspended into the subsurface exert an electrostatic pull on surface water molecules, causing an increase in surface tension. Examination of the amino acid sequence of phosvitin indicates that only a short segment at the C-terminus, containing a few nonpolar amino acid residues, might be involved in anchoring the protein to the interface. This suggests that proteins can easily adsorb and form a film at the air–water interface through attachment of a small peptide segment. ACKNOWLEDGMENT Financial support from the National Science Foundation (Grant BES9315123) is gratefully acknowledged.
REFERENCES 1. Xu, S., and Damodaran, S., Langmuir 8, 2021 (1992). 2. Xu, S., and Damodaran, S., J. Colloid Interface Sci. 159, 124 (1993). 3. Evans, M. T. A., Mitchell, J., Mussellwhite, P. R., and Irons, L., in ‘‘Surface Chemistry of Biological Systems’’ (M. Blank, Ed.), p. 1. Plenum Press, New York, 1970. 4. Birdi, K. S., Kolloid Z. Z. Polym. 250, 222 (1972). 5. Birdi, K. S., and Nikolov, A., J. Phys. Chem. 83, 365 (1979). 6. MacRitchie, F., and Alexander, A. E., J. Colloid Sci. 18, 464 (1963). 7. MacRitchie, F., and Alexander, A. E., J. Colloid Sci. 18, 453 (1963).
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8. Song, K. B., and Damodaran, S., Langmuir 7, 2737 (1991). 9. Graham, D. E., and Phillips, M. C., J. Colloid Interface Sci. 70, 403 (1979). 10. Ter-Minassian-Saraga, L., J. Colloid Interface Sci. 80, 393 (1981). 11. Damodaran, S., and Song, K. B., Biochim. Biophys. Acta 954, 253 (1988). 12. Tornberg, E., J. Colloid Interface Sci. 64, 391 (1978). 13. Norde, W., and Lyklema, J., J. Colloid Interface Sci. 66, 285 (1978). 14. Koutsoukos, P. G., Norde, W., and Lyklema, J., J. Colloid Interface Sci. 95, 385 (1983). 15. Byrne, B. M., van het Schip, A. D., van de Klundert, J. A. M., Arnberg, A. C., Gruber, M., and Geert, A. B., Biochemistry 23, 4275 (1984). 16. Clark, R. C., and Joubert, F. J., FEBS Lett. 13, 225 (1971). 17. Clark, R. C., Int. J. Biochem. 17, 983 (1985). 18. Renugopalakrishnan, V., Horowitz, P. M., and Glimcher, M. J., J. Biol. Chem. 260, 11406 (1985). 19. Prescott, B., Renugopalakrishnan, V., Glimcher, M. J., Bhushan, A., and Thomas, G. J., Jr., Biochemistry 25, 2792 (1986). 20. Yasui, S. C., Pancoska, P., Dukor, R. K., and Keiderling, T. A., J. Biol. Chem. 265, 3780 (1990). 21. Kozlowski, H., Mangani, S., Messori, L., Orioli, P. L., and Scozzafava, A., J. Inorg. Biochem. 34, 221 (1988). 22. Xu, S., and Damodaran, S., J. Colloid Interface Sci. 157, 485 (1993). 23. Johnson, W. C., in ‘‘Methods of Biochemical Analysis’’ (D. Glick, Ed.), p. 61. Wiley, New York, 1985. 24. Chang, C. T., Wu, C. S. C., and Yang, J. T., Anal. Biochem. 91, 13 (1978). 25. Ward, A. F. H., and Tordai, L., J. Chem. Phys. 14, 453 (1946). 26. Tanford, C., ‘‘Physical Chemistry of Macromolecules,’’ p. 358. Wiley, New York, 1967. 27. Grizzuti, K., and Perlmann, G. E., J. Biol. Chem. 245, 2573 (1970). 28. Hunter, J. R., Kilpatrick, P. K., and Carbonell, R. G., J. Colloid Interface Sci. 137, 462 (1990). 29. Kamtekar, S., Schiffer, J., Xiong, H., Babik, J. M., and Hecht, M. H., Science 262, 1680 (1993). 30. Xu, S., and Damodaran, S., Langmuir 10, 472 (1994). 31. Nozaki, Y., and Tanford, C., J. Biol. Chem. 246, 2211 (1972).
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0137
The Role of Electrostatic Forces in Anomalous Adsorption Behavior of Phosvitin at the Air/Water Interface SRINIVASAN DAMODARAN 1
AND
SHIQUIAN XU
Department of Food Science, University of Wisconsin—Madison, 1605 Linden Drive, Madison, Wisconsin 53706 Received February 16, 1995; accepted August 22, 1995
The adsorption of phosvitin at the air–water interface has been studied to elucidate the influence of electrostatic forces on protein adsorption at liquid interfaces. Because of its polyanionic character, adsorption of phosvitin at the air–water interface takes place only at low pH, but not near neutral pH. Phosvitin adsorbed at an initial pH of 2.0 is completely desorbed from the interface when the pH is increased to neutral pH. The saturated monolayer coverage for phosvitin is about 1.25 mg/m 2 at pH 2.0. However, in spite this significant amount of adsorption, no decrease in surface tension occurs. Instead, a consistent increase in surface tension of the solution occurs, which apparently violates the Gibbs adsorption equation. A model based on the configuration of phosvitin at the interface has been proposed to explain the thermodynamic reasons for this apparent violation of the Gibbs equation. It is shown that phosvitin is anchored to the interface only via a short C-terminus hydrophobic segment and the rest of the highly hydrophilic molecule is suspended in the subsurface. These suspended loops exert an electrostatic pull on surface water molecules, causing an increase in surface tension. However, the reduction in free energy resulting from removal of the hydrophobic segment from water to the interface is much greater than the increase in surface tension caused by charge–dipole interactions, so that there is actually a net reduction in free energy of the system. Thus, although adsorption of phosvitin apparently violates the Gibbs adsorption equation, it does not violate the basic thermodynamic principle. The results also show that proteins can adsorb to an interface against seemingly excessive electrostatic repulsive forces through attachment of only a small hydrophobic peptide segment. q 1996 Academic Press, Inc.
Key Words: phosvitin; adsorption; air–water interface; electrostatic forces; kinetics of adsorption.
(1, 2). Although it is recognized that the tendency of proteins to adsorb at phase boundaries, especially at air/water and oil/water interfaces, arises because of their amphiphilic nature, the exact role of electrostatic forces in the adsorption process is not well understood. Several studies relating to adsorbed and spread proteins films at various interfaces have been reported (3–14). However, only a few of those studies have dealt with the role of electrostatic forces in the kinetics of protein adsorption at the air–water interface (6, 8). MacRitchie and Alexander (6) reported on the effects of spread monolayers of various negatively and positively charged molecules on adsorption of lysozyme from a dilute solution to the air–water interface. Although this approach is useful for elucidating the effects of charge–charge interactions on protein adsorption to a charged monolayer, information regarding interaction of the force field of a clean interface with the electrostatic free energy of a protein on the latter’s rate of adsorption at the interface cannot be obtained from such an approach. The influence of electrostatic free energy of a protein on its adsorption at liquid interfaces can be best understood by studying the kinetics of adsorption of phosvitin. Phosvitin is a phosphoglycoprotein found in egg yolk of all avian species. At pH 7.0 it has a net charge of about 0179. The high net charge and the sensitivity of its conformation to changes in its electrostatic free energy make phosvitin an ideal candidate for studying the influence of electrochemical potential on adsorption of proteins at interfaces. MATERIALS AND METHODS
INTRODUCTION
Adsorption and film formation of proteins at interfaces is a complex phenomenon, influenced by the interaction of the interfacial force field with various molecular chemical potentials, such as hydrophobic, electrostatic, hydration, and conformational (entropic) chemical potentials, of proteins 1
To whom correspondence should be addressed.
0021-9797/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Phosvitin has a molecular weight of about 35,000 and contains 217 amino acid residues, of which 123 residues are serine residues (15, 16). Of the 123 serine residues, 118 are phosphorylated and 5 are ‘‘free’’ (17). The 4 threonine residues in phosvitin also are phosphorylated. The amino acid sequence of phosvitin (Fig. 1) shows that it contains only about 10% nonpolar amino acid residues, 6.5% acidic residues (Asp / Glu), and 18% basic residues (Lys / Arg / His). The region between residues 55 and 155 contains long stretches of phosphoserine residues interspersed with
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FIG. 1. Amino acid sequence of hen egg phosvitin (from Ref. (7)).
lysine, arginine, and asparagine residues. The only significant grouping of nonpolar amino acid residues is found at positions 205–216. The remainder of the molecule is extremely hydrophilic. Phosvitin carries a net charge of about 0179 at pH 7.0. Because of this high net negative charge, the physicochemical properties of phosvitin resemble that of a typical polyanionic polymer. At neutral pH, phosvitin predominantly exists in a random coil conformation, whereas at pH 2.0 its conformation is about 67% b-sheet and 11.5% a-helix (18–20). Hen egg phosvitin was obtained from Sigma Chemical Co. (St. Louis, MO). All salts used in this study were of ultrapure (Gold Label) grade from Aldrich Chemical Co. (Milwaukee, WI). The surface tensions of salt solutions were comparable to reported values and did not change during aging, which indicated that the salts were free of lowmolecular-weight organic impurities. [ 14C]Formaldehyde was from New England Nuclear Co. (Boston, MA). All other reagents were of analytical grade. Extreme care was taken in purifying water for adsorption studies. A Milli-Q ultrapure water purification system (Millipore Corp., Bedford, MA) with a Qpak1 cartridge package (composed of activated charcoal, reverse osmosis, ion exchange, and ultrafiltration cartridges) capable of removing inorganic and organic impurities was used to purify the water. The resistivity of water was usually 18.2 mV cm. To check the water quality, the surface tension of water was measured at 207C. If the surface tension of water was not 72.9 { 0.1 mN/m and did not remain constant during 24 h of aging, it was discarded. Phosvitin was radiolabeled by reductive methylation of amino groups with [ 14C]formaldehyde at pH 7.0 as described in detail elsewhere (1). Phosvitin concentration was determined using an E 1% value of 5.09 at 275 nm (21). The specific radioactivity of the labeled protein was about 0.608 mCi/mg. The kinetics of adsorption of phosvitin at the air–water interface was studied as described in detail elsewhere (1, 2,
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22). Briefly, protein solutions for adsorption studies were prepared by mixing an aliquot of a stock solution ( Ç0.55 mg/ml) of 14C-labeled phosvitin with 125 ml of water preadjusted to the required pH. The final pH of the dilute solution was noted. An aliquot of the final solution (120 ml) was poured into a Teflon trough (21 1 5.56 1 1.27 cm) and the surface was cleaned by sweeping with a fine capillary attached to an aspirator. The rate of change of surface concentration of the radiolabeled protein was monitored by measuring surface radioactivity using a rectangular gas proportional counter (8 1 4 cm) (Ludlum Measurements, Inc., Sweetwater, TX) as described elsewhere (1). The entire experimental setup was housed in a refrigerated incubator maintained at 24 { 0.27C and 90–95% relative humidity. A calibration curve relating cpm versus surface radioactivity ( mCi/m 2 ), constructed by spreading 14C-labeled b-casein on the air–water interface, was used to convert cpm to mCi/ m 2 . The methods of spreading, construction of the standard curve, and the rationale for using radiolabeled b-casein as a calibration standard have been described in detail elsewhere (22). The surface concentration (mg/m 2 ) was calculated by multiplying surface radioactivity with specific radioactivity ( mCi/mg) of the protein. The contribution of bulk radioactivity to cpm was corrected using a standard curve relating cpm versus specific radioactivity ( mCi/ml) of CH3 14COONa solutions. The rate of change of surface pressure was monitored by the Wilhelmy plate method using a thin sand-blasted platinum plate (1-cm width) hanging from an electrobalance (Cahn Instruments, Co., CA) as a probe. Both surface concentration and surface pressure were monitored simultaneously. The adsorption isotherm of phosvitin at the air–water interface was studied by incubating protein solutions for about 24 h at 247C, and measuring equilibrium surface concentration at various bulk concentrations. Circular dichroism (CD) spectra of phosvitin were taken using a computerized Olis Circular Dichroism spectrometer (On-Line Instrument Systems, Jefferson, GA). The instrument was calibrated with (1S)-( / )-10-camphorsulfonic acid (23). A quartz cell with a light path of 0.1 cm was used. Ten scans of each sample were averaged, and the mean residue ellipticity, [ u], values, expressed in deg cm2 dmol 01 , were calculated using a value of 115 for the mean residue molecular weight. The secondary structure parameters were estimated by the CDESTIMA software developed by Chang et al. (24). RESULTS
The CD spectra of phosvitin in water at various pH are presented in Fig. 2. At pH 7.0 the spectrum shows a strong negative band at 196 nm, which is indicative of a highly disordered conformation. The intensity of this negative band decreases and a broad negative trough appears in the 210-
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FIG. 2. Circular dichroic spectra of phosvitin at various solution pH. — , pH 7.0; – – – , pH 2.8; – r – r, pH 2.1; rrr, pH 1.5.
to 218-nm region as the pH is decreased from 7.0 to 2.1. At pH 1.5, the negative band at 196 nm disappears and a strong negative trough appears at 215 nm. The negative trough at 215 nm is characteristic of proteins with b-sheet structure. The secondary structure contents of phosvitin, estimated from the CD spectra using an algorithm developed by Chang et al. (24), at various pH are presented in Table 1. At pH 7, the conformation of phosvitin is predominantly random, with 64.5% random coil, 31% b-turns, and 4.5% b-sheet. Lowering of pH from 7.0 to 2.1 results in a dramatic increase in b-sheet content at the cost of reductions in random coil and b-turns. At pH 2.1, the conformation of phosvitin is 64.5% b-sheet, 1% b-turn, and 34.5% random coil. At pH 1.5, which is below the pK1 of phosphoserine residues, the b-sheet content increases to 71% and about 10% a-helix forms at the expense of a reduction in the random coil content to about 19%. These CD analyses indicate that a decrease in the electrostatic energy of phosvitin at acidic pH promotes conversion of random coil to b-sheet, and phosvitin predominantly exists as a b-type protein at pH 2. The effect of pH on adsorption of phosvitin from a dilute solution (1.5 mg/ml) to the air–water interface is shown in Fig. 3A. The rate and extent of adsorption increase progressively with decreasing pH. No significant adsorption occurs at pH 7.1. Although the rate and extent of adsorption increase significantly below pH 3.2, adsorption occurs only after a long lag period. At pH 3.2 and 2.5 the lag time is about 110 min, whereas at pH 2.0 it is only about 25 min. The effect of pH on adsorption of phosvitin is attributable to changes in its net charge as a function of pH. The net charge of phosvitin (calculated from its amino acid composition and assuming a value of 2.1 and 6.8 for pK1 and pK2 , respec-
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tively, of phosphoserine residues) at pH 2.0, 2.5, 3.2, and 7.1 is 015, 048, 071, and 0184, respectively. The inhibition of adsorption at pH 7.1 and an initiation of adsorption at pH 3.2 suggest that a net elimination of about 113 negative charges in phosvitin is required to initiate its adsorption at the interface. Further reduction of the net charge from 071 to 015, caused by decreasing the pH from 3.2 to 2.0, dramatically increases both the rate and the extent of adsorption at the interface. Although adsorption of phosvitin occurs at low pH, no decrease in surface tension of phosvitin solutions occurs as a result of adsorption (Fig. 3B). In fact, the surface tension actually increases slightly with adsorption time. The net increase of surface tension at pH 2.0 is about 0.6 mN/m, whereas at pH 7.1 it is only about 0.25 mN/m. This behavior, which is contrary to what one would expect for an adsorbed protein film at the air–water interface, was highly reproducible and was observed only in the case of phosvitin and not with other proteins, such as b-casein and bovine serum albumin. It must be pointed out that the initial surface tension (at zero time) of the solution was about 72 mN/m, which indicated absence of organic impurities at the surface. Thus, the net increase in surface tension caused by adsorption of phosvitin cannot be due to anything other than the affect of the adsorbed protein on surface water molecules. It appears that when phosvitin adsorbs to the air–water interface, attractive interaction between surface water molecules and charged (hydrophilic) segments of the protein in the subsurface causes a net increase of surface tension. This situation might be analogous to salt solutions, where ion–dipole interaction between surface water molecules and ionic solutes at the subsurface results in an increase in surface tension of salt solutions compared to that of pure water. Generally, protein adsorption at the air–water interface leads to a decrease in free energies of both the sorbate and the sorbent. This, however, need not always be the case. More often than not, only a part of the polypeptide chain lies in the interface, and the remainder is suspended into the subsurface. The thermodynamic state of the interface would be affected by the nature of its interaction with the adsorbed and suspended segments. Phosvitin is an atypical protein. The amino acid sequence clearly indicates that only the Cterminus stretch of about 17 amino acid residues (8% of the total amino acid residues) has the possibility of attaching itself to the interface. The rest of the molecule (92%) is highly charged, which requires it to be suspended into the subsurface. In this orientation, it is quite possible that the charge–dipole interaction between the suspended segments and surface water molecules may actually cause a slight increase in surface tension. As for all equilibrium processes, the only thermodynamic requirement for adsorption of a solute to an interface is a reduction in the free energy of the system. In this case, the system is composed of a sorbate and a sorbent. Thermodynamically, so long as the sum of
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FIG. 3. (A) Influence of bulk-phase pH on the kinetics of adsorption of phosvitin at the air–water interface. h, pH 2.0; L, pH 2.5; s, pH 3.2; n, pH 7.1. The bulk-phase protein concentration was 1.5 mg/ml. (B) Variation of surface pressure during adsorption of phosvitin at the air–water interface. Symbols are the same as above.
the free energy changes of the sorbate and the sorbent is negative, adsorption should proceed. Conversely, if the net reduction in free energy of the sorbate is greater than the net increase in free energy of the sorbent, then adsorption should occur. This appears to be the case for adsorption of phosvitin. The fact that positive adsorption takes place despite an apparent increase in surface tension indicates that the net decrease in the free energy of adsorbed phosvitin molecules is greater than the net increase in the free energy of the surface, such that the overall change in the free energy of the system is negative. In other words, it is probably the decrease in the free energy of the sorbate, and not that of the sorbent, that drives the adsorption process. The lack of surface pressure development also might indicate that even at 1.25 mg/m 2 surface concentration, the adsorbed phosvitin molecules do not form a continuous film, and probably might exist in a gaseous state because of lack of cohesive interaction between them. In other words, in the adsorbed state, only a small hydrophobic segment of each phosvitin molecule might be actually in the interface and TABLE 1 Secondary Structure Content of Phosvitin in Water at Various pH pH
% a-helix
% b-sheet
% b-turns
% aperiodic
7.0 2.8 2.1 1.5
0 0 0 10
4.5 32.5 64.5 71.0
31.0 18.5 1.0 0
64.5 49.0 34.5 19.0
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the bulk of the charged segments of the molecule might be suspended into the subsurface. The rate of adsorption of phosvitin was further analyzed using a diffusion-controlled adsorption model (7, 25) according to the relationship Gt Å 2C0 (D/3.1416) 1 / 2t 1 / 2 ,
[1]
where Gt is surface concentration at time t, D is the diffusion coefficient, C0 is the bulk protein concentration, and t is the time. It should be noted that Eq. [1] is valid only under irreversible adsorption conditions where there is no back diffusion from the interface. However, whether or not back diffusion of phosvitin occurs or an energy barrier to adsorption exists can be elucidated by analyzing the extent of deviation of the apparent diffusion coefficient (estimated from Eq. [1]) from that of the solution diffusion coefficient. The apparent diffusion coefficients of phosvitin at various pH (and net charge) were calculated from the slopes of the initial linear regions of G –t 1 / 2 plots (Fig. 3A). Figure 4 shows that the relationship between the apparent diffusion coefficient and the net charge of phosvitin follows an exponential model, with a correlation coefficient (r 2 ) of 0.995. The apparent diffusion coefficient of phosvitin at pH 7.1 is about 4 1 10 09 cm2 /s. This value is at least two orders of magnitude lower than the solution diffusion coefficient of a protein with a comparable molecular weight. This low apparent diffusion coefficient may be attributable to a high electrostatic energy barrier for adsorption of polyanionic phosvitin at the hydrophobic air–water interface. Decreasing the pH
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FIG. 4. Effect of bulk pH ( s ) and net charge ( h ) on the apparent diffusion coefficient of phosvitin. The apparent diffusion coefficient values were calculated from the slopes of linear regions of curves in Fig. 3A. The solid line is the fitting curve showing the exponential relationship (y Å 2.56 1 10 0.01 x , r 2 Å 0.996) between the net charge and the apparent diffusion coefficient.
from 7.1 to 2.0 increases the apparent diffusion coefficient to 2.06 1 10 07 cm2 /s. This value is still at least sixfold lower than that for a protein with comparable molecular weight and net charge (26). These lower-than-expected apparent diffusion coefficient values of phosvitin may be attributable to two possible phenomena. First, the hydrodynamic size of phosvitin at pH 7.1 is probably much larger than that of a protein with comparable molecular weight. Grizzuti and Perlmann (27) reported that at constant ionic strength the intrinsic viscosity of phosvitin increased dramatically in the pH range 5–7. This is due to ionization of the phosphoserine residues, resulting in extensive hydration and electrostatic repulsion-induced expansion of the molecule at pH 7.0. This is supported by the fact that phosvitin essentially exists in a random coil state at pH 7.0 (Table 1). Even though phosvitin assumes a b-sheet conformation at pH 2.0, it may still have a large hydrodynamic size as a result of extensive hydration of phosphoserine residues. Thus, the lower-than-expected apparent diffusion coefficient of phosvitin might be the result of its unusually large hydrodynamic size. Second, because of its high electrostatic free energy at pH 7.1, interaction of phosvitin with the low dielectric air–water interface would be thermodynamically unfavorable, resulting in a slowerthan-diffusion-controlled rate of adsorption (1). Incidentally, the hydrophilic protein lysozyme, but not by the hydrophobic protein b-casein, also has been shown to exhibit
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this behavior (2). Although the electrostatic free energy of the molecule is considerably lower at pH 2.0 than that at pH 7.1, the probability of its adsorption at the interface might be low because of its extensively hydrated state and high energy input required to dehydrate the molecule prior to its adsorption at the interface. In reality, both phenomena are likely to be responsible for the slow rate of adsorption of phosvitin to the air–water interface. The adsorption isotherm of phosvitin at pH 2.0 is presented in Fig. 5. The plot of Geq against equilibrium bulk concentration shows a plateau at 1.5–3.0 mg/ml, indicating formation of a saturated monolayer. Above 3.0 mg/ml bulk concentration, the surface concentration increases progressively, indicating formation of multilayers. The Ccrit value, that is, the minimum concentration above which formation of a saturated monolayer begins, is about 1.5 mg/ml. Since all adsorption studies were performed at 1.5 mg/ml bulk concentration, the rates of adsorption essentially reflect the kinetics at saturated monolayer coverage conditions. The saturated monolayer coverage (plateau region) for phosvitin at pH 2.0 is about 1.25 mg/m 2 . This value corresponds to ˚ 2 per molecule. an interfacial area of about 4516 A Adsorption of proteins at interfaces follows Langmuirian kinetics when the bulk concentration is below Ccrit (28), and under these conditions Geq is given by the relationship ln K 0 lG n Å ln
G , C(1 0 aG )
[2]
FIG. 5. Adsorption isotherm of phosvitin at pH 2.0. The bulk phase was water adjusted to pH 2.0 with 1 N HCl at 247C. Cb is equilibrium bulk concentration of phosvitin.
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FIG. 6. Plot of low bulk concentration adsorption isotherm data of phosvitin (Fig. 5) according to Eq. [2].
where K is the equilibrium constant, a is the average area occupied per protein molecule at monolayer coverage (i.e., 1/ G at saturated monolayer coverage), C is equilibrium bulk concentration of protein, G is the surface concentration at equilibrium, n is an exponent related to cooperativity among adsorbing protein molecules, and l is related to an activation energy barrier for adsorption. The adsorption isotherm of phosvitin was analyzed according to Eq. [2] in order to determine the equilibrium constant K. The right-hand side of Eq. [2] was plotted against G n for n Å 1, 2, and 3. The lowest value of n that gave a straight line with the highest correlation coefficient was selected as its value. Figure 6 shows that a best fit is obtained with n Å 1, indicating that there is no cooperativity among phosvitin molecules adsorbing at the interface. However, a negative value of l indicates that the activation energy barrier for adsorption decreases with increasing surface coverage. The equilibrium binding constant, determined from the intercept, is 293 mg/ m 2 wt%. The effects of NaCl and CaCl2 on adsorption of phosvitin at the air–water interface are shown in Fig. 7. At pH 7, neither NaCl nor CaCl2 facilitates adsorption of phosvitin, indicating that charge neutralization by counterions alone is not sufficient to promote adsorption of phosvitin. At pH 2.0, addition of 0.1 M NaCl has no significant effect on the rate and extent of adsorption of phosvitin compared to those of the pH 2.0 control. However, at pH 2.0, addition of increasing amounts of CaCl2 increases both the rate and the extent of adsorption of phosvitin at the interface. The apparent diffusion coefficient of phosvitin, calculated from the initial slopes of the curves in Fig. 7, increases with increasing concentrations of CaCl2 in the bulk phase (Table 2). In 0.1 M CaCl2 at pH 2.0, the apparent diffusion coefficient is about an order of magnitude greater than that in the absence of
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FIG. 7. Effects of CaCl2 and NaCl on the kinetics of adsorption of phosvitin at the air–water interface at pH 2.0 and 7.0: s, no salt, pH 2.0; l, 0.1 M CaCl2 , pH 7; /, 0.5 M CaCl2 , pH 7; , 0.1 M NaCl, pH 2.0; h, 0.001 M CaCl2 , pH 2.0; n, 0.01 M CaCl2 , pH 2.0; l, 0.1 M CaCl2 , pH 2.0. The bulk-phase protein concentration was 1.5 mg/ml.
CaCl2 , and is in the range expected for a protein of comparable molecular weight. In 0.1 M CaCl2 at pH 2.0, the surface concentration of phosvitin at equilibrium is about 2.5 mg/ m 2 , which is twice that of the value when no CaCl2 is present. However, even at this high surface concentration, the phosvitin film shows no positive surface pressure. Protein adsorption at interfaces is generally considered to be irreversible (7, 9, 27). However, the data in Fig. 8 show reversibility of adsorption of phosvitin at the air–water interface to changes in the pH of the bulk phase. In these experiments, phosvitin was first allowed to adsorb at the interface for 1000 min at pH 2.0. Aliquots (0.1–1.0 ml) of 1 M NaOH were then sequentially injected into the bulk phase. After each injection, the surface cpm of the adsorbed phosvitin film was monitored until it reached a new equilibrium value, TABLE 2 Effect of CaCl2 Concentration on the Apparent Diffusion Coefficient of Phosvitin Determined from the Rate of Adsorption at the Air–Water Interface CaCl2 concentration (M)
D 1 107 (cm7/s)
0 0.001 0.01 0.1
2.06 2.23 4.76 13.7
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DISCUSSION
The results presented here show that adsorption of phosvitin at the air–water interface exhibits several anomalous behaviors. First, adsorption occurs only at low pH, and complete and instantaneous desorption takes place when the pH of the bulk phase is increased to neutral pH. Second, adsorption causes a slight but reproducible increase, instead of a decrease, in surface tension. These anomalies must arise from the unusual charge and other physicochemical characteristics of phosvitin. Phenomenologically, the electrostatic charge of a protein molecule exerts two types of potential energy barriers for its adsorption at a hydrophobic interface, e.g., the air–water interface (8). In the initial stages of adsorption of a charged protein molecule at a clean air–water interface, if e is the net charge of the protein and e0 and e are dielectric constants of the aqueous phase and the gas (air) phase, respectively, then, according to the electrostatic theory, as the protein approaches the air–water interface an image charge equivalent to e * Å e( e0 0 e )/( e0 / e )
FIG. 8. pH-induced desorption of phosvitin from the air–water interface in the absence of CaCl2 in the bulk phase.
at which time a 0.5-ml aliquot of the bulk phase was withdrawn to check the final pH. Figure 8 shows that an increase in pH of the bulk phase causes a rapid decrease in surface cpm, indicating that an increase in the net charge of adsorbed phosvitin molecules causes instantaneous desorption of phosvitin from the interface. When the pH of the bulk phase is increased to 7.4, almost all adsorbed phosvitin is desorbed into the bulk phase. The effect of CaCl2 on pH-induced reversibility of adsorption of phosvitin at the air–water interface is shown in Fig. 9. In this case, phosvitin was first allowed to adsorb at the air–liquid interface from a solution containing 1.5 mg/ml phosvitin and 0.1 M CaCl2 , pH 2.0. After about 1400 min of adsorption, a predetermined amount of 1 M NaOH that would increase the pH of the bulk phase from 2 to about 8.2 was injected into the bulk phase using a syringe. The change in surface cpm was monitored with time. Figure 9 shows that an increase in pH of the bulk phase from an initial value of 2.0 to a final value of 8.2 causes desorption of phosvitin from the interface. However, the rate of desorption in the presence of 0.1 M CaCl2 is much slower than that in its absence (Figs. 8 and 9). This difference might arise from possible intermolecular crosslinking between adsorbed phosvitin molecules by divalent Ca 2/ ions. This Ca 2/ -induced cross bridging may occur between phosphoserine residues as well as between the sialic acid groups of the single carbohydrate chain in hen phosvitin.
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[3]
would appear in the low dielectric gas phase. As the protein approaches the interface, an electrostatic repulsive potential energy equivalent to
FIG. 9. pH-induced desorption of phosvitin from the air–water interface in the presence of 0.1 M CaCl2 in the bulk phase. s, surface cpm; h, surface pressure.
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Eele Å
ee * e 2 ( e0 0 e ) Å 2de0 2de0 ( e0 / e )
[4]
will develop, where d is the distance of the protein from the air–water interface. If the magnitude of this repulsive electrostatic potential is greater than any attractive hydrophobic potential, then no adsorption would take place. On the other hand, if the attractive hydrophobic potential is greater than the repulsive electrostatic potential, then adsorption would proceed, albeit at a slower rate. Once a certain amount of protein is adsorbed to the interface, the adsorbed protein would create an additional electrical barrier equivalent to Ep,ele Å
* e dc Å e c,
[5]
where c is the electrostatic potential in the two-dimensional plane of charged protein film at the interface. The kinetics of adsorption of phosvitin at the air–water interface might be affected by both these electrostatic energy barriers. Phosvitin essentially is a polyanionic polymer with a net charge of 0179 at neutral pH. The amino acid sequence of phosvitin shows that it has no discernible hydrophobic regions, except for a short stretch of nonpolar amino acid residues at positions 205–216 (Fig. 1). The lack of adsorption of phosvitin at pH 7.0 indicates that the increase in electrostatic repulsive potential energy of phosvitin as it approaches the interface is so large that it could not adsorb at the interface. For example, using Eq. [4] it can be shown that the electrostatic repulsive potential energy of phosvitin at pH 7 (with 0179 charges) would be equal to its thermal energy kT (where k is the Boltzman constant and T Å 297 K) when it is at a distance of 11.5 mm from the air–water interface. What this means is that, because of its high electrostatic free energy at pH 7, phosvitin would not be able to penetrate a subsurface layer 11.5 mm from the air–water interface by diffusion alone. Therefore, it is not surprising that it behaves purely as a macroion and lacks interfacial adsorptivity at pH 7. It is significant, however, that, even though phosvitin has a total of 93 charged residues (54 negatively charged and 39 positively charged residues) and a net charge of 015 at pH 2.0 and contains several uncharged hydrophilic residues, it is able to adsorb to a significant extent at this pH. Equation [4] predicts that the electrostatic potential energy of a molecule carrying a net charge of 015 will be equal to its thermal energy (at T Å 297 K) when it is about 0.158 mm from the air–water interface. This electrostatic potential energy barrier would not allow the molecule to adsorb at the interface. However, the fact that phosvitin is able to adsorb to the interface in spite of this electrostatic potential energy barrier indicates that a more favorable hydrophobic interaction that
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can more than offset the electrostatic barrier must exist. The C-terminus hydrophobic peptide segment 205–216 ought to be the segment that can hydrophobically interact with the air–water interface, since no other hydrophobic stretch can be found in the primary structure of phosvitin. The rest of the molecule might exist in the form of loops suspended into the subsurface. Previous studies have shown that initial anchoring of a protein to the air–water interface involves binding of only a small peptide segment of 4–6 amino acid residues (9–11). Recently, it has been shown that peptide segments with a repeating heptet sequence of –P–N–P–P– N–N–P– , where P and N are polar and nonpolar residues, respectively, readily form a-helices with amphiphilic surfaces in aqueous solutions (29). The binary code of peptide segment 204–216 resembles that of the above heptet with slight variations, suggesting that this segment might exist in the a-helix configuration at the interface. Construction of a helical wheel of the segment shows that one-half of the helix surface is hydrophobic with Val, Tyr, Ile, His, Leu, and Trp residues, and the other half of the helix surface is hydrophilic with Glu, Ser, Gly, Ser, Ile, Lys, and Arg residues. Based on these analyses, it appears that the hydrophobic surface of this short helical segment might be the site that anchors the protein to the air–water interface. The energetics of interaction of this surface with the interface appear to be favorable for overcoming the electrostatic repulsive interaction of the molecule with the interface. However, the fact that small changes in the pH of the subphase cause instantaneous desorption of phosvitin from the interface (Fig. 8) indicates that retention of the protein at the interface is highly sensitive to changes in the net charge of the molecule. Although phosvitin is able to adsorb to an extent of about 1.25 to 2.5 mg/m 2 at the air–water interface at pH 2, the surface tension of the solution apparently increases with increasing amounts of adsorption. It should be pointed out that at comparable surface concentrations, other proteins generally cause significant reductions in surface tension. For instance, at a monolayer coverage of 1.8 mg/m 2 , b-casein exerts a surface pressure of about 18 mN/m, and monolayers of lysozyme and bovine serum albumin display surface pressures of about 6.5 and 16 mN/m, respectively (9, 30). Although an increase in surface tension in spite of protein accumulation at the interface appears to be incongruous and seems to violate Gibbs’ adsorption equation, it can be explained thermodynamically. As pointed out earlier, the only thermodynamic requirement for adsorption is a reduction of free energy of the system. Whether this occurs from a loss of free energy of the interface or the adsorbed solute or both is irrelevant. In the case of phosvitin the overall decrease in free energy of the system seems to originate from the loss of free energy of adsorbed phosvitin and not from a reduction in the free energy of the interface. If we assume that the nonpolar residues of the C-terminus helix segment (Val, Tyr, Ile, Leu, and Trp) are involved in the attachment of
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negligible. It seems that even at this high surface coverage phosvitin does not exist as a film at the interface, but probably exists in a gaseous state because of a lack of cohesive interactions between the adsorbed segments. The contribution of electrostatic repulsive forces to the surface pressure of a protein film at the air–water interface is given by the Davies equation (5) Pele Å 6.1C 1 / 2[cosh sinh 01 (135/AeleC 1 / 2 ) 0 1], [7]
FIG. 10. Theoretical G versus Pele curves based on the Davies equation (Eq. [7]) for charged monolayers with z Å 015 at equivalent electrolyte concentrations of 0.001 ( s ) and 0.3 ( h ).
phosvitin to the interface, then, based on Tanford’s hydrophobicity scale (31), transfer of these nonpolar residues from the water phase to the interface would decrease the free energy of phosvitin by about 052 kJ/mol. For G Å 1.25 mg/m 2 (i.e., 3.6 1 10 012 moles/cm2 ), the reduction in free energy of the system, resulting from adsorbed phosvitin molecules, would be 01.89 erg/cm2 . In contrast, the increase in free energy of the system, resulting from an increase in the surface tension caused by charge–dipole interaction between suspended protein segments and surface water molecules, is only about 0.5 erg/cm2 . Therefore, in spite of an apparent increase in surface tension, the free energy of the system is decreased by about 01.4 erg/cm2 due to adsorption of phosvitin. The surface pressure of a protein film at any given surface concentration is given by (5) Pa / w Å Pkin / Pele / Pcoh ,
[6]
where Pkin is the surface pressure contribution from kinetic movement of molecules in the two-dimensional film, Pele is the contribution from electrostatic forces, and Pcoh is the contribution from cohesive interactions among molecules in the film. Since proteins are large, the contribution of Pkin to the surface pressure can be assumed to be negligible. The fact that the phosvitin film does not exhibit positive surface pressure even at a surface concentration of 1.25–2.5 mg/ m 2 indicates that the magnitudes of Pele and Pcoh also were
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where C is the concentration of equivalent electrolyte in the ˚ 2 ) per charge in bulk phase, Aele ( Å A/z) is the area (in A the surface of the protein film, and z is the net charge of the protein. Theoretical G versus Pele curves, calculated from Eq. [7], are shown in Fig. 10 for phosvitin at pH 2.0 (z Å 015) in water (C Å 0.001) and in 0.1 M CaCl2 (C Å 0.3). It should be noted that Eq. [7] predicts a value of Pele Å 2.5 mN/m for G Å 1.25 mg/m 2 (monolayer coverage when C Å 0.001), and a value of Pele Å 3 at G Å 2.5 mg/m 2 (monolayer coverage in the presence of 0.1 M CaCl2 , i.e., at C Å 0.3). The disagreement between experimental (negative surface pressure) and predicted surface pressure values indicates that, in the adsorbed state, the charged groups of phosvitin are not present in the interface. It is likely that all charged segments exist as loops suspended into the subphase. Hydration of charged segments and repulsive interactions between the loops via hydration-repulsion and electrostatic forces might prevent cohesive interactions between the adsorbed segments at the interface (shown schematically in Fig. 11). These might be the reasons for the lack of contribution of Pele and Pcoh to the surface pressure of the film. In addition, the highly charged loops in the subsurface might exert an electrostatic pull on surface water molecules, which might be the reason for the observed increase in surface tension. The results presented here show that, because of its high electrostatic free energy, phosvitin does not adsorb to the
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FIG. 11. Illustration of possible orientation of phosvitin at the air– water interface at pH 2.0.
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air–water interface at high pH. A decrease in electrostatic free energy at low pH increases the rate and extent of adsorption of phosvitin. A saturated monolayer of phosvitin formed in either the presence or the absence of CaCl2 at pH 2 exhibits a negative surface pressure, indicating that neither electrostatic repulsion nor cohesive interactions among the adsorbed segments occurs in the monolayer film, and the hydrophilic segments of phosvitin suspended into the subsurface exert an electrostatic pull on surface water molecules, causing an increase in surface tension. Examination of the amino acid sequence of phosvitin indicates that only a short segment at the C-terminus, containing a few nonpolar amino acid residues, might be involved in anchoring the protein to the interface. This suggests that proteins can easily adsorb and form a film at the air–water interface through attachment of a small peptide segment. ACKNOWLEDGMENT Financial support from the National Science Foundation (Grant BES9315123) is gratefully acknowledged.
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