Protein Sorption on Polymer Surfaces Measured by Fluorescence Labels E. BRYNDA, J. DROBNfK, J. VACfK, and J. KALAL, Institute of Macromolecular Chemistry, Czechoslovak Academy of Sciences, 162 06 Prague 6, Czechoslovakia
Summary Fluorescence labeling can be used in studying protein sorption on various surfaces with a sensitivity of about lows g/cm2, commensurate with radioactive labeling. Fluorescamine proved to be the most suitable compound for studying protein sorption on hydrophilic gels, because, unlike fluoresceine isothiocyanate and dansylchloride, free fluorochrome does not interfere with measurements. Sorption properties of labeled serum albumin were tested on poly(2-hydroxyethyl methacrylate), on the copolymer of 2-hydroxyethyl methacrylate with methyl methacrylate, and on polyethylene. Labeling does not cause aggregation of the protein, but, as expected, it shifts and somewhat broadens its electrophoretic band while at the same time slightly raising its affinity toward hydrophobic surfaces.
INTRODUCTION An important factor which is believed to influence interaction of blood or plasma with synthetic materials is protein sorption on surfaces of these rnaterial~.l-~It is essential in this case to determine which of the proteins present in the blood plasma is preferentially sorbed on the surface.4-6 This is why an investigation of sorptions of the individual proteins and of competitive sorptions from a mixture of proteins has been suggested as one of the methods of evaluation of alloplastic surfaces designated for contact with blood. The methods presently in use are predominantly based on radioactive labeling of proteins with 1125.It was the objective of this work to develop a method suitable for hydrogels swollen in water, which seem to be a promising group of compounds from the standpoint of biocompatibility, because their character is close to that of natural product^.^ In hydrogels an insignificant release of iodine or iodides may lead to artefacts, since unlike hydrophobic materials hydrogels Journal of Biomedical Materials Research, Vol. 12,55-65 (1978) 0 1978 John Wiley & Sons, Inc. 0021-9304/78/0012-0055$01.00
56
BRYNDA ET AL.
are permeable to iodine. A free radioactive isotope not only readily penetrates into them, but is accumulated there and forms stable complexes with some of them. We therefore attempted testing of an analogous method which, however, would employ fluorescence labeling of proteins. The experiments were based on a practice quite common in immunology, where fluorescence labeling is used.8 This practice has revealed that slight labeling does not in principle alter antigenic properties, which are a sensitive feature characterizing the intactness of the protein structure.8 We tested two fluorescent labels most in use a t present, namely, fluoresceinisothiocyanate and 1dimethylaminonaphthalene-5-sulfonylchloride,g and found them unsuitable for hydrogels. A suitable label was found in a recently described fluorescence agent-Fluorescamine.l0J1
MATERIALS A N D METHODS Bovine serum albumin (BSA) was obtained from a Laboratorni potfeby (Laboratory Instruments Work), Prague. Serum albumin labeled with fluoresceine isothiocyanate (FITC) was produced at the Institute of Serum and Vaccines, Prague. Fluorescamine (Fluram) (DANSC1) were and l-dimethylaminonaphthalene-5-sulfonylchloride Roche products. Triton X-100 used as detergent was manufactured by Packard. Albumin was sorbed from a Ringer's solution adjusted to pH 7.1 with NaHC03. Polyethylene was used in the form of tubes, inner diameter 4 mm, and as a film. Its surface was cleaned with methanol and distilled water prior to use. 2-Hydroxyethyl methacrylate (HEMA) was prepared by alkaline reesterification of methyl methacrylate with ethylene glycol and purified by employing the usual procedure;13 bp 352.15 Kl533.2 Pa, purity determined by gas chromatography: 99.1% HEMA, 0.13% ethylene dimethacrylate, 0.77% ethylene glycol. Methyl methacrylate (MMA) was obtained from East Bohemian Chemical Synthesia, Pardubice. Hydroquinone was removed by shaking into 5% Na2C03 (five times) followed by shaking with water (also five times). After that, MMA was dried and rectified twice. Concentration of the monomeric mixture during the polymerization was 20%. Ethanol was used as solvent. Concentration of initiator was 0.5% per monomeric mixture. The polymerization time was 16 hr a t 333 K. Polymers thus obtained were twice preprecipitated into ether, then dried in vacuo to constant weight. An 80120 mole % ratio was chosen in the
PROTEIN SORPTION AND FLUORESCENCE LABELS
57
preparation of the HEMA-MMA copolymer. BSA was labeled with DANSCl by using a modified method after Weber.12
Labeling with Fluorescarnine A 2.8-mg sample of Fluorescamine was dissolved in 7 ml acetone, cooled with ice, and added with rapid stirring to 25 ml of precooled solution containing 2 mg/ml of BSA in 0.05 M borax. After 3 min, the solution was dialyzed against ice-cool Ringer’s solution in a refrigerator for 20 hr (Ringer’s solution was changed twice during that time). The last dialysis was performed a t room temperature during 1hr. The final volume was adjusted to 50 ml so as to obtain a resulting 1 mg/ml of BSAF in Ringer’s solution which was used directly for sorptions. Preparation of Surfaces Sorptions of proteins were tested in glass tubes, inner diameter 4.5 mm, coated with a polymer film prepared as follows. A 10%solution of polymer in the acetone-methanol mixture (1:l)was poured into glass tubes cleaned with chromosulfuric acid and slowly let out. The procedure was repeated once more and the tubes were then dried a t room temperature. The film, swellable but insoluble in water, was rinsed with distilled water. Sorption experiments were carried out only using tubes coated with a completely transparent, optically homogeneous, smooth film. Prior to use, the tubes were filled with Ringer’s solution and the solution was left for 4 hr in order to reach equilibrium. Ringer’s solution was then squeezed out with a twofold volume of solution of labeled BSA. The sorption occurred a t 297 K. After the sorption had been completed, the protein solution was squeezed out with an eightfold volume of Ringer’s solution during 30 sec, and the tubes were additionally rapidly washed twice with two volumes of Ringer’s solution. A t the end of the process, the washing solutions did not contain any traces of proteins detectable by fluorescence. The sorbed proteins were desorbed with 1%solution of Triton X-100 in borax, pH 9.2 (0.2 M H3B03 was titrated with 10%NaOH) at room temperature for 24 hr, and their quantity was determined from fluorescence intensity measurements at 475 nm with excitation a t 390 nm by comparison with calibration curves. Since the fluorescence intensity of
58
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labeled proteins is not sufficiently stable and depends on time and on the mode of treatment of the labeled protein, calibration solutions were prepared separately for each series of experiments; these were placed in tubes coated with a polymer film and left under the same conditions as those used in desorption. Protein sorption in polyethylene tubes was tested in the same way. Sorption and desorption on polyethylene films was carried out in closed cells from which the sorption solution was squeezed out similiarly to the case where tubes were used. In all cases, the arrangement used ruled out any contact between the tested surface and the protein solution-air boundary. Fluorescence measurements were performed with a Hitachi Perkin-Elmer MPF-2A spectrofluorimeter. Multiple internal-reflection (MIR) infrared spectra were recorded with a Perkin-Elmer 621 IR spectrometer. Electrophoresis on acrylamide gel was carried out in a tris(hydroxymethy1)aminomethane-glycine buffer (pH 8.4). The efficiency of proteins desorption by the Triton solution was checked for films of polyHEMA and of the 20% HEMA-80% MMA copolymer by dissolving these films before and after desorption in a methanolacetone 1:l mixture and by comparing the fluorescence intensities of solutions thus obtained. For films of polyethylene, similar control was accomplished by comparing the MIR infrared absorption of sorbed and desorbed films. In both cases, more than 90%of sorbed protein was removed by the desorption procedure.
RESULTS AND DISCUSSION
BSA Labeled with DANSCl (BSAD) In measurements of calibration solutions, the BSAD concentrations could be detected starting from g/ml. After BSAD had been sorbed on HEMA, it was possible to desorb fluorescence compounds also without detergents. Two types of emission spectra were observed. When the solution of BSAD was dialyzed, the solution inside the bag exhibited the fluorescence maximum a t 500 nm (all maxima are given in noncorrected wavelength), whereas the maximum for the outside solution appeared a t 488 nm. The substances eluted from the hydrogel surface previously treated with BSAD showed also a maximum a t 488 nm. Therefore, we concluded that the solution obtained by desorption
PROTEIN SORPTION AND FLUORESCENCE LABELS
59
contained mostly free fluorochrome which had migrated inside the gel (and, probably, had accumulated in it) during the treatment by the solution of BSAD. The samples were therefore purified from free fluorochrome by precipitation and repeated dialyses. The content of free fluorochrome was determined by an equilibrium dialysis of the BSAD solution into the same volume of Ringer’s solution. In a purified 10 mg % BSAD solution the fluorescence intensity of free fluorochrome was about 4% of the fluorescence of BSAD, which is too much for a reliable investigation of sorption on the hydrogel surface. Neither is DANSCl very suitable if combined with Triton X-100 solutions used for washing away sorbed proteins. The fluorescence of BSAD with an excitation maximum near 340 nm was disturbed by the long-wave edge of fluorescence of the Triton X-100 solution (emission maximum at 380 nm) excited in the same region (excitation maximum a t 300 nm).
BSA Labeled with FITC (BSA-FITC) Concentrations of calibration solutions starting from g BSA-FITC/ml could be detected. After BSA-FITC had been sorbed on polyHEMA, the fluorescence compounds were desorbed from the gel even without detergent. The emission fluorescence spectrum of desorption solutions with a maxiimum at 514 nm was somewhat shifted compared to the spectrum of the BSA FITC solution with its maximum at 518 nm. In both cases, the excitation maximum lay near 460 nm. The fluorescence intensity increased almost linearly with the time of sorption; there was no perceptible saturation even after 24 hr. The above characteristics show that the values obtained are in this case determined by diffusion of the low molecular fluorochrome into the gel rather than by sorption of BSA-FITC on its surface. In repeated dialyses of the BSA-FITC solutions, the low molecular weight fluorochrome was released into the dialyzate without considerable slowing-down. In attempts at determination of the relative concentration of free label by dialysis into the same volume (similarly to BSAD), the state of equilibrium could not be reached even after several days, and the fluorochrome concentration in the dialyzate continued to increase. It may be inferred, therefore, that FITC bonded on BSA is gradually released into solution because of the instabiIity of the bond.
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BSA Labeled with Fluorescarnine (BSAF) Excitation and emission maxima were found at 390 nm and 472 nm, respectively. Concentration of calibration solutions starting from g BSAF/ml could be detected. When tested in the dialysis cell, the BSAF solution did not release any low molecular weight fluorescence compounds even after a few days. Also, if used in sorption experiments, it did not give rise to effects characteristic of diffusion of free fluorochrome into the gel, such as washing out of fluorescence compounds without detergents, slow increase in concentration of fluorescence compounds in the gel not leading to saturation with increasing time of sorption, and high fluorescence intensities of the desorption solutions. All these facts indicate that in the case of BSAF, the fluorescence determination of sorption is not distorted by the diffusion of low molecular weight fluorescence compounds into the gel. With films of pure polyHEMA, the detected sorption of BSAF from Ringer’s solution was about g/cm2 at the times used within 1-24 hr. Such low values were already at the limits of applicability of the method; with respect to errors, which will be discussed below, no characteristics of sorptions could be checked. The method was therefore tested on similar films of the copolymer 20% HEMA-80% MMA, prepared and tested similarly to those of pure polyHEMA. An addition of the hydrophobic component considerably raised the sorption of BSAF from Ringer’s solution. Figure 1shows the amount of BSAF per cm2 of the surface of the 20% HEMA-80% MMA copolymer as a function of the sorption time. One can see that the equilibrium is established at a slow rate, and that the amount of sorbed BSAF goes on increasing even after several tens of hours. Figure 2 shows the amount of BSAF sorbed on the surface of the 20% HEMA-80% MMA copolymer after sorption lasting 4 hr as a function of the BSAF concentration in the sorption solution. Similarly to the time dependence, the concentration dependence does not exhibit saturation of the sorption capacity within the range under investigation. The effect of the fluorescence label on the sorption of BSA was evaluated using competitive sorption between BSAF and BSA. The amount of BSAF sorbed from the solution of 50 mg % BSAF 50 mg % BSA in Ringer’s solution was ca. 5% of the amount of BSAF sorbed from a solution of 100 mg % BSAF; the ratio was independent of the sorption time. It may be concluded, therefore, that in the case
+
PROTEIN SORPTION AND FLUORESCENCE LABELS
0
I
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I
I
10
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Lo
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time [hours]
Fig. 1. Time dependence of the concentration of BSAF sorbed on the surface of the 20% HEMA-8O?? MMA copolymer from 100 mg 96 of solution.
I
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0.10
01
a /
0.25
I
0.50 0.75 1.00 sobtion concentration, mglml
Fig. 2. Concentration of BSAF sorbed on the surface of the 20% HEMA-gO?? MMA copolymer after sorption a t 297 K for 4 hr vs. BSAF concentration in solution.
of competitive sorption, there is no preferential sorption of BSA compared with BSAF. On the other hand, however, one cannot decide to what extent BSAF is preferentially sorbed, since (as shown by Fig. 2) the concentrations used could not be chosen in the saturation range, and the amount of BSAF sorbed depended on the BSAF concentration in the sorption solution even if BSA was absent. By examining the amount of BSAF sorbed on the surface of polyethylene as a function of the sorption time (Table I), it was found that
BRYNDA ET AL.
62
TABLE I Competitive Sorption between BSAF and BSA on Polyethylene and Kinetics Sorption a t 297 K Composition of Solution 50 mg % BSAF 25 mg % BSAF 25 mg % BSA 100 mg % BSAF 50 mg % BSAF 50 mg % BSA 100 mg % BSAF
+ +
50 mg % BSAF
+ 50 mg % BSA
Surface Relative Sorption Concentration Sorption of Time (hr) of BSAF (fig/cm2) BSAF in Mixture (%) 2 2 2 2 0.25 0.5 1.0 10 0.25 0.5 1.0 10
0.372 0.208 0.373 0.238 0.368 0.371 0.373 0.373 0.231 0.236 0.238 0.236
56 100 64
after a short time the sorption capacity for BSAF became saturated. Table I shows the amount of BASF sorbed on polyethylene from solutions with various contents of BSAF and BSA after 2 hr of sorption as well as the kinetics of sorption. One can see that with the concentrations used, i.e., 50 mg % and 100 mg % BSAF, the equilibrium is established virtually within 30 min and is independent of the BSAF concentration in solution. If this value (0.373 pg/cm2 BSAF) is regarded as loo%, then the amount of BSAF sorbed from the solution of 25 mg % BSA 25 mg % BSAF is about 56% of this value, and the amount of BSAF sorbed from the solution 50 mg % BSA 50 mg % BSAF is about 64%. Consequently, BSA labeled with Fluorescamine is sorbed more strongly than unlabeled BSA. A shift in the electrophoretic band of BSAF with respect to BSA (Fig. 3) shows that the protein labeled with Fluorescamine has a lower number of positive charges than the unlabeled one, obviously due to the label being bonded on the amino groups of the protein. On the other hand, one can see that the labeling did not lead to an aggregation of the proteins. The broadening of the zone for BSAF can probably be assigned to the bonding of Fluorescamine on various types and various numbers of amino groups of the protein, so that the labeled BSAF sample is heterogeneous in this respect. The procedure used in labeling, i.e., addition of acetone to the solution of BSA and sub-
+
+
PROTEIN SORPTION AND FLUORESCENCE LABELS
63
Fig. 3. Electrophoresis on acrylamide gel (1)BSA; (2) BSAF; ( 3 ) BSAD; (4) BSA-FITC.
sequent dialyses, did not have any effect on the electrophoretic mobility of BSA. In conclusion, we shall try to summarize the advantages and disadvantages of the fluorescence labeling of proteins in the investigation of sorption. The advantage consists in a simpler technique compared with work involving radioisotopes and in the independence of the half-life period and of the radiochemical damage, that is, in the long-term applicability of the same labeled sample. The main difficulties encountered in applying fluorescence labels in the determination of protein sorption on polymeric, and particularly hydrophilic, surfaces are as follows. 1)With hydrophilic gels, the difficulties consist in the presence of a free label or in the gradual release of the latter due to the insufficient stability of the bond with the protein, as demonstrated by experiments with DANSCl and FITC. A signal due to the low molecular weight label diffused into the bulk of the gel may exceed the measured signal
64
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due to the labeled protein sorbed on the gel surface, even if the quantity of the free label in the sorption solution is very small (accumulation of the label in the gel mass). However, similar difficulties could arise in hydrophilic gels also if radioactive-labeled proteins were used. 2) Instability of fluorescence of labeled protein was apparent. The fluorescence intensity of calibration BSAF solutions decreased with time and was also dependent on the conditions and materials used in storing the solutions. Special attention should therefore be devoted to the way in which the standards are stored. 3) Change of the protein properties after labeling was noted. Labeling has always led to a change in the electrophoretic mobility, and in the case of BSAF investigated here, it also led to a change in the sorption properties. Preferential sorption of BSAF on polyethylene and on the 20% HEMA-80% MMA copolymer is probably due to an increase in the hydrophobic character of the protein surface due to cancellation of the charged amino group and to the bonding of a hydrophobic label. It will be necessary, therefore, when investigating competitive sorptions, to characterize this effect due to the presence of the label by applying alternate labeling of the individual proteins. We believe that the fluorescence method could be improved; to a considerable extent better dispersion, low-temperature measurements, or polarization measurements would undoubtedly make possible a reliable detection of the low molecular weight fluorochrome released in the reaction, which is impossible in the case of a released radioactive label. At present, we are introducing multireflexion technique of fluorescence measurements directly on the gel surface (analogously to the MIR infrared technique),14 which offers some further possibilities of extending the methods already in use. References 1. R. E. Baier and R. C. Dutton, J . Riomed. Mater. Res., 3,191 (1969). 2. E. W. Salzman, Bull. N . Y. Acad. Med., 48,225 (1972). 3. L. Vroman, A. L. Adams, M. Klings, and G. Fischer, Adu. Chem. Ser., 145,255 (1975). 4. M. A. Packham, G . Evans, M. F. Glynn, and J. F. Mustard, J . Lab. Clin. Med., 73, 686 (1969). 5. C. S. P. Jenkins, M. A. Packham, M. A. Guccione, and J. F. Mustard, J. Lab. Clin. Med., 81,280 (1973).
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6. D. J. Lyman, K. G. Klein, J. L. Bash, B. K. Fritzinger, J. D. Andrade, and F. Bonomo, Thromb. Hemorrh. Suppl., 42,109 (1971). 7. J. D. Andrade, H. B. Lee, M. S. Jhon, S. V. Kim, and J. B. Hibbs, Jr., Trans. Am. SOC.Artif. Intern. Organs, 19,l (1973). 8. R. C . Nairn, in Fluorescent Protein Tracing, E. and S. Livingstone Ltd., London, 1964. 9. T. P. Murtha, Disertation, University Microfilms, Ann Arbor, Mich., 69-9879 (1968). 10. M. Weigele, S. L. DeBernardo, J. P. Tengi, and W. Leimgruber, J. Am. Chem. Soc., 94,5927 (1972). 11. P. Bohlen, S. Stein, W. Dairman, and S. Undenfriend, Arch. Biochem. Biophys., 155,213 (1973). 12. G. Weber, Biochem. J., 51,155 (1952). 13. J. KopeEek, J. Jokl, and D. Lim, in Macromolecular Chemistry, Prague 2965 ( J . Polym. Sci. C, 16), 0. Wichterle and B. SedlBEek, Eds., Interscience, New York, 1968, Part 7, pp. 3877. 14. G. I. Loeb and N. J. Harrick, Anal. Chem., 45,687 (1973).
Received February 4,1977
Summary Fluorescence labeling can be used in studying protein sorption on various surfaces with a sensitivity of about lows g/cm2, commensurate with radioactive labeling. Fluorescamine proved to be the most suitable compound for studying protein sorption on hydrophilic gels, because, unlike fluoresceine isothiocyanate and dansylchloride, free fluorochrome does not interfere with measurements. Sorption properties of labeled serum albumin were tested on poly(2-hydroxyethyl methacrylate), on the copolymer of 2-hydroxyethyl methacrylate with methyl methacrylate, and on polyethylene. Labeling does not cause aggregation of the protein, but, as expected, it shifts and somewhat broadens its electrophoretic band while at the same time slightly raising its affinity toward hydrophobic surfaces.
INTRODUCTION An important factor which is believed to influence interaction of blood or plasma with synthetic materials is protein sorption on surfaces of these rnaterial~.l-~It is essential in this case to determine which of the proteins present in the blood plasma is preferentially sorbed on the surface.4-6 This is why an investigation of sorptions of the individual proteins and of competitive sorptions from a mixture of proteins has been suggested as one of the methods of evaluation of alloplastic surfaces designated for contact with blood. The methods presently in use are predominantly based on radioactive labeling of proteins with 1125.It was the objective of this work to develop a method suitable for hydrogels swollen in water, which seem to be a promising group of compounds from the standpoint of biocompatibility, because their character is close to that of natural product^.^ In hydrogels an insignificant release of iodine or iodides may lead to artefacts, since unlike hydrophobic materials hydrogels Journal of Biomedical Materials Research, Vol. 12,55-65 (1978) 0 1978 John Wiley & Sons, Inc. 0021-9304/78/0012-0055$01.00
56
BRYNDA ET AL.
are permeable to iodine. A free radioactive isotope not only readily penetrates into them, but is accumulated there and forms stable complexes with some of them. We therefore attempted testing of an analogous method which, however, would employ fluorescence labeling of proteins. The experiments were based on a practice quite common in immunology, where fluorescence labeling is used.8 This practice has revealed that slight labeling does not in principle alter antigenic properties, which are a sensitive feature characterizing the intactness of the protein structure.8 We tested two fluorescent labels most in use a t present, namely, fluoresceinisothiocyanate and 1dimethylaminonaphthalene-5-sulfonylchloride,g and found them unsuitable for hydrogels. A suitable label was found in a recently described fluorescence agent-Fluorescamine.l0J1
MATERIALS A N D METHODS Bovine serum albumin (BSA) was obtained from a Laboratorni potfeby (Laboratory Instruments Work), Prague. Serum albumin labeled with fluoresceine isothiocyanate (FITC) was produced at the Institute of Serum and Vaccines, Prague. Fluorescamine (Fluram) (DANSC1) were and l-dimethylaminonaphthalene-5-sulfonylchloride Roche products. Triton X-100 used as detergent was manufactured by Packard. Albumin was sorbed from a Ringer's solution adjusted to pH 7.1 with NaHC03. Polyethylene was used in the form of tubes, inner diameter 4 mm, and as a film. Its surface was cleaned with methanol and distilled water prior to use. 2-Hydroxyethyl methacrylate (HEMA) was prepared by alkaline reesterification of methyl methacrylate with ethylene glycol and purified by employing the usual procedure;13 bp 352.15 Kl533.2 Pa, purity determined by gas chromatography: 99.1% HEMA, 0.13% ethylene dimethacrylate, 0.77% ethylene glycol. Methyl methacrylate (MMA) was obtained from East Bohemian Chemical Synthesia, Pardubice. Hydroquinone was removed by shaking into 5% Na2C03 (five times) followed by shaking with water (also five times). After that, MMA was dried and rectified twice. Concentration of the monomeric mixture during the polymerization was 20%. Ethanol was used as solvent. Concentration of initiator was 0.5% per monomeric mixture. The polymerization time was 16 hr a t 333 K. Polymers thus obtained were twice preprecipitated into ether, then dried in vacuo to constant weight. An 80120 mole % ratio was chosen in the
PROTEIN SORPTION AND FLUORESCENCE LABELS
57
preparation of the HEMA-MMA copolymer. BSA was labeled with DANSCl by using a modified method after Weber.12
Labeling with Fluorescarnine A 2.8-mg sample of Fluorescamine was dissolved in 7 ml acetone, cooled with ice, and added with rapid stirring to 25 ml of precooled solution containing 2 mg/ml of BSA in 0.05 M borax. After 3 min, the solution was dialyzed against ice-cool Ringer’s solution in a refrigerator for 20 hr (Ringer’s solution was changed twice during that time). The last dialysis was performed a t room temperature during 1hr. The final volume was adjusted to 50 ml so as to obtain a resulting 1 mg/ml of BSAF in Ringer’s solution which was used directly for sorptions. Preparation of Surfaces Sorptions of proteins were tested in glass tubes, inner diameter 4.5 mm, coated with a polymer film prepared as follows. A 10%solution of polymer in the acetone-methanol mixture (1:l)was poured into glass tubes cleaned with chromosulfuric acid and slowly let out. The procedure was repeated once more and the tubes were then dried a t room temperature. The film, swellable but insoluble in water, was rinsed with distilled water. Sorption experiments were carried out only using tubes coated with a completely transparent, optically homogeneous, smooth film. Prior to use, the tubes were filled with Ringer’s solution and the solution was left for 4 hr in order to reach equilibrium. Ringer’s solution was then squeezed out with a twofold volume of solution of labeled BSA. The sorption occurred a t 297 K. After the sorption had been completed, the protein solution was squeezed out with an eightfold volume of Ringer’s solution during 30 sec, and the tubes were additionally rapidly washed twice with two volumes of Ringer’s solution. A t the end of the process, the washing solutions did not contain any traces of proteins detectable by fluorescence. The sorbed proteins were desorbed with 1%solution of Triton X-100 in borax, pH 9.2 (0.2 M H3B03 was titrated with 10%NaOH) at room temperature for 24 hr, and their quantity was determined from fluorescence intensity measurements at 475 nm with excitation a t 390 nm by comparison with calibration curves. Since the fluorescence intensity of
58
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labeled proteins is not sufficiently stable and depends on time and on the mode of treatment of the labeled protein, calibration solutions were prepared separately for each series of experiments; these were placed in tubes coated with a polymer film and left under the same conditions as those used in desorption. Protein sorption in polyethylene tubes was tested in the same way. Sorption and desorption on polyethylene films was carried out in closed cells from which the sorption solution was squeezed out similiarly to the case where tubes were used. In all cases, the arrangement used ruled out any contact between the tested surface and the protein solution-air boundary. Fluorescence measurements were performed with a Hitachi Perkin-Elmer MPF-2A spectrofluorimeter. Multiple internal-reflection (MIR) infrared spectra were recorded with a Perkin-Elmer 621 IR spectrometer. Electrophoresis on acrylamide gel was carried out in a tris(hydroxymethy1)aminomethane-glycine buffer (pH 8.4). The efficiency of proteins desorption by the Triton solution was checked for films of polyHEMA and of the 20% HEMA-80% MMA copolymer by dissolving these films before and after desorption in a methanolacetone 1:l mixture and by comparing the fluorescence intensities of solutions thus obtained. For films of polyethylene, similar control was accomplished by comparing the MIR infrared absorption of sorbed and desorbed films. In both cases, more than 90%of sorbed protein was removed by the desorption procedure.
RESULTS AND DISCUSSION
BSA Labeled with DANSCl (BSAD) In measurements of calibration solutions, the BSAD concentrations could be detected starting from g/ml. After BSAD had been sorbed on HEMA, it was possible to desorb fluorescence compounds also without detergents. Two types of emission spectra were observed. When the solution of BSAD was dialyzed, the solution inside the bag exhibited the fluorescence maximum a t 500 nm (all maxima are given in noncorrected wavelength), whereas the maximum for the outside solution appeared a t 488 nm. The substances eluted from the hydrogel surface previously treated with BSAD showed also a maximum a t 488 nm. Therefore, we concluded that the solution obtained by desorption
PROTEIN SORPTION AND FLUORESCENCE LABELS
59
contained mostly free fluorochrome which had migrated inside the gel (and, probably, had accumulated in it) during the treatment by the solution of BSAD. The samples were therefore purified from free fluorochrome by precipitation and repeated dialyses. The content of free fluorochrome was determined by an equilibrium dialysis of the BSAD solution into the same volume of Ringer’s solution. In a purified 10 mg % BSAD solution the fluorescence intensity of free fluorochrome was about 4% of the fluorescence of BSAD, which is too much for a reliable investigation of sorption on the hydrogel surface. Neither is DANSCl very suitable if combined with Triton X-100 solutions used for washing away sorbed proteins. The fluorescence of BSAD with an excitation maximum near 340 nm was disturbed by the long-wave edge of fluorescence of the Triton X-100 solution (emission maximum at 380 nm) excited in the same region (excitation maximum a t 300 nm).
BSA Labeled with FITC (BSA-FITC) Concentrations of calibration solutions starting from g BSA-FITC/ml could be detected. After BSA-FITC had been sorbed on polyHEMA, the fluorescence compounds were desorbed from the gel even without detergent. The emission fluorescence spectrum of desorption solutions with a maxiimum at 514 nm was somewhat shifted compared to the spectrum of the BSA FITC solution with its maximum at 518 nm. In both cases, the excitation maximum lay near 460 nm. The fluorescence intensity increased almost linearly with the time of sorption; there was no perceptible saturation even after 24 hr. The above characteristics show that the values obtained are in this case determined by diffusion of the low molecular fluorochrome into the gel rather than by sorption of BSA-FITC on its surface. In repeated dialyses of the BSA-FITC solutions, the low molecular weight fluorochrome was released into the dialyzate without considerable slowing-down. In attempts at determination of the relative concentration of free label by dialysis into the same volume (similarly to BSAD), the state of equilibrium could not be reached even after several days, and the fluorochrome concentration in the dialyzate continued to increase. It may be inferred, therefore, that FITC bonded on BSA is gradually released into solution because of the instabiIity of the bond.
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BSA Labeled with Fluorescarnine (BSAF) Excitation and emission maxima were found at 390 nm and 472 nm, respectively. Concentration of calibration solutions starting from g BSAF/ml could be detected. When tested in the dialysis cell, the BSAF solution did not release any low molecular weight fluorescence compounds even after a few days. Also, if used in sorption experiments, it did not give rise to effects characteristic of diffusion of free fluorochrome into the gel, such as washing out of fluorescence compounds without detergents, slow increase in concentration of fluorescence compounds in the gel not leading to saturation with increasing time of sorption, and high fluorescence intensities of the desorption solutions. All these facts indicate that in the case of BSAF, the fluorescence determination of sorption is not distorted by the diffusion of low molecular weight fluorescence compounds into the gel. With films of pure polyHEMA, the detected sorption of BSAF from Ringer’s solution was about g/cm2 at the times used within 1-24 hr. Such low values were already at the limits of applicability of the method; with respect to errors, which will be discussed below, no characteristics of sorptions could be checked. The method was therefore tested on similar films of the copolymer 20% HEMA-80% MMA, prepared and tested similarly to those of pure polyHEMA. An addition of the hydrophobic component considerably raised the sorption of BSAF from Ringer’s solution. Figure 1shows the amount of BSAF per cm2 of the surface of the 20% HEMA-80% MMA copolymer as a function of the sorption time. One can see that the equilibrium is established at a slow rate, and that the amount of sorbed BSAF goes on increasing even after several tens of hours. Figure 2 shows the amount of BSAF sorbed on the surface of the 20% HEMA-80% MMA copolymer after sorption lasting 4 hr as a function of the BSAF concentration in the sorption solution. Similarly to the time dependence, the concentration dependence does not exhibit saturation of the sorption capacity within the range under investigation. The effect of the fluorescence label on the sorption of BSA was evaluated using competitive sorption between BSAF and BSA. The amount of BSAF sorbed from the solution of 50 mg % BSAF 50 mg % BSA in Ringer’s solution was ca. 5% of the amount of BSAF sorbed from a solution of 100 mg % BSAF; the ratio was independent of the sorption time. It may be concluded, therefore, that in the case
+
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time [hours]
Fig. 1. Time dependence of the concentration of BSAF sorbed on the surface of the 20% HEMA-8O?? MMA copolymer from 100 mg 96 of solution.
I
"E
0.10
01
a /
0.25
I
0.50 0.75 1.00 sobtion concentration, mglml
Fig. 2. Concentration of BSAF sorbed on the surface of the 20% HEMA-gO?? MMA copolymer after sorption a t 297 K for 4 hr vs. BSAF concentration in solution.
of competitive sorption, there is no preferential sorption of BSA compared with BSAF. On the other hand, however, one cannot decide to what extent BSAF is preferentially sorbed, since (as shown by Fig. 2) the concentrations used could not be chosen in the saturation range, and the amount of BSAF sorbed depended on the BSAF concentration in the sorption solution even if BSA was absent. By examining the amount of BSAF sorbed on the surface of polyethylene as a function of the sorption time (Table I), it was found that
BRYNDA ET AL.
62
TABLE I Competitive Sorption between BSAF and BSA on Polyethylene and Kinetics Sorption a t 297 K Composition of Solution 50 mg % BSAF 25 mg % BSAF 25 mg % BSA 100 mg % BSAF 50 mg % BSAF 50 mg % BSA 100 mg % BSAF
+ +
50 mg % BSAF
+ 50 mg % BSA
Surface Relative Sorption Concentration Sorption of Time (hr) of BSAF (fig/cm2) BSAF in Mixture (%) 2 2 2 2 0.25 0.5 1.0 10 0.25 0.5 1.0 10
0.372 0.208 0.373 0.238 0.368 0.371 0.373 0.373 0.231 0.236 0.238 0.236
56 100 64
after a short time the sorption capacity for BSAF became saturated. Table I shows the amount of BASF sorbed on polyethylene from solutions with various contents of BSAF and BSA after 2 hr of sorption as well as the kinetics of sorption. One can see that with the concentrations used, i.e., 50 mg % and 100 mg % BSAF, the equilibrium is established virtually within 30 min and is independent of the BSAF concentration in solution. If this value (0.373 pg/cm2 BSAF) is regarded as loo%, then the amount of BSAF sorbed from the solution of 25 mg % BSA 25 mg % BSAF is about 56% of this value, and the amount of BSAF sorbed from the solution 50 mg % BSA 50 mg % BSAF is about 64%. Consequently, BSA labeled with Fluorescamine is sorbed more strongly than unlabeled BSA. A shift in the electrophoretic band of BSAF with respect to BSA (Fig. 3) shows that the protein labeled with Fluorescamine has a lower number of positive charges than the unlabeled one, obviously due to the label being bonded on the amino groups of the protein. On the other hand, one can see that the labeling did not lead to an aggregation of the proteins. The broadening of the zone for BSAF can probably be assigned to the bonding of Fluorescamine on various types and various numbers of amino groups of the protein, so that the labeled BSAF sample is heterogeneous in this respect. The procedure used in labeling, i.e., addition of acetone to the solution of BSA and sub-
+
+
PROTEIN SORPTION AND FLUORESCENCE LABELS
63
Fig. 3. Electrophoresis on acrylamide gel (1)BSA; (2) BSAF; ( 3 ) BSAD; (4) BSA-FITC.
sequent dialyses, did not have any effect on the electrophoretic mobility of BSA. In conclusion, we shall try to summarize the advantages and disadvantages of the fluorescence labeling of proteins in the investigation of sorption. The advantage consists in a simpler technique compared with work involving radioisotopes and in the independence of the half-life period and of the radiochemical damage, that is, in the long-term applicability of the same labeled sample. The main difficulties encountered in applying fluorescence labels in the determination of protein sorption on polymeric, and particularly hydrophilic, surfaces are as follows. 1)With hydrophilic gels, the difficulties consist in the presence of a free label or in the gradual release of the latter due to the insufficient stability of the bond with the protein, as demonstrated by experiments with DANSCl and FITC. A signal due to the low molecular weight label diffused into the bulk of the gel may exceed the measured signal
64
BRYNDA ET AL.
due to the labeled protein sorbed on the gel surface, even if the quantity of the free label in the sorption solution is very small (accumulation of the label in the gel mass). However, similar difficulties could arise in hydrophilic gels also if radioactive-labeled proteins were used. 2) Instability of fluorescence of labeled protein was apparent. The fluorescence intensity of calibration BSAF solutions decreased with time and was also dependent on the conditions and materials used in storing the solutions. Special attention should therefore be devoted to the way in which the standards are stored. 3) Change of the protein properties after labeling was noted. Labeling has always led to a change in the electrophoretic mobility, and in the case of BSAF investigated here, it also led to a change in the sorption properties. Preferential sorption of BSAF on polyethylene and on the 20% HEMA-80% MMA copolymer is probably due to an increase in the hydrophobic character of the protein surface due to cancellation of the charged amino group and to the bonding of a hydrophobic label. It will be necessary, therefore, when investigating competitive sorptions, to characterize this effect due to the presence of the label by applying alternate labeling of the individual proteins. We believe that the fluorescence method could be improved; to a considerable extent better dispersion, low-temperature measurements, or polarization measurements would undoubtedly make possible a reliable detection of the low molecular weight fluorochrome released in the reaction, which is impossible in the case of a released radioactive label. At present, we are introducing multireflexion technique of fluorescence measurements directly on the gel surface (analogously to the MIR infrared technique),14 which offers some further possibilities of extending the methods already in use. References 1. R. E. Baier and R. C. Dutton, J . Riomed. Mater. Res., 3,191 (1969). 2. E. W. Salzman, Bull. N . Y. Acad. Med., 48,225 (1972). 3. L. Vroman, A. L. Adams, M. Klings, and G. Fischer, Adu. Chem. Ser., 145,255 (1975). 4. M. A. Packham, G . Evans, M. F. Glynn, and J. F. Mustard, J . Lab. Clin. Med., 73, 686 (1969). 5. C. S. P. Jenkins, M. A. Packham, M. A. Guccione, and J. F. Mustard, J. Lab. Clin. Med., 81,280 (1973).
PROTEIN SORPTION AND FLUORESCENCE LABELS
65
6. D. J. Lyman, K. G. Klein, J. L. Bash, B. K. Fritzinger, J. D. Andrade, and F. Bonomo, Thromb. Hemorrh. Suppl., 42,109 (1971). 7. J. D. Andrade, H. B. Lee, M. S. Jhon, S. V. Kim, and J. B. Hibbs, Jr., Trans. Am. SOC.Artif. Intern. Organs, 19,l (1973). 8. R. C . Nairn, in Fluorescent Protein Tracing, E. and S. Livingstone Ltd., London, 1964. 9. T. P. Murtha, Disertation, University Microfilms, Ann Arbor, Mich., 69-9879 (1968). 10. M. Weigele, S. L. DeBernardo, J. P. Tengi, and W. Leimgruber, J. Am. Chem. Soc., 94,5927 (1972). 11. P. Bohlen, S. Stein, W. Dairman, and S. Undenfriend, Arch. Biochem. Biophys., 155,213 (1973). 12. G. Weber, Biochem. J., 51,155 (1952). 13. J. KopeEek, J. Jokl, and D. Lim, in Macromolecular Chemistry, Prague 2965 ( J . Polym. Sci. C, 16), 0. Wichterle and B. SedlBEek, Eds., Interscience, New York, 1968, Part 7, pp. 3877. 14. G. I. Loeb and N. J. Harrick, Anal. Chem., 45,687 (1973).
Received February 4,1977
