Direct Measurement of Protein Adsorption on Latex Particles by

Chapter 29

Direct Measurement of Protein Adsorption on Latex Particles by Sedimentation FieldFlow Fractionation 1

1,3

2

Yong Jiang , J. Calvin Giddings , and Ronald Beckett

Downloaded by COLUMBIA UNIV on March 10, 2013 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0602.ch029

1

Department of Chemistry, Field-Flow Fractionation Research Center, University of Utah, Salt Lake City, UT 84112 Department of Chemistry, Water Studies Centre, Monash University, P.O. Box 197, Caulfield East, Victoria 3145, Australia

2

In previous work from this group it was shown that sedimentation field-flow fractionation (SdFFF) is capable of the direct measurement of quantities as small as a few attograms (10-18 g) of various materials, including γ-globulin, adsorbed on latex beads. Here, following a review of the principles of adsorbed mass measurements by SdFFF, the previous work is extended to include numerous measurements of immunoglobulin (IgG) adsorbed on polystyrene latex beads under diverse conditions. A few additional γ-globulin measurements are reported as well. Adsorption of IgG on latex was quite rapid with adsorption times usually and d, respectively. When the particle adsorbs a coating of mass m having a density p and a density increment Ap , F becomes the sum of two terms, the first for the particle and the second an incremental term for the coatings (7) p

p

c

p

c

c

If the particle diameter is d and the mean film thickness is h, F becomes F = F + Kd hAp G 2


p

+ hAp j

2

= d 7cG^£*-

c

c

(6)

providing h « d. (A spherical particle with an idealized uniform film is shown in Figure 3.) Since F is obtained by F F F retention measurements and is thus a known quantity (via equation 1), it remains only to invert the above expressions for F to obtain values for the coating mass and thickness, m and ft, respectively. We obtain V) c

m

„

Pc

F

-

PP

Pc

A

m

and F "nGd Ap

h h

dAp 6A

p

2

c

w

Pc

These expressions are valid when the particles and their external coating are both denser than the carrier medium and sink to the outer wall, as is the case here. A more general treatment accounting for negative Ap values and "floating" particles is given in the original publication (7). If the adsorbed mass is small compared to the bare particle mass, (mp «m ), then t may be only slightly increased by adsorption, that is, by the addition of m to m . A small error in the final terms of equations 7 or 8 would then cause a large error in m or ft, respectively. Thus it is perhaps more accurate, and generally preferable in practice, to obtain F (by numerical means) from equation 1 for the bare particle rather than rely on equation 4 to get F from particle parameters. The latter (less accurate) approach underlies equations 7 and 8. The proposed improvement is implemented by rearranging equations 5 and 6 as follows p

r

c

p

c

p

p

m m

_Pc(F-F ) GA p

c

~

Pc

and

In Proteins at Interfaces II; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

( 9 )

409

410

PROTEINS AT INTERFACES II

where force F on the coated particles and force F on the bare particles are to be obtained using equation 1 from the respective t values measured at the same field strength G . If different G values are used for the two t measurements, (F - F )/G would be replaced by (FIG) - (FplG ). Steric perturbations to retention (6, 8), although small under most circumstances, might best be compensated by adjusting G and G values such that F = F . However, i f these perturbations are significant, a sterically corrected version of equation 1 should be used. For cases in which equation 2 is a good approximation to equation 1, m and h can be expressed explicitly in terms of the time increment At between the elution of coated and uncoated particles. For this purpose we use equation 2 to obtain p

r

r

p

p

p

p

c

r


F-Fp=^*r

(ID

The substitution of equation 11 into equations 9 and 10 yields

*-=gfc* and 6

h= wt°7cd !SiGAp At 6

2

A

(13)

r

c

Equation 12 will be used later to show that At is highly sensitive to small adsorbed masses. Specifically, even with G values as small as 544 gravities (as used here), only 10-20 IgG molecules are needed to give measurable shifts in At . r

r

Experimental Equipment and Materials. The sedimentation FFF system used in this work has been described in the literature (70). The device is similar to the Model S101 Colloid/Particle Fractionator from FFFractionation, Inc. (Salt Lake City, UT). The channel has dimensions of 90 cm in length, 2 cm in breadth, and 0.0127 cm in thickness. A l l work was done at room temperature (22 ± 1°C). The spin rate used in these experiments was 1800 rpm, producing 544 gravities of acceleration. The FFF system was equipped with a Kontron H P L C pump (Kontron, London, U K ) to pump the carrier liquid into the channel. The eluted sample components were detected by a U V detector (Model 757 Absorbance Detector, Applied Biosystems, Ramsey, NJ) with the wavelength set at 280 nm. The detector signal was recorded by a chart recorder, stored in a computer, and analyzed by a special program written in this laboratory. The particles used for the coating work were 0.215 |im polystyrene latex beads with surfaces specially cleaned for protein coating (Polyscience, Inc., Warrington, PA). Human IgG was purchased from Sigma Chemical Company (St. Louis, MO). The carrier was a 10 mmol Tris[hydroxy methyl] aminomethane (United States Biochemical Corp., Cleveland, OH) solution. The p H of the solution was adjusted to 9.0 using H N O 3 (J. T. Baker Chemical Co., Phillipburg, NJ). Phosphate buffer solutions of various pH values were used for sample preparation in this work. They were prepared by using proper ratios of Na2HP04 and NaH2P04 (Mallinckrodt Specialty Chemicals Co., Paris, K Y ) . The actual p H of the solutions were deter mined using a Model 630 pH meter (Fisher Scientific, Fair Lawn, NJ).


29. JIANG ET AL.

Measurement of Protein Adsorption on Latex Particles


Preparation of Coated Particles for FFF Analysis. The 2.5% (w/v) original particle suspension was first diluted to 0.0125% with triple distilled water. The IgG sample was dissolved in 20 m M phosphate buffer solution of the required p H value to yield 20 mg/mL stock solutions. The stock solutions were then diluted to the desired concentration in the same buffer solution. A 50 p L aliquot of each protein solution was added to a 50 p L 0.0125% polystyrene latex suspension. The mixture was then allowed to stand at room temperature (22 ± 1°C) for various specified intervals before sedimentation FFF analysis. Approximately 10 or 20 p L of sample was injected directly into the F F F channel. Following the injection, the flow was stopped for 4 minutes for relax ation, a process to allow the particles to reach an equilibrium state in the channel. After relaxation, the flow was resumed and the run proceeded under various specified conditions. Results and Discussion Precision in Measurement of Coating Mass. The mass of protein adsorbed m is calculated from the measured SdFFF retention time (t ) for the coated bead using equations 7 and 12. In order to assess the errors in m and the minimum adsorption level detectable by the method, 37 runs were made over 3 days on a 0.215 pm diameter polystyrene under the same operating conditions of field strength (544 gravities, 1800 rpm) and flowrate (2.0 mL/min) as used generally in these studies. The measured t values and standard deviations obtained for each day's work are given in Table I. The standard deviation for the entire data set was 0.06 min. This shows that the precision in measuring t is less than 1%. There was a small (0.08 min) difference in the mean of die results for day 2 compared to the mean for days 1 and 3, which were identical. Equation 12 can be used to obtain an expression for the standard deviation of the calculated coating mass a f

r

c

r

r

mc

Table I. Retention Time t and Standard Deviation c (Both in Minutes) of 0.22 pm PS at 1800 RPM and a Flowrate of 2.0 mLVmin r

t (min) r

t (min) (average) o, (min) r

r

t

Davl 7.233 7.167 7.267 7.300 7.200 7.200 7.300 7.233 7.267 7.233 7.253 7.233 7.237 7.24

Dav2 7.330 7.333 7.300 7.265 7.330 7.267 7.330 7.267 7.360 7.330 7.400 7.400

Dav3 7.267 7.267 7.267 7.267 7.233 7.233 7.233 7.267 7.200 7.200 7.233 7.167

7.32

7.24

0.038

0.046

0.033


411


412

This yields a value for (T« of 1.5 X 10* g for our experimental conditions, assuming p = 1.37 g/mL. For human IgG, which has an average molecular weight of 158,500, corresponds to ~6 molecules. If the measured mass of the coated particles is ^3 0^ greater than the mass of the uncoated particles, then the difference in mass which we attribute to the adsorbed protein is highly significant statistically. Thus the coating mass that can be determined with relative certainty by the SdFFF technique at a field strength of 544 gravities is only 4.5 x 10" g, a mass that corresponds to about 17 protein molecules. This value indicates the high sensitivity of the method to the adsorbed amount. Under conditions of adsorbate and adsorbent concentrations where the change of the protein concentration is small, the conventional depletion method for studying adsorption has limitations. In SdFFF, the adsorbed mass is determined direcdy from the retention time or retention time increment and not indirecdy by the difference in the concentration of protein remaining in the solution. The high sensitivity of SdFFF makes the method suitable for the investigation of protein adsorption over a large range of conditions which should be particularly useful in probing the low adsorption-density end of the isotherm and for adsorption isotherms where the adsorption is strong but the equilibrium concentration in solution is below the detection limit for the analytical method being used. 18

c


18

Determination of Adsorption Isotherms. As the concentration of IgG added to the latex suspension increases, the peak generated by the latex is shifted to higher retention times, indicating an increase in mass of the beads. We attribute this increase to the adsorption of IgG on the particle surface. Using equation 7 or 12, the adsorbed mass m may be calculated either from the measured force (F) exerted on the particles in each of the experiments or from the time increment At . The adsorption density X , expressed as mass adsorbed per unit area, may also be cal culated based on m and the estimated surface area of the particle (1.45 x 10" m ). In addition, we also calculated the thickness of the adsorbed layer by means of equation 13 (or equation 8). (The calculated h is, of course, only a mean thickness for a compact coating of protein.) These data are summarized in Table n. The adsorption isotherm (X vs C ) of IgG on the surface of PS latex beads is displayed in Figure 4a. The equilibrium concentration of IgG, C q, remaining in the solution after the adsorption is finished was obtained by subtracting the mass adsorbed to the particles in unit volume of solution from the initial concentration. In most cases, the initial concentration and the equilibrium concentration differ only slighdy. The isotherm obtained can be fitted to the Langmuir equation c

r

13

2

c

eq

e

* = r a ^

15

where a and b are constants. Equation 15 can be rearranged to give ^ - = bC + \ eq


(16)

29. JIANG ET AL.



adsorbed

Figure 3. Illustration of an idealized particle with adsorbed coating of thickness h. (Reprinted with permission from Langmuir. Copyright 1991 American Chemical Society.)

C

e q

(mg/mL)

Q (mg/mL) q

Fig 4. Adsorption isotherm plots for human IgG onto PS latex particles: (a) adsorption density X versus equilibrium solution concentration; (b) Langmuir plot C qlX versus Ceq- The line in Figure 4a is a Langmuir model curve plotted using the parameters a and b obtained from the regression line in Figure 4b. e


413


414

Table II. Compilation of Mass m Adsorption Density X, and Thickness h of Film of IgG on Polystyrene Latex Beads for Different Protein Concentrations


Cy

0.05 0.10 0.20 0.40 0.60 0.80 1.0 2.0 3.0 4.0 5,0

A

m (X1016)

X (mg/m )

h (A)

0.0164 0.0152 0.0147 0.0137 0.0137 0.0136 0.0136 0.0135 0.0135 0.0136 0.0137

3.97 5.04 5.54 6.65 6.65 6.77 6.77 6.89 6.89 6.77 6.65

2.75 3.47 3.81 4.58 4.58 4.66 4.66 4.74 4.74 4.66 4.58

19.9 25.3 27.9 33.4 33.4 34.0 34.0 34.6 34.6 34.0 33.4

c

2

Thus the Langmuir constants a and b can be obtained from the intercept and slope, respectively, of a plot of C PC versus C q. The straight line plot shown in Figure 4b demonstrates that the data follow the Langmuir form. Using the constants so obtained (a = 109, b = 0.20) the solid line in Figure 4a was plotted. The plateau in the Langmuir adsorption isotherm is lib. For the case of IgG adsorbed to polystyrene at p H 7 and ionic strength 0.01 M, the maximum (plateau) adsorption level was found experimentally to be 4.75 mg/m . This is in agreement with the values found for adsorption of globular proteins, which usually have a value of a few mg/m depending on the nature of the protein and the particle surface (77, 72). Assuming an average molecular weight of 158,500 Dalton, the adsorption density above corresponds to about 2600 IgG molecules per particle. The average area occupied by a protein molecule is thus about 56 nm , which is also within the range reported in the literature of about 10-100 n m (77). This area is equivalent to a square with a side length of 7.4 nm. Since a spherical particle of the same mass and density as an IgG molecule will have a diameter of about 7.2 nm, we concluded that the protein molecules are rather tightly packed on the particle surface. In our earlier work on protein adsorption (7), PS latex beads were used without any further clean-up procedure. For these beads, residual surfactant (sodium lauryl sulfate) is probably present on the surface due to the procedure used in the preparation of the latex particles (75). The adsorption behavior of ^-globulin on these particles was studied. We found that, although the isotherm is also similar to that of a Langmuir type, the plateau adsorption was reached at a much higher concentration. In the present study we have used particles prepared without surfactant. The isotherms of these two type of particles are shown in Figure 5. The figure shows that the plateau adsorption values on the surface of particles with and without the surfactant are fairly close, being 2.94 and 3.10 mg/m , respectively. However, the concentrations of proteins required to reach the plateau adsorption differ by about one order of magnitude, approximately 0.3 mg/mL for the clean surface and 4 mg/mL for the surface with surfactant. The results suggest that the protein and the surfactant compete for adsorption sites on the surface of the latex beads. eq

e

2

2

2

2

2

Influence of p H on the Adsorption Maximum. The maximum adsorption density for the IgG-polystyrene system was measured at different solution p H values but



29. JIANG ETAL.


with the ionic strength maintained constant at 0.007 M . The results, which are plotted in Figure 6, display a small but distinct peak in the adsorption maximum occurring near p H 7. The adsorption density at the p H of this peak is at least 25% above the values obtained outside the p H range 5.5-9.5. Since the isoelectric point of various IgG proteins is reported to be in the p H range 5.8-7.3 (14) it is likely that the maximum in the adsorption density measured here occurs at the isoelectric point for the sample. A number of reasons could contribute to this observation. For example, intermolecular charge repulsion would be minimized at the isoelectric point which would lead to less charge repulsion between adsorbed protein molecules on the surface. It is also possible that reduced intramolecular repulsion could lead to a contraction of the protein molecule, leading to a smaller cross sectional area per molecule. In addition, protein-surface charge repulsion could also contribute to the reduction in adsorption density at any p H above the isoelectric point as the latex surface would be negatively charged under the conditions studied. Adsorption Characteristics. The adsorption of IgG onto polystyrene is quite rapid with adsorption times generally being less than 10 minutes, sometimes much less. With increasing IgG concentration, the adsorption density increases in a series of quasi-equilibrium steps. Figure 7 illustrates that the same adsorption density is obtained irrespective of whether the IgG is added in one step or two. In this series of experiments, 0.05 mg/mL IgG was equilibrated with polystyrene beads for 60 min and then the IgG concentration was increased to 1 mg/mL. The adsorption process was followed by making SdFFF runs after specific elapsed times. The results for this two step adsorption were then compared (Figure 7) with those from a single adsorption with 1 mg/mL IgG. Protein adsorption can be accompanied by a relaxation process where the molecular conformation changes over time in such a way that the proteins occupy a higher area per molecule than on initial adsorption (75). This could possibly lead to a lower amount adsorbed in the two step adsorption experiment. The fact that the same adsorption density was achieved in two steps as in one would appear to indicate that either this surface conformational change of the IgG molecules is reversible or that the adsorbed molecules can undergo a further rearrangement which reduces the area occupied per molecule when the surface becomes crowded. Protein Desorption. The SdFFF adsorption method will only measure relatively strongly bound protein as during elution (typically 5-20 min) the particle surface is continually washed with fresh carrier solution. Desorption of the protein over longer time periods was tested in a series of experiments where initial adsorption was carried out in solutions of 1 mg/mL IgG and then the suspension was diluted to give 0.025 mg/mL. After various periods of time at the lower concentration, SdFFF runs were performed to measure the amount of IgG remaining on the surface. The results of these experiments are plotted in Figure 8. Slow desorption to about 20% of the original IgG adsorbed mass occurred over about 2 hours. However, negligible desorption occurred after this for periods up to 6 hours. Conclusions New results are presented here on the direct measurement of human IgG adsorption on polystyrene latex beads using sedimentation FFF. The results confirm the high sensitivity and overall efficacy of sedimentation FFF used as a tool for measuring adsorption on colloidal particles.


415

416



clean surface

0'

0

1

1

1

5

10

15

C

e q

'

20

(mg/mL)

Figure 5. Comparison of isotherm of human y-globulin on the surface of PS latex spheres with and without surfactant on the surface. The plateau adsorp tion is reached at a much lower protein concentration on the clean surface.

Figure 6. Maximum adsorption density X for human IgG on PS latex particles versus solution pH. Ionic strength was maintained constant at 0.007 M .


29. JIANG ET AL.


add 1 mg/mL IgG in one step

X

/

GO

1 Downloaded by COLUMBIA UNIV on March 10, 2013 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0602.ch029

o add 0.05 mg/mL IgG first, lh later add IgG to 1 mg/mL

o GO

I

100

50

'

~~1— 150

200

TIME (min)

Figure 7. Adsorption density X versus time for 0.05 mg/mL and 1 mg/mL IgG additions (open symbols) and for a two-step adsorption in which the concentration was increased to 1 mg/mL after a preliminary adsorption for 1 hour at 0.05 mg/mL (closed symbols).

4.0'

. lms/mL IgG

3.5-

\

1 mg/mL IgG for lh, then diluted to 0.025mg/mL IgG

Z £ g o

2.5

0.025mg/mL IgG coated

O oo

Q

Direct Measurement of Protein Adsorption on Latex Particles by

Recommend Documents