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Measurement of protein adsorption to gold surface by radioiodination methods: suppression of free iodide sorption
Colloids and Surfaces B: Biointerfaces 17 (2000) 59 – 67 www.elsevier.nl/locate/colsurfb
Measurement of protein adsorption to gold surface by radioiodination methods: suppression of free iodide sorption Y.J. Du, R.M. Cornelius, J.L. Brash * Departments of Chemical Engineering, Pathology and Molecular Medicine, McMaster Uni6ersity, Hamilton, ON, L8S 4L7, Canada Received 16 February 1999
Abstract The adsorption of albumin and fibrinogen to gold metal using proteins radiolabeled with 125I is reported. Previous studies indicated that the interaction of the metal surface with free 125I− ion, present in trace amounts during protein adsorption experiments, resulted in substantial uptake, thus giving erroneously high estimates of adsorbed protein. Different approaches were investigated either to suppress the sorption of 125I− ion to gold or to differentiate between the sorption of labeled protein and 125I−. In the latter case an appropriate correction to the radioactivity on the surface should allow a valid estimate of protein adsorption. It was found that the addition of small amounts of nonradioactive iodide to the protein solution effectively suppresses the binding of 125I− ion (present in trace amounts relative to nonradioactive iodide) to the gold surface. An alternative approach involving pre-exposure to sodium iodide was not effective in suppressing the sorption of 125I− to gold. It was also found that treatment of gold surface with SDS is not 100% efficient in eluting adsorbed protein. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Free iodide; Radiolabeled proteins; Protein adsorption to metals
1. Introduction The rapid adsorption of proteins is regarded as the first major event following contact of surfaces with blood and other biological systems [1].While an extensive literature exists on the adsorption of proteins to polymer surfaces [2], less knowledge is available on protein adsorption to metals. Interest has recently focused on gold as a surface which can be readily modified by chemisorption of thiol* Corresponding author. Tel.: +1-905-5259140; fax: +1905-5211350. E-mail address: [email protected] (J.L. Brash)
containing compounds under ambient conditions [3,4]. The organization of alkane thiols into structured monolayers through self assembly provides a means of preparing surfaces with well defined chemistry. In particular fine control of chemical and physical properties can be achieved using alkane thiols with different terminal functional groups [5–7]. Adsorption experiments on gold using radiolabeled proteins, reported previously, indicated that trace amounts of unbound or free radioactive iodide ion present in the protein solution can lead to serious overestimation of protein adsorption due to uptake of the free radioactive iodide ion on
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the gold surface [8]. The surface radioactivity following exposure of gold surface to solutions of the same protein concentration, but differing amounts of free radioactive iodide, clearly demonstrated that an increase of free radioactive iodide in the working solution results in a corresponding increase in surface radioactivity. Gold – iodide interactions are known to occur via chemical reaction forming complex ionic species such as AuI− 2 and AuI− 4 [9,10]. It was found that typically 60– 95% of the total radioactivity associated with the surface is due to 125I− [8]. In the same study, SDS was used to elute adsorbed protein from gold surface, and the radioactivity in the eluates and on the surface following elution were determined. The free iodide and protein uptake were estimated on the assumption that sorbed iodide ion is unaffected by SDS while adsorbed protein is eluted quantitatively. This initial work thus underlined the problem of free iodide interaction with metal surfaces, and indicated that caution should be used when interpreting adsorption data obtained using 125I− labeled proteins. The objective of the present work was to more fully characterize SDSelutability, and develop alternative approaches to the suppression of 125I− sorption to gold. Two procedures to suppress 125I− sorption, used singly and in combination, were investigated. The first is based on the hypothesis that pre-exposure to nonradioactive iodide should saturate the surface, thus minimizing the subsequent binding of free radioactive iodide present in protein adsorption experiments. A potential disadvantage of this method is that pre-exposure to NaI may change the nature of the metal surface such that its adsorption behavior is changed. The second approach was to include nonradioactive iodide in the buffer used for the adsorption experiments. It was hypothesized that if the ratio of nonradioactive to radioactive iodide is high, the radioactive iodide taken up during protein adsorption experiments should be significantly decreased. In addition, the elution of adsorbed proteins from gold-based surfaces using SDS was further investigated as a means of differentiating between protein and 125I− bound to the gold surface.
2. Materials and methods
2.1. Surfaces The polyurethane used as a substrate for gold deposition was synthesized according to methods described previously [8,11]. 4,4%-Diphenylmethane diisocyanate (MDI), polytetramethylene oxide (PTMO) of molecular weight 980 and ethylene diamine (ED) were reacted in a 2:1:1 molar ratio. Polymer films of about 0.5 mm thickness were cast from dimethylformamide (DMF), dried under vacuum, and rinsed with methanol prior to gold coating. Gold was thermally evaporated, to a thickness of about 100 nm, onto both sides of the polymer films. The gold coated polymers were stored covered at room temperature. Prior to use, the gold surfaces were cleaned by immersion in a peroxide–ammonia solution at 85°C for 5 min. The surfaces were then rinsed extensively and incubated for 4 h in Milli-Q water prior to adsorption experiments.
2.2. Buffer preparation Protein adsorption experiments were carried out using phosphate buffered saline (PBS) pH 7.4, or alternatively phosphate buffered saline containing nonradioactive sodium iodide (PBS–NaI). The concentration of nonradioactive iodide in PBS–NaI ranged from 0.01 to 5 mol% of the NaCl concentration, and the total molar concentration of NaCl and NaI was the same as that of NaCl in PBS. Thus 1 l of PBS–NaI (0.01%) buffer contained 0.002 g NaI and 8.5 g NaCl while the other components were the same as in PBS. Illustrative buffer formulations, including osmolarity data, are shown in Table 1. It should be noted that typical isotonic buffers used in protein adsorption studies have osmolarities in the range of 3009 20 mOsm.
2.3. Proteins Human albumin (Behringwerke AG, Marburg, Germany) was reconstituted with phosphate buffered saline (PBS) pH 7.4. Human fibrinogen (Calbiochem, La Jolla, California, USA) was dia-
Y.J. Du et al. / Colloids and Surfaces B: Biointerfaces 17 (2000) 59–67
lyzed against isotonic Tris buffered saline, aliquoted, and stored at − 70°C. Proteins were labeled with 125I (ICN Pharmaceuticals Inc, Radiochemical Division, Irvine, CA, USA) using the iodogen technique [12]. The labeled protein was dialyzed overnight against phosphate buffered saline, with three changes of dialysate, to remove most of the unbound radioactive iodide. Trace amounts of free radioactive iodide, still present in the resulting labeled protein solution, were measured by counting the supernatant following precipitation of the protein with trichloroacetic acid (TCA, 20% w/v in water). For fibrinogen, the free radioactive iodide content was generally less than 1%, and for albumin, less than 3%. Protein solutions (1% labeled, 99% unlabeled protein) of about 2 mg ml − 1 in the appropriate buffer were prepared for adsorption experiments. Solutions containing a mixture of unlabeled protein at a concentration of 2 mg ml − 1, and free 125I-iodide ion at a concentration equivalent to that in the labeled protein solutions, were used to investigate the contribution of free iodide to the radioactivity on the gold surfaces in the labeled protein experiments.
2.4. Adsorption procedure Metal surfaces (0.6 cm2) were exposed to 250 ml of protein solution for 2 h at room temperature under static conditions. Following adsorption, samples were rinsed three times in buffer, wicked onto filter paper to remove residual adherent buffer, and their radioactivity determined. Table 1 Composition, pH and osmolarity of PBS and PBS–NaI (5%) buffers PBS Disodium hydrogen phosphate (g l−1) Sodium dihydrogen phosphate (g l−1) Sodium chloride (g l−1) Sodium iodide (g l−1) pH (adjusted) Osmolarity (mOsm)
PBS–NaI (5%)
1.32
1.32
0.345
0.345
8.5 – 7.38 3039 5
8.08 1.09 7.40 293 95
61
The surfaces and the eluates were counted. A minimum of four replicates was measured for each set of experimental conditions. All experiments were done in duplicate.
2.5. Suppression of
125 −
I
sorption to gold surface
The methods investigated to minimize the binding of free 125-iodide during protein adsorption were as follows: (1) pre-exposure of surfaces to a concentrated (0.5 M) nonradioactive NaI solution; (2) addition of nonradioactive iodide to the buffer used for the protein adsorption experiment. The combination of both procedures was also investigated. In addition, a study of the elution of adsorbed proteins from surfaces using SDS was carried out. The rationale for each of these approaches and a description of the protocols used are as follows. It was hypothesized that incubation of gold with cold NaI would saturate the surface with iodide ions, thus pre-empting any subsequent binding of radioactive iodide during protein adsorption experiments. To determine whether this approach was effective, the radioactivity associated with the surface following protein adsorption, with and without pre-exposure to NaI, was measured. It was expected that substitution of a small amount of NaI for NaCl in PBS would not change significantly the general properties of the buffer (see Table 1) but would suppress the adsorption of radioactive iodide, present at a much lower concentration. For this approach to be effective, the concentration of NaI in the buffer should be in considerable excess over that of Na125I. The ratio used was of the order of 5× 105:1.
2.6. Elution with sodium dodecyl sulfate (SDS) The removal of protein from gold using SDS, a well-known protein eluent, was also investigated. Following protein adsorption, surfaces were incubated overnight with 2% (w/v) aqueous SDS. The surface and eluate radioactivities were determined. It was hypothesized that this treatment would remove the adsorbed proteins from the
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Table 2 Comparison of surface radioactivity on gold following contact (2 h) with 125I-labeled albumin containing 2% free 125I-iodide, or unlabeled albumin containing the equivalent amount of free 125 I-iodide
125 I-albumin Albumin+125I−
Surface radioactivity (cpm cm−2)
S.D. (n =4)
26 960 24 800
1815 1990
surface, but would have no effect on the chemically bound free iodide. This approach was also followed in previous work [8].
3. Results and discussion In a first set of experiments, gold surfaces were incubated with a labeled albumin solution in which 2% of the radioactivity was present as free 125-iodide, or with an unlabeled albumin solution containing the same amount of free 125-iodide. The results are shown in Table 2, and indicate that the small amount of free iodide present in the protein solution contributes most of the radioactivity found on the surface. Additional results showed that an increase in free iodide concentration in the protein solution resulted in a corresponding increase in surface radioactivity (data not shown). Thus it is clearly important to remove as much as possible of the free 125-iodide from labeled protein preparations. Trace amounts of free 125-iodide are, however, inevitable, and means must be devised to reduce its uptake on gold surfaces, or to distinguish between iodide ion and protein on the surface.
3.1. In6estigation of the effect of NaI on adsorption beha6ior of albumin The methods used to suppress iodide uptake were evaluated to determine whether the adsorption behavior of proteins would be changed. Glass was used as a test surface for this purpose. From previous work in this laboratory it is known that the trace amounts of free iodide present in radiolabeled protein solutions do not adsorb to glass so
that any effect on protein adsorption of inclusion of NaI in the buffer should be readily apparent. The effects of the procedural modifications on protein adsorption were investigated using four different protocols. For protocols 1 and 2, surfaces were incubated in water, and for protocols 3 and 4 in 0.5 M aqueous NaI solution for 4 h. The surfaces from 2 and 4 were then incubated for 2 h in radiolabeled albumin/PBS–NaI; those from 1 and 3 were incubated in radiolabeled albumin/ PBS. Following adsorption, the surfaces were rinsed three times in PBS, and the radioactivity determined. The surfaces were then incubated in 2% aqueous SDS overnight and the radioactivity measured again. The data are presented in Table 3. It appears that a reduction in the adsorption of albumin on glass of the order of 7–10% occurs when the surface is pretreated with NaI or when iodide is included in the buffer. The differences, however, are not significant. The combination of NaI pre-exposure and use of PBS–NaI buffer results in a somewhat greater reduction of the order of 25%, suggesting an additive effect of the two procedures. Glass surfaces were also exposed to solutions containing unlabeled albumin and free iodide (albumin+ 125I−) to confirm that iodide does not bind significantly. As shown in Table 4, the surface radioactivity is of the order of background, clearly demonstrating that free iodide does not bind to glass, and indicating that the radioactivity associated with the surfaces when labeled protein is present (Table 3) is entirely due to the adsorption of the protein. From these experiments it may be concluded that on glass the effects of NaI pre-exposure and PBS–NaI buffer on protein adsorption behavior are relatively small. It is expected that this would hold true for gold also in the case of inclusion of cold NaI but not in the case of pre-exposure to cold NaI.
3.2. Effect of NaI pre-exposure and PBS–NaI buffer on sorption of 125I− to Gold It was expected that both pre-exposure of the surface to NaI and addition of NaI to the buffer
Protocol
NaI in PBS buffer
Pre-exposure to 0.5 M NaI (4 h)
Surface radioactivity (cpm cm−2)
S.D. (n =4)
Surface radioactivity after SDS exposure (cpm cm−2)
S.D (n= 4)
1 2 3 4
No Yes No Yes
No No Yes Yes
44 200 39 200 41 200 33 100
7350 4870 3320 3860
3700 2800 2900 2100
440 700 890 340
a 1, control (normal protocol); 2, use of PBS–NaI (1%); 3, pre-exposure of surface to 0.5 M NaI; 4, use of PBS–NaI (1%) and pre-exposure of surface to 0.5 M NaI. Albumin concentration, 2 mg ml−1, adsorption time, 2 h.
Y.J. Du et al. / Colloids and Surfaces B: Biointerfaces 17 (2000) 59–67
Table 3 Data on albumin adsorption to glass demonstrating effect of different experimental protocolsa
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would suppress free iodide sorption to gold. The same four protocols discussed above for glass were used to investigate these two approaches. Typical data for albumin are shown in Table 5. It is seen that the inclusion of a small amount of cold NaI in the buffer causes a large decrease in surface radioactivity (2 versus 1), presumably due to the suppression of 125I− iodide binding to gold by competition of the cold iodide. Assuming that the radioactivity on the surface in protocol 2 is entirely due to protein, the fraction of surface radioactivity in protocol 1 due to free iodide sorption is approximately 90%, similar to data we reported previously [8]. Pre-exposure of the surfaces to NaI (protocol 3) also resulted in a decrease in surface radioactivity, but much less than for protocol 2. The combined effect of pre-exposure to NaI and inclusion of NaI in the buffer (protocol 4) was similar to inclusion of NaI in the buffer only (protocol 2). Thus it appears that inclusion of a small amount of cold NaI is effective in suppressing 125I− iodide binding to gold.
Similar effects were found by Bohnert et al. [13] in studies of the adsorption of 125I− labeled proteins to hydrogels. It was shown that the inclusion of nonradioactive iodide in the buffer greatly reduced the uptake of 125I− iodide by these materials. Horbett [14] has also reported on the combined effects of pre-exposure to NaI and inclusion of NaI in the buffer to minimize sorption of 125I− to hydrogels.
3.3. Effect of NaI content of PBS –NaI buffer on free iodide sorption The concentration of NaI in the experiments discussed above was 1% (mol/mol) of the NaCl concentration. To determine the effect of NaI concentration, experiments were carried out in which the degree of substitution of NaI for NaCl in the buffer was varied. As can be seen from Fig. 1, the radioactivity associated with the gold surface increases rapidly with decreasing NaI content
Table 4 Radioactivity associated with glass surface following exposure (2 h) to unlabeled albumin solutions containing added 125-iodidea Protocol
NaI in PBS buffer
Pre-exposure to 0.5 M NaI (4 h)
Surface radioactivity (cpm cm−2)
S.D. (n =4)
1 2 3 4
No Yes No Yes
No No Yes Yes
80 130 40 70
30 50 20 30
a 1, control (normal protocol); 2, use of PBS–NaI (1%); 3, pre-exposure of surface to 0.5 M NaI; 4, use of PBS–NaI (1%) and pre-exposure of surface to 0.5 M NaI. Albumin concentration, 2 mg ml−1.
Table 5 Surface radioactivity on gold following incubation (2 h) with radiolabeled albumin (2 mg ml−1) using different approaches to minimize 125I− binding to golda Protocol
NaI in PBS buffer
Pre-exposure to 0.5 M NaI (4 h)
Surface radioactivity (cpm cm−2)
SD (n = 4)
1 2 3 4
No Yes No Yes
No No Yes Yes
34 020 3220 24 780 2860
2850 350 3300 400
a
1, control (normal protocol); 2, use of PBS–NaI (1%); 3, pre-exposure of surface to 0.5 M NaI; and 4, use of PBS–NaI (1%) and pre-exposure of surface to 0.5 M NaI.
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Fig. 1. Surface radioactivity associated with glass and gold following exposure of surfaces to buffers containing radiolabeled albumin and various amounts of NaI. Albumin concentration, 2 mg ml − 1; adsorption time, 2 h.
at substitutions less than 1%. The high surface radioactivity on gold when no NaI is present in the buffer is due to the binding of radioactive free iodide to the surface as discussed above. The data for 1 and 5% NaI substitution are not significantly different, suggesting that a level of 1% is appropriate for minimization of 125-iodide–gold interactions. Data for glass are also shown in Fig. 1. There is no variation in albumin adsorption on glass with NaI concentration over the entire range studied. Also, since the radioactivity on glass is due entirely to protein adsorption (Table 3), the data in Fig. 1 support the conclusion that the addition of NaI to the working buffer does not significantly affect the adsorption behavior of the protein.
3.4. SDS elution of albumin from surfaces SDS, an anionic surfactant, is recognized to be an efficient protein eluent [15,16]. SDS elution of radiolabeled protein from metal surfaces was previously reported [8], and it was suggested that this procedure provides a means of separating the contributions of 125I-protein from 125I-iodide, assuming that the protein is 100% elutable and the iodide is unaffected by SDS. As mentioned previously, the elutability of proteins may differ depending on the nature of the surface and the protein. It was of interest to determine the effect of the PBS–NaI buffer on the SDS-elutability of proteins from glass and gold. Data on the adsorption of albumin to glass,
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Table 6 Radioactivity on gold surface following incubation (2 h) with radiolabeled albumin (2 mg ml−1) before and after elution with SDS NaI in PBS buffer
Surface radioactiv- Surface radioactivity (cpm cm−2) ity post SDS (cpm cm−2)
No Yes
26 960 2780
26 000 1850
measured pre- and post-SDS treatment, are shown in Table 3. Since free 125I− does not bind to this surface, residual radioactivity after exposure to SDS is presumably due to protein that is nonelutable. The fraction of albumin adsorbed to glass that cannot be removed by SDS elution can be estimated by comparing the surface radioactivity before and after SDS elution, and is approximately 7%. This may be considered minimal. In similar experiments on gold, the non-elutable
radioactivity after incubating with SDS is about 70% when PBS–NaI is used and about 96% without NaI in the buffer (Table 6). Therefore it appears that much of the protein adsorbed to gold is strongly attached and cannot be removed by SDS. This finding is in accordance with the results of Murray et al. [17] who found that a non-ionic surfactant was not able to remove proteins from unmodified gold whereas it did remove some proteins from gold surface modified by a hydrophobic layer. Therefore SDS elution may be used as a method to evaluate the quality of modifications of gold surface. Analogous experiments to the above were done for fibrinogen and show a similar effect using PBS–NaI buffer (Fig. 2). Residual radioactivity following SDS treatment is also shown. If the PBS–NaI buffer is effective in suppressing the binding of free 125I− to gold, these data suggest that not all the fibrinogen adsorbed on the gold surface is elutable.
Fig. 2. Surface radioactivity associated with gold following exposure of surfaces to radiolabeled fibrinogen. " with solid line, normal protocol; , with solid line, use of PBS–NaI (1%) buffer; " with dashed line, normal protocol + SDS elution; with dashed line, use of PBS – NaI (1%) buffer +SDS elution. Adsorption time, 2 h.
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4. Conclusions Different approaches (pre-exposure to NaI, addition of NaI to the buffer, and SDS elution) have been investigated to eliminate 125I− binding, or to differentiate between 125I− and 125I-protein on gold surface. The addition of small amounts of NaI to the buffer has the greatest effect in suppressing 125I− sorption to gold. Data on albumin adsorption to glass, a surface which was shown not to sorb 125I−, indicate that the presence of NaI in the buffer does not affect protein adsorption. Thus the addition of small amounts of NaI to the working buffer may permit reliable protein adsorption measurements on gold using 125I-labeled proteins. Work reported previously [8] indicated that the SDS treatment did not have a significant effect on the surface bound iodide ion, and emphasized that caution should be exercised in measuring protein adsorption to metal surfaces using radioiodinated proteins due to complications such as surface bound iodide, potentially incomplete elution of protein by SDS, and possible variation in elutability from different surfaces. The work reported here, while confirming that the SDS treatment does not result in removal of surface bound free iodide, indicates that SDS does not elute protein completely from gold surfaces. Elution by SDS is clearly less reliable as a means of estimating protein adsorption to gold surfaces than the addition of cold iodide to the buffer as reported here.
Acknowledgements Financial support of this work by the Natural
.
67
Sciences and Engineering Research Council of Canada, and the Medical Research Council of Canada, is gratefully acknowledged.
References [1] J.L. Brash, in: C.P. Sharma, et al. (Eds.), Blood Compatible Materials and Devices, Technomic, Lancaster, PA, 1991, p. 3. [2] T.A. Horbett, J.L. Brash (Eds.), Proteins at Interfaces II: Fundamentals and Applications, vol. 602, ACS Symposium Series, 1995. [3] G.M. Whitesides, J.P. Mathias, C.T. Seto, Science 254 (1991) 1312. [4] A. Ulman, in: A. Ulman (Ed.), An Introduction to Ultrathin Organic Films: from Langmuir – Blodgett to Self-Assembly, Academic, New York, 1991, p. 279. [5] K.L. Prime, G.M. Whitesides, J. Am. Chem. Soc. 115 (1993) 10714. [6] M. Schneegans, E. Menzel, J. Colloid Interface Sci. 88 (1982) 97. [7] S.E. Creager, L.A. Hockett, G.K. Rowe, Langmuir 8 (1992) 854. [8] H. Sheardown, R.M. Cornelius, J.L. Brash, Colloids Surf. B 10 (1997) 29. [9] A. Davis, T. Trau, D.R. Young, Hydrometallurgy 32 (1993) 143. [10] F.A. Letnikov, N.V. Vilor, in: D.I. Groves et al. (Eds.), Gold in the Hydrothermal Process, The University of Western Australia, 1990, p. 9. [11] J.P. Santerre, Ph.D. thesis, McMaster University, 1990. [12] L.C. Knight, A.Z. Budzynski, S.A. Olexa, Thrombos. Haemostas. 46 (1981) 593. [13] J.L. Bohnert, T.A. Horbett, B.D. Ratner, F.H. Royce, Invest. Ophthalmol. Vis. Sci. 29 (1988) 362. [14] T.A. Horbett, in: D.F. Williams (Ed.), Techniques for Protein Adsorption Studies, CRC, Boca Raton, FL, 1986, p. 183. [15] S. Maulik, P. Dutta, D.K. Chattoraj, S.P. Moulik, Colloids Surf. B 11 (1998) 1. [16] R.J. Rapoza, T.A. Horbett, J. Colloid Interface Sci. 136 (1990) 480. [17] B.S. Murray, L. Cros, Colloids Surf. B 10 (1998) 227.
Measurement of protein adsorption to gold surface by radioiodination methods: suppression of free iodide sorption Y.J. Du, R.M. Cornelius, J.L. Brash * Departments of Chemical Engineering, Pathology and Molecular Medicine, McMaster Uni6ersity, Hamilton, ON, L8S 4L7, Canada Received 16 February 1999
Abstract The adsorption of albumin and fibrinogen to gold metal using proteins radiolabeled with 125I is reported. Previous studies indicated that the interaction of the metal surface with free 125I− ion, present in trace amounts during protein adsorption experiments, resulted in substantial uptake, thus giving erroneously high estimates of adsorbed protein. Different approaches were investigated either to suppress the sorption of 125I− ion to gold or to differentiate between the sorption of labeled protein and 125I−. In the latter case an appropriate correction to the radioactivity on the surface should allow a valid estimate of protein adsorption. It was found that the addition of small amounts of nonradioactive iodide to the protein solution effectively suppresses the binding of 125I− ion (present in trace amounts relative to nonradioactive iodide) to the gold surface. An alternative approach involving pre-exposure to sodium iodide was not effective in suppressing the sorption of 125I− to gold. It was also found that treatment of gold surface with SDS is not 100% efficient in eluting adsorbed protein. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Free iodide; Radiolabeled proteins; Protein adsorption to metals
1. Introduction The rapid adsorption of proteins is regarded as the first major event following contact of surfaces with blood and other biological systems [1].While an extensive literature exists on the adsorption of proteins to polymer surfaces [2], less knowledge is available on protein adsorption to metals. Interest has recently focused on gold as a surface which can be readily modified by chemisorption of thiol* Corresponding author. Tel.: +1-905-5259140; fax: +1905-5211350. E-mail address: [email protected] (J.L. Brash)
containing compounds under ambient conditions [3,4]. The organization of alkane thiols into structured monolayers through self assembly provides a means of preparing surfaces with well defined chemistry. In particular fine control of chemical and physical properties can be achieved using alkane thiols with different terminal functional groups [5–7]. Adsorption experiments on gold using radiolabeled proteins, reported previously, indicated that trace amounts of unbound or free radioactive iodide ion present in the protein solution can lead to serious overestimation of protein adsorption due to uptake of the free radioactive iodide ion on
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Y.J. Du et al. / Colloids and Surfaces B: Biointerfaces 17 (2000) 59–67
the gold surface [8]. The surface radioactivity following exposure of gold surface to solutions of the same protein concentration, but differing amounts of free radioactive iodide, clearly demonstrated that an increase of free radioactive iodide in the working solution results in a corresponding increase in surface radioactivity. Gold – iodide interactions are known to occur via chemical reaction forming complex ionic species such as AuI− 2 and AuI− 4 [9,10]. It was found that typically 60– 95% of the total radioactivity associated with the surface is due to 125I− [8]. In the same study, SDS was used to elute adsorbed protein from gold surface, and the radioactivity in the eluates and on the surface following elution were determined. The free iodide and protein uptake were estimated on the assumption that sorbed iodide ion is unaffected by SDS while adsorbed protein is eluted quantitatively. This initial work thus underlined the problem of free iodide interaction with metal surfaces, and indicated that caution should be used when interpreting adsorption data obtained using 125I− labeled proteins. The objective of the present work was to more fully characterize SDSelutability, and develop alternative approaches to the suppression of 125I− sorption to gold. Two procedures to suppress 125I− sorption, used singly and in combination, were investigated. The first is based on the hypothesis that pre-exposure to nonradioactive iodide should saturate the surface, thus minimizing the subsequent binding of free radioactive iodide present in protein adsorption experiments. A potential disadvantage of this method is that pre-exposure to NaI may change the nature of the metal surface such that its adsorption behavior is changed. The second approach was to include nonradioactive iodide in the buffer used for the adsorption experiments. It was hypothesized that if the ratio of nonradioactive to radioactive iodide is high, the radioactive iodide taken up during protein adsorption experiments should be significantly decreased. In addition, the elution of adsorbed proteins from gold-based surfaces using SDS was further investigated as a means of differentiating between protein and 125I− bound to the gold surface.
2. Materials and methods
2.1. Surfaces The polyurethane used as a substrate for gold deposition was synthesized according to methods described previously [8,11]. 4,4%-Diphenylmethane diisocyanate (MDI), polytetramethylene oxide (PTMO) of molecular weight 980 and ethylene diamine (ED) were reacted in a 2:1:1 molar ratio. Polymer films of about 0.5 mm thickness were cast from dimethylformamide (DMF), dried under vacuum, and rinsed with methanol prior to gold coating. Gold was thermally evaporated, to a thickness of about 100 nm, onto both sides of the polymer films. The gold coated polymers were stored covered at room temperature. Prior to use, the gold surfaces were cleaned by immersion in a peroxide–ammonia solution at 85°C for 5 min. The surfaces were then rinsed extensively and incubated for 4 h in Milli-Q water prior to adsorption experiments.
2.2. Buffer preparation Protein adsorption experiments were carried out using phosphate buffered saline (PBS) pH 7.4, or alternatively phosphate buffered saline containing nonradioactive sodium iodide (PBS–NaI). The concentration of nonradioactive iodide in PBS–NaI ranged from 0.01 to 5 mol% of the NaCl concentration, and the total molar concentration of NaCl and NaI was the same as that of NaCl in PBS. Thus 1 l of PBS–NaI (0.01%) buffer contained 0.002 g NaI and 8.5 g NaCl while the other components were the same as in PBS. Illustrative buffer formulations, including osmolarity data, are shown in Table 1. It should be noted that typical isotonic buffers used in protein adsorption studies have osmolarities in the range of 3009 20 mOsm.
2.3. Proteins Human albumin (Behringwerke AG, Marburg, Germany) was reconstituted with phosphate buffered saline (PBS) pH 7.4. Human fibrinogen (Calbiochem, La Jolla, California, USA) was dia-
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lyzed against isotonic Tris buffered saline, aliquoted, and stored at − 70°C. Proteins were labeled with 125I (ICN Pharmaceuticals Inc, Radiochemical Division, Irvine, CA, USA) using the iodogen technique [12]. The labeled protein was dialyzed overnight against phosphate buffered saline, with three changes of dialysate, to remove most of the unbound radioactive iodide. Trace amounts of free radioactive iodide, still present in the resulting labeled protein solution, were measured by counting the supernatant following precipitation of the protein with trichloroacetic acid (TCA, 20% w/v in water). For fibrinogen, the free radioactive iodide content was generally less than 1%, and for albumin, less than 3%. Protein solutions (1% labeled, 99% unlabeled protein) of about 2 mg ml − 1 in the appropriate buffer were prepared for adsorption experiments. Solutions containing a mixture of unlabeled protein at a concentration of 2 mg ml − 1, and free 125I-iodide ion at a concentration equivalent to that in the labeled protein solutions, were used to investigate the contribution of free iodide to the radioactivity on the gold surfaces in the labeled protein experiments.
2.4. Adsorption procedure Metal surfaces (0.6 cm2) were exposed to 250 ml of protein solution for 2 h at room temperature under static conditions. Following adsorption, samples were rinsed three times in buffer, wicked onto filter paper to remove residual adherent buffer, and their radioactivity determined. Table 1 Composition, pH and osmolarity of PBS and PBS–NaI (5%) buffers PBS Disodium hydrogen phosphate (g l−1) Sodium dihydrogen phosphate (g l−1) Sodium chloride (g l−1) Sodium iodide (g l−1) pH (adjusted) Osmolarity (mOsm)
PBS–NaI (5%)
1.32
1.32
0.345
0.345
8.5 – 7.38 3039 5
8.08 1.09 7.40 293 95
61
The surfaces and the eluates were counted. A minimum of four replicates was measured for each set of experimental conditions. All experiments were done in duplicate.
2.5. Suppression of
125 −
I
sorption to gold surface
The methods investigated to minimize the binding of free 125-iodide during protein adsorption were as follows: (1) pre-exposure of surfaces to a concentrated (0.5 M) nonradioactive NaI solution; (2) addition of nonradioactive iodide to the buffer used for the protein adsorption experiment. The combination of both procedures was also investigated. In addition, a study of the elution of adsorbed proteins from surfaces using SDS was carried out. The rationale for each of these approaches and a description of the protocols used are as follows. It was hypothesized that incubation of gold with cold NaI would saturate the surface with iodide ions, thus pre-empting any subsequent binding of radioactive iodide during protein adsorption experiments. To determine whether this approach was effective, the radioactivity associated with the surface following protein adsorption, with and without pre-exposure to NaI, was measured. It was expected that substitution of a small amount of NaI for NaCl in PBS would not change significantly the general properties of the buffer (see Table 1) but would suppress the adsorption of radioactive iodide, present at a much lower concentration. For this approach to be effective, the concentration of NaI in the buffer should be in considerable excess over that of Na125I. The ratio used was of the order of 5× 105:1.
2.6. Elution with sodium dodecyl sulfate (SDS) The removal of protein from gold using SDS, a well-known protein eluent, was also investigated. Following protein adsorption, surfaces were incubated overnight with 2% (w/v) aqueous SDS. The surface and eluate radioactivities were determined. It was hypothesized that this treatment would remove the adsorbed proteins from the
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Table 2 Comparison of surface radioactivity on gold following contact (2 h) with 125I-labeled albumin containing 2% free 125I-iodide, or unlabeled albumin containing the equivalent amount of free 125 I-iodide
125 I-albumin Albumin+125I−
Surface radioactivity (cpm cm−2)
S.D. (n =4)
26 960 24 800
1815 1990
surface, but would have no effect on the chemically bound free iodide. This approach was also followed in previous work [8].
3. Results and discussion In a first set of experiments, gold surfaces were incubated with a labeled albumin solution in which 2% of the radioactivity was present as free 125-iodide, or with an unlabeled albumin solution containing the same amount of free 125-iodide. The results are shown in Table 2, and indicate that the small amount of free iodide present in the protein solution contributes most of the radioactivity found on the surface. Additional results showed that an increase in free iodide concentration in the protein solution resulted in a corresponding increase in surface radioactivity (data not shown). Thus it is clearly important to remove as much as possible of the free 125-iodide from labeled protein preparations. Trace amounts of free 125-iodide are, however, inevitable, and means must be devised to reduce its uptake on gold surfaces, or to distinguish between iodide ion and protein on the surface.
3.1. In6estigation of the effect of NaI on adsorption beha6ior of albumin The methods used to suppress iodide uptake were evaluated to determine whether the adsorption behavior of proteins would be changed. Glass was used as a test surface for this purpose. From previous work in this laboratory it is known that the trace amounts of free iodide present in radiolabeled protein solutions do not adsorb to glass so
that any effect on protein adsorption of inclusion of NaI in the buffer should be readily apparent. The effects of the procedural modifications on protein adsorption were investigated using four different protocols. For protocols 1 and 2, surfaces were incubated in water, and for protocols 3 and 4 in 0.5 M aqueous NaI solution for 4 h. The surfaces from 2 and 4 were then incubated for 2 h in radiolabeled albumin/PBS–NaI; those from 1 and 3 were incubated in radiolabeled albumin/ PBS. Following adsorption, the surfaces were rinsed three times in PBS, and the radioactivity determined. The surfaces were then incubated in 2% aqueous SDS overnight and the radioactivity measured again. The data are presented in Table 3. It appears that a reduction in the adsorption of albumin on glass of the order of 7–10% occurs when the surface is pretreated with NaI or when iodide is included in the buffer. The differences, however, are not significant. The combination of NaI pre-exposure and use of PBS–NaI buffer results in a somewhat greater reduction of the order of 25%, suggesting an additive effect of the two procedures. Glass surfaces were also exposed to solutions containing unlabeled albumin and free iodide (albumin+ 125I−) to confirm that iodide does not bind significantly. As shown in Table 4, the surface radioactivity is of the order of background, clearly demonstrating that free iodide does not bind to glass, and indicating that the radioactivity associated with the surfaces when labeled protein is present (Table 3) is entirely due to the adsorption of the protein. From these experiments it may be concluded that on glass the effects of NaI pre-exposure and PBS–NaI buffer on protein adsorption behavior are relatively small. It is expected that this would hold true for gold also in the case of inclusion of cold NaI but not in the case of pre-exposure to cold NaI.
3.2. Effect of NaI pre-exposure and PBS–NaI buffer on sorption of 125I− to Gold It was expected that both pre-exposure of the surface to NaI and addition of NaI to the buffer
Protocol
NaI in PBS buffer
Pre-exposure to 0.5 M NaI (4 h)
Surface radioactivity (cpm cm−2)
S.D. (n =4)
Surface radioactivity after SDS exposure (cpm cm−2)
S.D (n= 4)
1 2 3 4
No Yes No Yes
No No Yes Yes
44 200 39 200 41 200 33 100
7350 4870 3320 3860
3700 2800 2900 2100
440 700 890 340
a 1, control (normal protocol); 2, use of PBS–NaI (1%); 3, pre-exposure of surface to 0.5 M NaI; 4, use of PBS–NaI (1%) and pre-exposure of surface to 0.5 M NaI. Albumin concentration, 2 mg ml−1, adsorption time, 2 h.
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Table 3 Data on albumin adsorption to glass demonstrating effect of different experimental protocolsa
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would suppress free iodide sorption to gold. The same four protocols discussed above for glass were used to investigate these two approaches. Typical data for albumin are shown in Table 5. It is seen that the inclusion of a small amount of cold NaI in the buffer causes a large decrease in surface radioactivity (2 versus 1), presumably due to the suppression of 125I− iodide binding to gold by competition of the cold iodide. Assuming that the radioactivity on the surface in protocol 2 is entirely due to protein, the fraction of surface radioactivity in protocol 1 due to free iodide sorption is approximately 90%, similar to data we reported previously [8]. Pre-exposure of the surfaces to NaI (protocol 3) also resulted in a decrease in surface radioactivity, but much less than for protocol 2. The combined effect of pre-exposure to NaI and inclusion of NaI in the buffer (protocol 4) was similar to inclusion of NaI in the buffer only (protocol 2). Thus it appears that inclusion of a small amount of cold NaI is effective in suppressing 125I− iodide binding to gold.
Similar effects were found by Bohnert et al. [13] in studies of the adsorption of 125I− labeled proteins to hydrogels. It was shown that the inclusion of nonradioactive iodide in the buffer greatly reduced the uptake of 125I− iodide by these materials. Horbett [14] has also reported on the combined effects of pre-exposure to NaI and inclusion of NaI in the buffer to minimize sorption of 125I− to hydrogels.
3.3. Effect of NaI content of PBS –NaI buffer on free iodide sorption The concentration of NaI in the experiments discussed above was 1% (mol/mol) of the NaCl concentration. To determine the effect of NaI concentration, experiments were carried out in which the degree of substitution of NaI for NaCl in the buffer was varied. As can be seen from Fig. 1, the radioactivity associated with the gold surface increases rapidly with decreasing NaI content
Table 4 Radioactivity associated with glass surface following exposure (2 h) to unlabeled albumin solutions containing added 125-iodidea Protocol
NaI in PBS buffer
Pre-exposure to 0.5 M NaI (4 h)
Surface radioactivity (cpm cm−2)
S.D. (n =4)
1 2 3 4
No Yes No Yes
No No Yes Yes
80 130 40 70
30 50 20 30
a 1, control (normal protocol); 2, use of PBS–NaI (1%); 3, pre-exposure of surface to 0.5 M NaI; 4, use of PBS–NaI (1%) and pre-exposure of surface to 0.5 M NaI. Albumin concentration, 2 mg ml−1.
Table 5 Surface radioactivity on gold following incubation (2 h) with radiolabeled albumin (2 mg ml−1) using different approaches to minimize 125I− binding to golda Protocol
NaI in PBS buffer
Pre-exposure to 0.5 M NaI (4 h)
Surface radioactivity (cpm cm−2)
SD (n = 4)
1 2 3 4
No Yes No Yes
No No Yes Yes
34 020 3220 24 780 2860
2850 350 3300 400
a
1, control (normal protocol); 2, use of PBS–NaI (1%); 3, pre-exposure of surface to 0.5 M NaI; and 4, use of PBS–NaI (1%) and pre-exposure of surface to 0.5 M NaI.
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65
Fig. 1. Surface radioactivity associated with glass and gold following exposure of surfaces to buffers containing radiolabeled albumin and various amounts of NaI. Albumin concentration, 2 mg ml − 1; adsorption time, 2 h.
at substitutions less than 1%. The high surface radioactivity on gold when no NaI is present in the buffer is due to the binding of radioactive free iodide to the surface as discussed above. The data for 1 and 5% NaI substitution are not significantly different, suggesting that a level of 1% is appropriate for minimization of 125-iodide–gold interactions. Data for glass are also shown in Fig. 1. There is no variation in albumin adsorption on glass with NaI concentration over the entire range studied. Also, since the radioactivity on glass is due entirely to protein adsorption (Table 3), the data in Fig. 1 support the conclusion that the addition of NaI to the working buffer does not significantly affect the adsorption behavior of the protein.
3.4. SDS elution of albumin from surfaces SDS, an anionic surfactant, is recognized to be an efficient protein eluent [15,16]. SDS elution of radiolabeled protein from metal surfaces was previously reported [8], and it was suggested that this procedure provides a means of separating the contributions of 125I-protein from 125I-iodide, assuming that the protein is 100% elutable and the iodide is unaffected by SDS. As mentioned previously, the elutability of proteins may differ depending on the nature of the surface and the protein. It was of interest to determine the effect of the PBS–NaI buffer on the SDS-elutability of proteins from glass and gold. Data on the adsorption of albumin to glass,
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66
Table 6 Radioactivity on gold surface following incubation (2 h) with radiolabeled albumin (2 mg ml−1) before and after elution with SDS NaI in PBS buffer
Surface radioactiv- Surface radioactivity (cpm cm−2) ity post SDS (cpm cm−2)
No Yes
26 960 2780
26 000 1850
measured pre- and post-SDS treatment, are shown in Table 3. Since free 125I− does not bind to this surface, residual radioactivity after exposure to SDS is presumably due to protein that is nonelutable. The fraction of albumin adsorbed to glass that cannot be removed by SDS elution can be estimated by comparing the surface radioactivity before and after SDS elution, and is approximately 7%. This may be considered minimal. In similar experiments on gold, the non-elutable
radioactivity after incubating with SDS is about 70% when PBS–NaI is used and about 96% without NaI in the buffer (Table 6). Therefore it appears that much of the protein adsorbed to gold is strongly attached and cannot be removed by SDS. This finding is in accordance with the results of Murray et al. [17] who found that a non-ionic surfactant was not able to remove proteins from unmodified gold whereas it did remove some proteins from gold surface modified by a hydrophobic layer. Therefore SDS elution may be used as a method to evaluate the quality of modifications of gold surface. Analogous experiments to the above were done for fibrinogen and show a similar effect using PBS–NaI buffer (Fig. 2). Residual radioactivity following SDS treatment is also shown. If the PBS–NaI buffer is effective in suppressing the binding of free 125I− to gold, these data suggest that not all the fibrinogen adsorbed on the gold surface is elutable.
Fig. 2. Surface radioactivity associated with gold following exposure of surfaces to radiolabeled fibrinogen. " with solid line, normal protocol; , with solid line, use of PBS–NaI (1%) buffer; " with dashed line, normal protocol + SDS elution; with dashed line, use of PBS – NaI (1%) buffer +SDS elution. Adsorption time, 2 h.
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4. Conclusions Different approaches (pre-exposure to NaI, addition of NaI to the buffer, and SDS elution) have been investigated to eliminate 125I− binding, or to differentiate between 125I− and 125I-protein on gold surface. The addition of small amounts of NaI to the buffer has the greatest effect in suppressing 125I− sorption to gold. Data on albumin adsorption to glass, a surface which was shown not to sorb 125I−, indicate that the presence of NaI in the buffer does not affect protein adsorption. Thus the addition of small amounts of NaI to the working buffer may permit reliable protein adsorption measurements on gold using 125I-labeled proteins. Work reported previously [8] indicated that the SDS treatment did not have a significant effect on the surface bound iodide ion, and emphasized that caution should be exercised in measuring protein adsorption to metal surfaces using radioiodinated proteins due to complications such as surface bound iodide, potentially incomplete elution of protein by SDS, and possible variation in elutability from different surfaces. The work reported here, while confirming that the SDS treatment does not result in removal of surface bound free iodide, indicates that SDS does not elute protein completely from gold surfaces. Elution by SDS is clearly less reliable as a means of estimating protein adsorption to gold surfaces than the addition of cold iodide to the buffer as reported here.
Acknowledgements Financial support of this work by the Natural
.
67
Sciences and Engineering Research Council of Canada, and the Medical Research Council of Canada, is gratefully acknowledged.
References [1] J.L. Brash, in: C.P. Sharma, et al. (Eds.), Blood Compatible Materials and Devices, Technomic, Lancaster, PA, 1991, p. 3. [2] T.A. Horbett, J.L. Brash (Eds.), Proteins at Interfaces II: Fundamentals and Applications, vol. 602, ACS Symposium Series, 1995. [3] G.M. Whitesides, J.P. Mathias, C.T. Seto, Science 254 (1991) 1312. [4] A. Ulman, in: A. Ulman (Ed.), An Introduction to Ultrathin Organic Films: from Langmuir – Blodgett to Self-Assembly, Academic, New York, 1991, p. 279. [5] K.L. Prime, G.M. Whitesides, J. Am. Chem. Soc. 115 (1993) 10714. [6] M. Schneegans, E. Menzel, J. Colloid Interface Sci. 88 (1982) 97. [7] S.E. Creager, L.A. Hockett, G.K. Rowe, Langmuir 8 (1992) 854. [8] H. Sheardown, R.M. Cornelius, J.L. Brash, Colloids Surf. B 10 (1997) 29. [9] A. Davis, T. Trau, D.R. Young, Hydrometallurgy 32 (1993) 143. [10] F.A. Letnikov, N.V. Vilor, in: D.I. Groves et al. (Eds.), Gold in the Hydrothermal Process, The University of Western Australia, 1990, p. 9. [11] J.P. Santerre, Ph.D. thesis, McMaster University, 1990. [12] L.C. Knight, A.Z. Budzynski, S.A. Olexa, Thrombos. Haemostas. 46 (1981) 593. [13] J.L. Bohnert, T.A. Horbett, B.D. Ratner, F.H. Royce, Invest. Ophthalmol. Vis. Sci. 29 (1988) 362. [14] T.A. Horbett, in: D.F. Williams (Ed.), Techniques for Protein Adsorption Studies, CRC, Boca Raton, FL, 1986, p. 183. [15] S. Maulik, P. Dutta, D.K. Chattoraj, S.P. Moulik, Colloids Surf. B 11 (1998) 1. [16] R.J. Rapoza, T.A. Horbett, J. Colloid Interface Sci. 136 (1990) 480. [17] B.S. Murray, L. Cros, Colloids Surf. B 10 (1998) 227.