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Adsorption of Gelatin to a Polystyrene/Water Interface as a Function of Concentration, pH, and Ionic Strength† S. Fiona Turner,‡ Stuart M. Clarke,§ Adrian R. Rennie,*,| Paul N. Thirtle,⊥ Zhi Xin Li,⊥ Robert K. Thomas,⊥ Sean Langridge,# and Jeffery Penfold# Faculty of Life Sciences, University of Manchester, Sackville Street, Manchester M60 1QD, U.K., BP Institute, University of Cambridge, Madingley Rise, Cambridge CB3 OEZ, U.K., Neutron Research Laboratory, Uppsala University, Studsvik, SE-61182 Nyko¨ ping, Sweden, Physical and Theoretical Chemistry Laboratory, South Parks Road, Oxford OX1 3QZ, U.K., and Rutherford Appleton Laboratory, Chilton, Didcot, Oxon OX11 0QX, U.K. Received January 28, 2005. In Final Form: May 4, 2005 The technique of neutron reflection has been used to investigate the adsorption of R-enriched gelatin from aqueous solution onto spun polystyrene substrates. Neutron reflection can provide information about the distribution of material perpendicular to an interface as well as total adsorbed amounts. The adsorbed layers were found to have maximum density at the surface, decaying with distance into solution. The adsorbed amount, layer thickness, and density were all seen to increase with solution concentration. Temperature was found to have little effect on adsorption. Thicker, less dense layers were observed at high pH and thinner, denser layers were observed at low pH, but the total adsorbed amount did not change significantly. The presence of sodium chloride had little effect on the adsorbed layers. The results are discussed in the context of other studies and the known amino acid sequence of R-gelatin.
Introduction Gelatin adsorbs readily to most surfaces under a broad range of conditions. Its adsorption is particularly interesting because the structure of the adsorbed layer varies from surface to surface and with the pH, temperature, ionic strength, and concentration of the solution. In addition, several different types of gelatin can be obtained from different animal sources and extraction treatments. It is widely used in the food, pharmaceutical, and photographic industries to stabilize colloidal dispersions, yet much remains unknown about the characteristics of adsorbed gelatin layers. Gelatin. Gelatin is a biopolymer produced from the protein collagen, which forms the stress-bearing network in the skin, bone, cartilage, tendons, and ligaments of higher animals. A small percentage of collagen can be extracted into cold aqueous solutions of salt or dilute acid.1 This is known as acid-stabilized collagen or tropocollagen. Tropocollagen molecules are ∼2850 Å long and 14 Å in diameter2 with three parallel strands forming a triple helix structure. Thermal denaturing of the tropocollagen produces a mixture of single- (R) and double- (β) stranded gelatins, with a few triple (γ) strands. Pure R-gelatin consists of a single long strand of 609 amino acid groups or residues on a peptide backbone
†
Part of the Bob Rowell Festschrift special issue. * Corresponding author. E-mail:
[email protected]; Tel: +46 155 221831. ‡ University of Manchester. § University of Cambridge. | Uppsala University. ⊥ Physical and Theoretical Chemistry Laboratory. # Rutherford Appleton Laboratory. (1) Ramachandran, G. N. Treatise on Collagen; Academic Press: New York, 1967; Vol. 1.
where R1-n represents the amino acid groups. These amino acid side groups determine the characteristics of the molecule because many of them will have a charge, depending on the pH of the solution. The probability that a group will be dissociated at a certain solution pH can be calculated from its pK value: the probability, Pr, of dissociation of an acid group HA f H+ + A- is given by
Pr )
10(pH - pKa) 10(pH - pKa) + 1
(1)
For basic groups, the published pK value refers to dissociation such as NH3+ f NH2 + H+. The probability of association to give a positively charged site is given by 1 - Pr (dissociation). Every amino acid has an amino and a carboxylic acid group that ionize to give equal and opposite electrical charges, and certain amino acids have an additional acid or basic group that, if ionized, gives the site a net charge. Each amino acid group behaves independently. The product of probability and electronic charge determines the probable charge at each dissociated site; the sum of these probable charges determines the overall charge on the molecule. The pattern of charges along the molecule will determine its conformation and behavior. The interfacial behavior of polyelectrolytes has been the subject of many studies.3,4 Adsorption of Gelatin. A large amount of literature is available on the adsorption of gelatin, including studies of adsorption to polystyrene latices. The surface of polystyrene latices is curved and usually highly charged, so comparison with the flat, uncharged polystyrene film used in the present study is interesting. The adsorption of different types of gelatin to polystyrene latices has been (2) Rose, P. I. In The Theory of the Photographic Process, 4th ed.; James, T. H., Ed.; Macmillan: New York, 1977; p 51. (3) Fleer, G. J.; Stuart, M. A. Cohen; Scheutjens, J. M. H. M.; Cosgrove, T.; Vincent, B. Polymers at Interfaces, 1st ed.; Chapman & Hall: London, 1993. (4) Goddard E. D. J. Colloid Interface Sci. 2002, 256, 228.
10.1021/la050256o CCC: $30.25 © 2005 American Chemical Society Published on Web 07/19/2005
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Table 1. Previous Studies of Gelatin Adsorption to Latices
studied by various techniques. Details of the techniques, conditions, and results of these studies are summarized in Table 1. There is general agreement that the adsorbed amount increases with solution concentration to a plateau value. Vaynberg et al.,5,6 Hu et al.,7 and Cosgrove et al.8 quote plateau values of around 1.3-2.5 mg m-2 for gelatin close
to its IEP. Much larger values are reported by Kellaway and Najib9 as well as increased adsorption at lower temperatures, although this is attributed to the solubility of the food gelatins used. Hu et al.7 report almost identical adsorption isotherms at 25 and 40 °C in 2 wt % formamide solution and results in pure aqueous solution at 25 °C that are comparable to those of groups working at 40 °C.5,6,8
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It is difficult to compare the concentrations at which plateau values were reached because of the different units used. In Vaynberg et al.’s5,6 comparison of adsorption to polystyrene and acrylic latices, they conclude that there is reduced adsorption to highly charged surfaces. This may also be the cause of the variation in the results of Ngothai et al.,10,11 whereby adsorption is far higher onto larger latices because smaller latices produced using the same method are likely to have a higher surface charge. If so, the surface charge on the latices prepared by Ngothai et al. must be high compared to the small latices used by Cosgrove, Vaynberg, and Hu et al.5-8 Kellaway and Najib9 report similar adsorption maxima around the IEP of both acid and alkaline processed food gelatins, decreasing to around 75% of the maxima at high and low pH. Other groups measured the effect of pH on the adsorbed amount of photographic gelatin but only at and above the IEP of the gelatin. Vaynberg et al.5,6 report decreasing adsorption onto acrylic latex as pH is increased above the IEP but no change in adsorption onto polystyrene latex. Cosgrove et al.8 report decreased adsorption at high pH onto polystyrene latex. It is possible that the latex used by Cosgrove et al. may have had a higher surface charge than the polystyrene latex of Vaynberg et al. It is also possible that the sodium acrylate buffer used by Vaynberg et al. screened some electrostatic effects. Both groups report thicker layers on polystyrene latex at high pH. The change reported by Vaynberg et al. is a much smaller effect, again possibly due to the sodium acrylate buffer. The hydrodynamic layer thickness represents the tail of the distribution rather than the decay length, so the order-of-magnitude difference in the values is not unexpected. Cosgrove et al.8 refer to previous work where the adsorbed amount increased predictably with increasing salt concentration from ∼3 mg m-2 in pure water to ∼4 mg m-2 in 10 mM NaCl. In the results that they publish, however, they report a reduced adsorbed amount and reduced layer thickness in 1 mM NaCl solution compared to those in pure gelatin solution. The layer thickness increases, but the adsorbed amount in 10 mM NaCl still has values between 1.1 and 2.1 mg m-2. Vaynberg et al. report a slight increase in the adsorbed amount and a decrease in layer thickness on both polystyrene and acrylic latices as the salt concentration was increased up to 100 mM, causing aggregation of the acrylic latex at 2 M but with the polystyrene remaining polydisperse until >4 M. Both groups obtained gelatin with similar characteristics from Kodak, so the contrasting results must be due to the different latices used. Hu et al.7 performed many of their experiments using 2 wt % aqueous solution of formamide, an organic solvent that offers good solubility of gelatin at room temperature. The authors believe that the adsorption mechanism in formamide solution is less dependent on electrostatic effects because they noted a resemblance of the relative adsorption behavior to that of other hydrophobic systems. (5) Vaynberg, K. A.; Wagner, N. J.; Sharma, R.; Martic, P. J. Colloid Interface Sci. 1998, 205, 131. (6) Vaynberg, K. A.; Wagner, N. J., Sharma, R Biomacromolecules 2000, 1, 466. (7) Hu, T.; Gao, J.; Auweter, H.; Iden, R.; Lueddecke, E.; Wu, C. Polymer 2002, 43, 5545. (8) Cosgrove, T.; Hone, J. H. E.; Howe, A. M.; Heenan, R. K. Langmuir 1998, 14, 5376. (9) Kellaway, I. W.; Najib, N. M. Int. J. Pharm. 1980, 6, 285. (10) Ngothai, Y.; Bhattacharya, S. N.; Coopes, I. H. J. Colloid Interface Sci. 1997, 193, 307. (11) Ngothai, Y.; Ur’ev, N. B.; Bhattacharya, S. N. Colloid J. 1995, 57, 202.
Turner et al. Table 2. Amino Acid Groups in the r-Gelatin Molecule amino acida
no. per R-gelatin strand
glycine proline alanine hydroxyproline glutamic acid arginine aspartic acid serine lysine valine glutamine leucine threonine phenylalanine isoleucine asparagine methionine histidine
203 76 75 69 33 30 23 20 18 12 11 11 9 7 6 3 2 1
pK12
4.15 12.10 3.71 10.67
6.04
a
The formulas for each amino acid group can be found in refs 12 and 13.
If so, then their results may be relevant to adsorption to uncharged polystyrene film. In formamide solution, the plateau adsorbed amount remained at 2.5 mg m-2, but the plateau was reached at higher concentration. The hydrodynamic layer thickness was significantly higher, at around 20 nm compared to 4 nm in pure water. Experimental Section Materials. The gelatin used for this study was extracted after alkaline pretreatment from calf skin gelatin and underwent further fractional precipitation to remove material with high molecular weight. The sample had a gelation temperature of 38 °C. Solutions had to be heated to above 38 °C to dissolve the gelatin, and solutions of more than 1% gelatin by weight would gel on cooling below 38 °C. The radius of gyration of the molecule was measured at 173 ( 5 Å by small-angle neutron scattering from a 0.5 wt % solution at 20 °C. An amino acid sequence for calf skin R-gelatin is given in ref 2, but many of the glutamine and aspartane side groups would have been hydrolyzed to their respective acids during alkaline pretreatment. The proportion that undergoes hydrolysis determines the isoelectric point (IEP) of the molecule, pH 4.9 for the sample used in this study. The numbers of each amino acid side group given in Table 2 have been calculated from the sequence in ref 2 with the number of acid groups adjusted to give an IEP of 4.9. These numbers correspond to a relative molecular mass (RMM) of 55 000 for pure R-gelatin. The R-enriched gelatin used in this study had Mn ) 85 000 and Mw ) 114 000.14 Because there were few triple-helix γ strands in animal gelatin, it can be assumed that the sample contained ∼45% (by number) single R strands and ∼55% double β strands. The amino acid sequences of the two R strands in β-gelatin have not been documented, but it is assumed2 that they remain similar. The number of ionizations along an R-gelatin molecule and the net charge on the molecule have been calculated from the pK values in Table 2. These are shown as a function of pH in Figure 1. The charge on the molecule is dominated by the dissociation of approximately 50 basic and 50 acidic residues along the R-gelatin molecule. At low pH, only the basic lysine and arginine residues are ionized, with positive charge. There is a sharp change below the IEP as the glutamic and aspartic acid groups ionize, making the number of positive and negative sites more equal. Above the IEP, the molecule has a slight negative charge because there are eight more acidic than basic groups. At very high pH, (12) Handbook of Chemistry and Physics, 76th ed.; Lide, D. R., Ed.; CRC Press: London, 1996. (13) Merck Index, 12th ed.; Budavari, S., Ed.; Merck: Whitehouse Station, NJ, 1996. (14) We are grateful to Kodak Research, Harrow, U.K. (Dr. E. A. Simister) for this information.
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Figure 1. Number of ionized groups on the R-gelatin molecule (open squares) and net charge (filled triangles) as a function of solution pH. The arrow denotes the IEP. Table 3. Densities and Neutron Scattering Length Densities for the Materials Used in This Study density/g cm-3
material water PS silicon silicon oxide amorphous gelatin a
scattering length density/10-6 Å-2
H2O D2O hydrogenous deuterated
1.0012 1.1012 1.112 1.2a 2.3312 2.2012
-0.56 6.35 1.4 6.5 2.07 3.40
in H2O in D2O
1.412 1.4a
2.1 3.6
Calculated from the value for hydrogenous material.
the lysine and arginine residues cease to ionize, and the negative charge on the molecule increases. Neutron Reflection. Neutron reflection determines the composition profile normal to an interface from the ratio of reflected to incident intensity. The variation in specular intensity with angle and wavelength depends on the neutron refractive index profile normal to the surface. The refractive index, n, for neutrons of wavelength λ is given by
n2 ) 1 -
λ2F π
Q)
(4πλ)sin θ
(3)
where λ is the wavelength and θ is the angle of incidence. The three sample angles covered a range of momentum transfer of 0.012 < Q/Å-1 < 0.5. The slits defining the incident and reflected beams were opened during the angular scan to optimize the illumination of the sample and maintain the angular resolution at 5%. For any given profile, the reflectivity can be calculated exactly using the optical matrix methods of Abeles.18,19 The data were compared with model calculations of the reflectivity using an iterative least-squares technique. Care was taken that each model of the layer composition fit all data sets measured, with different contrasts obtained by mixing normal water, H2O, and D2O. Interfacial roughness was included in the model, and the fitting parameters were thickness, t, scattering length density, F, and interfacial roughness for each layer, whose density profile could be rectangular, exponential, or half-Gaussian. The results for F can be interpreted as chemical compositions using the data in Table 3.
Results and Analysis (2)
where F is the neutron scattering length density of the medium and depends only on the atomic and isotopic compositions. The values of F for the materials used in the present study are given in Table 3. The scattering length densities of deuterated and protonated materials are very different. This enables different contrasts to be used to provide independent data sets to check the validity of a model or to label otherwise indistinguishable components of a mixture to determine the location of each material. The scattering length densities for R-gelatin have been calculated from the composition outlined in Table 2. Similar substrates were used for all experiments: silicon crystals covered with thin films of polystyrene prepared by spincoating. The silicon crystals were first cleaned of organic contamination by immersion in RCA115 (basic hydrogen peroxide) at ∼75 °C for 10 min, followed by rinsing. They were then treated with a buffered hydrofluoric acid solution (Isoform, 7:1 NH4F/ HF for 3 min) and rinsed with water. HF etches the silicon oxide layer and leaves a surface that is hydrogen-passivated and hydrophobic. This reduces the tendency for the polystyrene to de-wet on heating. Deuterated PS films were prepared by spinning from a 1% w/w solution in toluene at ∼70 °C onto a large glass slide, floating on the surface of water, and were then lowered onto the large surface of the block cut as a (100) face. The films were annealed on the crystals for 2-3 h under vacuum at ∼160 °C, which is well above the glass-transition temperature of the polystyrene (∼100 °C). The layers on the neutron reflection crystals were uniform, but the thickness varied between experiments from 290 to 450 Å. (15) Kern, W. J. Electrochem. Soc. 1990, 137, 1887.
Neutron reflection experiments were conducted using the timeof-flight reflectometer SURF at the ISIS facility as described elsewhere.16 The crystal substrate was mounted horizontally clamped above a PTFE cell containing the solution to be measured. Care was taken to avoid air bubbles in the cell because they would collect on the reflection surface and affect the measurements. Each substrate was initially characterized using four water contrasts: H2O, D2O, contrast matched to silicon (CMSi) with a scattering length density of 2.07 × 10-15 Å-2, and contrast matched to a scattering length density of 4.0 × 10-15 Å-2 (CM4). Measurements were then performed on adsorbate solutions. Each gelatin solution was allowed to equilibrate in contact with the surface for at least 30 min before measurement. An experiment using FTIR-ATR spectroscopy17 demonstrated that almost all adsorption was complete within 30 min. Measurements were made at three sample angles: 0.35, 0.8, and 1.8°. It is usual to express the reflection data as a function of the momentum transfer normal to the surface, Q, given by
All gelatin layers measured by neutron reflection were found to have a maximum density at the polystyrene surface, decreasing with distance into solution. Data were fit with models using rectangular, half-Gaussian, and exponential density profiles. Rectangular profiles gave visibly worse fits, but there was very little difference between fits with half-Gaussian and exponential density profiles for the data in this study. Two-layer models were not found to offer any advantage. The results presented in this paper are from fits with half-Gaussian profiles because these were thought to be more natural, starting with zero gradient rather than infinite gradient at the surface. Cosgrove et al.8 also reported adsorbed layers with maximum density at the surface, decreasing exponentially into solution. The decreasing density profile is further supported by comparison with viscometry and dynamic light scattering studies.5-7,11 These techniques measure the furthest point from the surface at which adsorbed gelatin can be detected rather than the standard deviation or exponential decay length, and the layer (16) Penfold, J.; Richardson, R. M.; Zarbakakhsh, A.; Webster, J. R. P.; Bucknall, D. G.; Rennie, A. R.; Jones, R. A. L.; Cosgrove, T.; Thomas R. K.; Higgins, J. S.; Fletcher, P. D. I.; Dickinson, E.; Roser, S. J.; McLure, I. A.; Hillmann, A. R.; Richards, R. W.; Staples, E. J.; Burgess, A. N.; Simister, E. A.; White, J. W. J. Chem. Soc., Faraday Trans. 1997, 93, 3899. (17) Turner, S. F. Ph.D. Thesis, University of Cambridge, Cambridge, U.K., 1998. (18) Abeles, F. Ann. Phys. 1948, 3, 504. (19) Heavens, O. S. In Optical Properties of Thin Solid Films; Dover: New York, 1991.
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Figure 2. (a) Adsorbed amount of gelatin as a function of solution concentration on the d-polystyrene surface (filled triangles) and the h-polystyrene surface (open squares). The error bars are larger for the measurements on h-polystyrene because the scattering length density of h-polystyrene offers less contrast with that of gelatin. (b) Adsorbed layer thickness (filled shapes) and composition (open shapes) as a function of solution concentration. Triangles represent measurements on d-polystyrene, and squares represent measurements on hpolystyrene. A model with a half-Gaussian density profile has been used to fit the data, so the values presented are the widths of the half-Gaussian density profiles and the maximum density at the surface; the composition of gelatin decreases with distance into solution.
thicknesses reported are an order of magnitude higher than those in the present study and those in Cosgrove et al. Adsorption as a Function of Concentration. Adsorption isotherms for gelatin adsorption onto two different polystyrene surfaces are shown in Figure 2a. One silicon crystal had a coating of d-polystyrene, and one had a coating of h-polystyrene. The variations in volume fraction and adsorbed layer thickness with concentration are shown in Figure 2b. The adsorbed layers formed on the h-polystyrene surface were significantly less dense than those formed on the d-polystyrene surface and contained less material. Because isotopic labeling should not affect the surface properties of a polystyrene surface, it was suspected that the difference was due to a change in the experimental conditions. Both samples had been kept at 34 ( 1 °C. In another neutron reflection experiment, adsorbed layers were heated and cooled between 25 and 55 °C, with no change observed in the reflectivity profiles. Variation of the temperature of addition of solutions to the cells was also found to have little effect on the adsorbed layers. The solution pH had not been controlled, but the gelatin acts as a buffer, reducing the pH of the solution toward its IEP (pH 4.9). Solutions were made shortly before addition to the d-polystyrene surface but then stood for around 10-14 h before use on the h-polystyrene surface. Sealed flasks were used, but it was observed that the pH of solutions of 0.02% gelatin was around 5.5 initially, decreasing to around 5 after ∼5 h because of the absorption of carbon dioxide from the air in the flask. This decrease would have been enhanced, and further decrease prevented, by the gelatin that is self-buffering toward its IEP. The first solution added to each block would have had a particularly different pH: the initial solution added to the d-polystyrene surface was only 0.0001% gelatin by weight, which would have had little buffering effect, giving a pH of around 6.5. In contrast, the first solution added to the h-polystyrene surface was 0.001% gelatin that had been standing for hours. The pH would have been around 5. Increasing adsorption with solution concentration is in agreement with previous studies, although plateaus have been observed at higher concentrations, as discussed in the Introduction. The adsorbed amounts observed in the present study are much higher than those reported for
Turner et al.
polystyrene latices under similar conditions. For example, the R-gelatin studied by Cosgrove et al.8 gave coverage onto polystyrene latex of about a fifth of that observed for 1% solution onto polystyrene film during the present study, although the reported layer thickness is similar. This is likely to reflect the charged nature of the latex in comparison to the nature of the uncharged polystyrene film and is further supported by the findings of Vaynberg et al.5 of reduced adsorption onto more highly charged latices. Polystyrene film offers a more favorable surface for hydrophobic interactions. Increased adsorption to the polystyrene film implies that hydrophobic interaction is a significant mechanism for the adsorption of gelatin onto polystyrene. This is in agreement with the opinion of Hu et al.,7 although the observation of increased adsorbed amount rather than layer thickness does not agree with their results using formamide solution to encourage hydrophobic bonding. Adsorption as a Function of pH. A second experiment was conducted to investigate the effect of solution pH on the adsorption from 0.02% gelatin solutions onto two d-polystyrene-coated substrates. Both sets of measurements were initiated with measurements of pure gelatin solution, with pH not adjusted (pH 5). Sodium hydroxide and hydrochloric acid were then used to adjust the solution pH, as 0.1 and 1 M solutions in H2O and D2O. These were added dropwise to give the required pH change. The concentrations of gelatin were not reduced by more than 1% of their original values. BDH narrow-range pH paper was initially used to measure pH but was found to be inaccurate for gelatin solution measurements when a solution measured as pH 6 etched a silicon surface. This solution was measured as pH 10 by a pH meter and by an indicator liquid. The pH meter was used for all further measurements and supplied all of the pH measurements in this paper. Figure 3a shows the amounts of gelatin adsorbed onto polystyrene surfaces from 0.02% solution as a function of pH. The error bars are large for the measurement at high pH because the solutions had begun to etch the silicon surface, rendering the model from the characterization data less accurate. Re-characterization was not possible owing to limited beam time and the fact that the gelatin could not be rinsed off. There was also some uncertainty in the pH of this measurement because the BDH narrowrange pH paper used for the measurement did not give accurate measurements at high pH. The variations in adsorbed layer thickness and density are shown in Figure 3b. It can be seen that there is no significant variation in the adsorbed amount with pH. There is, however, a trend toward thicker, less dense layers at high pH and thinner, more dense layers at low pH. This is in agreement with the findings of Vaynberg et al.6 for adsorption to polystyrene latices. Cosgrove et al.8 report increased layer thickness but with a reduced adsorbed amount at high pH. This may be due to the higher surface charge on their latex, as discussed earlier. A change in layer thickness with no accompanying change in adsorbed amount suggests that the conformation of each molecule changes with pH, with the molecules lying closer to the surface at low pH, with more loops into solution at high pH. This must be due to changes in the pattern of charges along the molecule as a function of solution pH. In Figure 1, it was shown that there are significantly more ionized groups above the IEP of R-gelatin, although the net charge on the molecule is low. Solvation will be favored by a section of the molecule with a high density of charges, and a position close to the
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Figure 3. (a) Adsorbed amount of gelatin from 0.02% solutions as a function of pH, measured on two separate d-polystyrene surfaces. The experimental reflectivity profiles have been fitted with a half-Gaussian model for the adsorbed layer density profile. (b) Thickness (filled shapes) and composition (open shapes) of the adsorbed layer as a function of pH. The values plotted are the standard deviations of half-Gaussian density profiles for the adsorbed gelatin layers used to fit the reflectivity data and the maximum density at the surface. The squares and triangles represent measurements performed on different d-polystyrene surfaces.
Figure 4. Schematic diagrams of predicted conformation of R-gelatin molecules at pH 3, 5, and 8. The diagrams show the maximum distance from the surface (if the molecule were fully extended) plotted against residue number. Filled diamonds represent positive charges, and open squares represent negative charges.
hydrophobic surface will be preferred by an uncharged section of the molecule. Thus, an adsorbed molecule is likely to have more loops or tails penetrating into solution at high pH. Figure 4 shows a schematic diagram of the pattern of charged groups along an adsorbed R-gelatin molecule at pH 3, 5, and 8 and a first-level prediction of its conformation. The molecule is spread out along the x axis and numbered from 1 to 609 to represent the position of each residue within the molecule. Positively charged sites are shown as diamonds, and negatively charged sites, as squares. Below about pH 3, around 50 of the 609 amino acid groups along the molecule are ionized. The molecule has a high positive charge. Between pH 5 and 10, around 100 groups are ionized, but the distribution of positive and negative charges is more even. The molecule has very little charge at pH 5 and a small negative charge at pH 10. As a first approximation, a section of the molecule with a high density of charged sites will seek a position in solution; an uncharged section will prefer a position at the hydrophobic/aqueous interface. If there are more than 5 charged residues in a section of 20, then that section of the molecule has been plotted in Figure 4 as a loop or tail penetrating into solution. The y axis shows the maximum displacement that a loop could reach from the surface if
it were fully extended. This is not likely to happen, but it provides a scale for the comparison of different graphs. The distances have been calculated according to a distance between alternate carbon atoms in a chain of 2.53 Å20 and therefore a distance between amino groups of around 3.8 Å. The condition of 5 charged groups in 20 to make an area of the chain hydrophilic has been found empirically to give loops with maximum penetration into solution of ∼75 Å. This is of the correct order of magnitude to give the distribution widths measured for the adsorbed layers. It can be seen from the graphs that a single molecule lies close to the surface at pH 3, giving a thin, dense layer. There are a few loops in solution at pH 5 and more still at pH 8, giving a thicker, less dense layer. The hydrophilicity condition can be varied from 3 to 7 in 20 or from 6 to 10 in 30 without affecting this trend. Only the size and number of loops are affected, as would be expected. The representation used in Figure 4 is a simplification to one dimension for the convenience of portrayal; in reality, the molecule is likely to adopt a random, coiled conformation in three dimensions. It would be interesting to generate a three-dimensional model using secondary structure prediction techniques, but an accurate model was considered to be beyond the scope of the current work. The validity of the ionization predictions was tested by measurements of the surface tension of gelatin solutions at different pH values as a function of concentration.17 If the adsorbed amounts are assumed to be similar to those reported below for the polystyrene/water interface, then the Gibbs equation can be applied in reverse to give the number of species at the interface. For 0.02% solution, assuming an adsorbed amount of ∼2 mg m-2, this gave 17 species at the surface for pure solution, 18 at pH 10, and 25 at pH 3. This is in accord with Figure 1: if the net charge on the molecule is higher, then more counterions will be retained in the adsorbed layer to screen the electrostatic repulsion between neighboring positively charged amino acid groups. Adsorption as a Function of Ionic Strength. The addition of sodium chloride had no significant effect on the total amount of gelatin adsorbed and very little effect on the adsorbed layer thickness and density, as shown in Figure 5. The measurements are all within the error of each other, but there is a slight trend toward a compressed layer at high ionic strength. Again, these results are supported by those of Vaynberg et al.6 for adsorption to relatively low surface charge polystyrene latex. The effect is not simply due to pH because the addition of sodium chloride to gelatin was found to increase its pH slightly, (20) Tanford, C. J. Phys. Chem. 1972, 76, 3020.
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Figure 5. (Bottom) Thickness (open squares) and proportion of gelatin (filled triangles) in the layer adsorbed from a 0.02% solution of gelatin in H2O with added NaCl. The parameters plotted are the standard deviation and maximum density of a model with a half-Gaussian density profile decaying into solution. (Top) Solution pH measured by an ATI Orion 520A pH meter (solid line) and a BDH 4080 indicator liquid (dotted line).
as shown in Figure 5. Thicker, less dense layers were observed at high pH in the previous experiment. Conclusions The adsorption of R-gelatin has been studied primarily by neutron reflection measurements. Adsorbed layers were found to have maximum density at the surface, decaying
Turner et al.
with distance into solution. The adsorbed amount, layer thickness, and density were all seen to increase with solution concentration. Comparison with adsorption studies performed using polystyrene substrates with some charge5-11 indicates that hydrophobic interactions are the main adsorption mechanism for gelatin; lower coverage is seen on more highly charged surfaces. Thicker, less dense layers were observed at high pH and thinner, denser layers were observed at low pH, but the total adsorbed amount did not change significantly. This implies that as the pH changes the same number of molecules are adsorbed to the polystyrene surface but their conformation is altered with changes in the solution pH. In addition, surface tension measurements at the air/solution interface indicated that many more counterions were retained at the interface at pH 3 than at pH 5 or 10. Explanations are proposed for both of these pH effects in terms of the amino acid sequence of the R-gelatin molecule. At low pH, a higher proportion of the molecule is uncharged and hydrophobic, so the molecules lie closer to the surface, forming thinner, denser adsorbed layers. Twice as many groups ionize at pH 10, causing the molecule to extend into solution. The net charge on the molecule is lower, however, limiting the number of counterions retained. More counterions will be retained in the adsorbed layers at low pH because the molecules have a higher net charge and are more closely packed. Acknowledgment. S.F.T. thanks the Oppenheimer Fund of Cambridge University and Kodak European Research for a research studentship. We are grateful to the CCLRC ISIS facility for neutron beam time. LA050256O
