J . Phys. Chem. 1994,98, 5376-5381
5376
Effect of Ionic Strength on Protein Adsorption Kinetics J. J. Ramsden'Jl$ and J. E. Prenosilt Department of Chemical Engineering, E TH, Universitatsstrasse 6, 8092 Zurich, Switzerland, and Department of Biophysical Chemistry, Biozentrum, 4056 Basle, Switzerland Received: November 29, 1993; In Final Form: February 8, 1994" The kinetics of the protein apotransferrin adsorbing onto Si(Ti)Oz surfaces from solution is accurately measured using a n integrated optics technique. Specifically, the influence of salt and buffer concentrations on the adsorption rate is investigated. Analysis of the kinetic curves allows the separate evaluation of the diffusivity and the area occupied per adsorbed molecule. As the ionic strength of the solution increases, the lateral diffusivity decreases and the effective area per molecule increases. These observations are analyzed in terms of the surface charge model of Healy and White. It is shown that increasing ionic strength diminishes the negative surface potential but increases surface p H and hence net protein charge near the surface. Including these parameters in the adsorption kinetic equations leads to goodquantitative agreement with the experimentaldata over an intermediate range of ionic strengths, but not a t the highest values investigated, where nonelectrostatic factors begin to play a role.
Introduction Since protein adsorption at the solid-solution interface can and does take place over a wide range of ionic strengths, it is important to understand whether the kinetics measured under one set of conditions apply to others and, if not, how to predict the kinetics a t other ionic strengths. It is generally recognized that the adsorption process comprises a number of distinct stages, notably transport of proteins to the surface, their adhesion at the surface, and postadsorption conformational changes which may bind the proteins more or less strongly to the surface. In this last stage one should also include the possibility of lateral mobility of the adsorbed protein. Each of these stages is likely, and in some cases known, to be affected in different ways and to differing extents by the ionic strength of the solution. Nevertheless, the overall mechanism of adsorption and its dependence on ionic strength are not yet quantitatively, and maybe not even qualitatively, understood. Given that reported data in the area are fragmentary and present an incomplete picture,l-4 it is a priority to improve the experimental state of affairs. In this work, a novel integrated optics method is used to accurately measure the kinetics of the adsorption of transferrin, a well-characterized protein, dissolved in buffered salt solutions of various ionic strengths, on a silicon titanium dioxide surface. This surface is an optical waveguide. The adsorbed protein molecules cause a measurable shift in the mode indices of guided waves excited in the waveguide. From this shift, the number of adsorbed molecules per unit area can be determined to an accuracy of f 7 0 molecules/km2, with a temporalresolutionof 10s. Thekineticdatasoobtainedprovide an extremely valuable starting point for elucidating the mechanism of the process. It will be shown how the parameter characterizing the transport stage (the lateral diffusion coefficient) can be separately evaluated from that characterizing the adsorption stage (the area occupied per molecule).
-
N = n sin a
Experimental Section Planar optical waveguides made from Sil,Tiy02,y = 0.4, with a diffraction grating (line density A-1 = 2400 "-1) incorporated in their surface, were obtained from AS1 AG, Zurich, Switzerland. Before use, they were cleaned by standing in hot (ca. 100 "C)
* Author for correspondence. f
8
concentrated permonosulfuric acid (H2SOs) for 20-30 min, rinsing extensively in distilled water, and standing overnight in the same buffered salt solution in which the protein was to be dissolved (see below). Since the isoelectric points of Si02 and Ti02 are about 2.5 and 5.8, re~pectively,~ the waveguides are expected to be negatively charged. Human apotransferrin (>97% pure) was obtained from Sigma and used without further purification. Solutions were made up in tris(hydroxymethy1)aminomethaneHCl (Tris), pH 8.0, at various concentrations and containing various amounts of NaCl (see Table 1). The isoelectric point (pHi,,) of apotransferrin is 6.1.6 Therefore, at pH 8 the protein is negatively charged. Stock solutions had a concentraton of 8-12 mg/mL and were diluted to the bulk protein concentration, c, of 100 pg/cm3 (11.25 X 10-6 M f 1015 molecules/cmj) immediately before each experiment. It was found that fresh stock solutions (less than 3 h old) gave slightly different kinetics from older solutions, but after this initial period of change the solutions were stable and could be used for many days (provided they remained sterile). Therefore, all stock solutions were aged for at least 3 h before use. A small silicone rubber (RTV-M533, Wacker Chemie, Munich, Germany) flow-through cuvette was fixed over the waveguide (Figure l), and protein solutions were impelled across the grating surface by a peristaltic pumpat thedownstream end of the cuvette. The waveguide and cuvette were mounted in a goniometer scanning device (10s-I, AS1 AG, Zurich), with which the angle between a linearly polarized external beam (He-Ne laser, X = 632.8 nm) and the grating can be varied while measuring the power coupled into the waveguide with a photodiode situated at the end of the waveguide (Figure 1). At certain discrete values of the angle between the beam and the grating normal, the diffracted beam matches a possible guided mode, incoupling takes place, and a sharp maximum is seen in the plot of incoupled power vs angle. The angle a corresponding to maximum power incoupled into the waveguide is given by7
ETH.
Biozentrum. Abstract published in Aduance ACS Abstracts, April 15, 1994
+tX/A
(1)
where n is the refractive index of air and C the diffraction order. a was measured and used to determine N, the index of an incoupled mode, for the zeroth order transverse electric (TE) and transverse magnetic (TM) modes. Measurements of a were repeated every 25 s (as the asymptotic region was approached, this interval was extended, because the number of adsorbed molecules then increases very slowly). Adsorption was allowed to continue well into the asymptotic
0022-3654/94/2098-5376%04.50/0 0 1994 American Chemical Society
The Journal of Physical Chemistry, Vol. 98, No. 20, 1994 5371
Protein Adsorption Kinetics
TABLE 1: Parameters ( I and alm) Fitting the Experimental Data to Ea 9' [buffer]/ [salt]/ I/ng D/cm2 (a/m)/ Mwt/ mM mM s-l s-l cm2pg-' a/nm2 pg cm-2 13.4 0.54 7.05 1.72X 10-6 1.01 purewater 0 1.73 23.1 0.32 4.08 7.57 X 2mM Tris 0 2.10 28.0 0.26 0.73 5.73 X 1@ 10 mM Tris 0 39.4 0.19 2.96 10 mM Tris 20 0.40 2.29 X 3.98 53.1 0.14 0.22 9.60X 10mM Tris 100 7.67 102 0.07 0.15 5.94 X 10mM Tris 500 D was calculated from I using (5) and (6),with c = 100 pg cm-), and a from a / m using m = 0.133ag. The value for D estimated from the hydrodynamic radius R, of 3.03 nmi7 using the Stokes-Einstein formula is 8.07 X IO-' cm2/s,and a calculated from T R , is ~ 29 nm2.Msat was calculated from eq 10 using the experimentallydetermined values = 0.55.13 of a and
I
I
1
0.3 N
u 0.2 0
2
.
.
y
a
I
I
-F
(----.' ++
1 0 1 -+ d
"."
-
.
0
2000
1000
(1
tls Figure 2. Kinetics of aprotransferrin adsorption. The different curves refer to different solvent conditions: (a) 1 mM Tris-HC1; (b) 10 mM Tris-HC1; (c) 10 mM Tris-HC1 100 mM NaCI, (d) 10 mM Tris-HC1 + 500 mM NaCI, all at pH 8.0.
+
concentration CA in the layer,iO i.e. nA = nc
I
",
" /i
I
center of the inlet tube (distance x ) .
region, attaining a coverage of more than 95% of saturation. Afterwards, buffered salt solution without protein flowed through the cuvette, and the mass of protein removed was recorded. All measurements were carried out a t 25.2 f 0.2 OC.
Results The mode indices N are related to the optogeometric parameters of the waveguide (see Figure 1) according to7,*
(3)
where the coefficient dn/dc depends on the refractivity of protein molecules and has practically the same value of 0.182 cm3/g for all proteins.iO According to the Gibbs convention, the surface excess of proteins is defined as M C A ~ A(for CA >> c), which becomeslo
kf
/L
Figure 1. The measuring cell. The silicone rubber cuvette C self-seals to the waveguiding film F, which in turn is supported on glass S. A denotes the layer of adsorbed protein molecules. The protein solution is drawn through the cuvette at a flow rate 3 by a peristaltic pump at the downstream end. Light L from an external source is coupled into the layer F by means of the grating G, and the incoupled power is measured by photodiode P. The radius R of the cuvette is 1 mm, and its crosssectional area is 10-2 cm2. The center of the grating is 3.5 mm from the
CA(dn/dC)
dA(nA - nc)/(dn/dc)
(4)
upon eliminating CA using (3). Since apart from the different N all the other parameters are independent of the polarization of the guided wave, the two mode equations (2) can be solved simultaneously to yield both d A and n~ and hence, using (4), M . Figure 2 shows some typical kinetic curves. Note that for each salt concentration a definite plateau is attained; Le., it does not appear that the curves merely differ in the rate of protein adsorption. Transport. Hydrodynamic factors control transport in the flow cell. The flow rate 3 used throughout was 1.43 X 10-4 cm3/s. Given the dimensions of the cell (Figure l ) , we calculate a mean velocity u of fluid in the cell of 1.4 X l e 2 cm/s. The Reynolds number is then only 0.16. On the other hand, the diffusion coefficient of the protein is very small, of the order of 10-6 cmz/s, and the Peclet number is therefore large. Under these conditions transport to the surface is governed by convection down to a region close to the surface, through which transport is diffusive;li the flux Z to the surface is given byiz
I = DC/dh
(5)
where dh is the thickness of the layer through which diffusion occurs and which depends on the hydrodynamic characteristics of the cell. The parabolic Poiseuille flow profile is not fully developed in the grating region in which the adsorption is monitored, and the appropriate expression for dh to be substituted into ( 5 ) is11 where p = 0 and 1 for the T E and T M modes, respectively, and m is the mode number. d and n denote thickness and refractive index, respectively, and are identified by the subscripts S (support), F (waveguiding film), A (adsorbed adlayer), and C (cover). This linearized form7 of the full mode equation is valid for d A
