Langmuir 1991, 7, 1755-1764
1755
Infrared Spectroscopic Studies of Time-Dependent Changes in Fibrinogen Adsorbed to Polyurethanes Thomas J. Lenk,tJ Thomas A. Horbett,tJ and Buddy D. Ratner*JJ Department of Chemical Engineering and Center for Bioengineering, BF-10, University of Washington, Seattle, Washington 98195
Krishnan K. Chittur Department of Chemical Engineering, University of Alabama in Huntsville, Huntsville, Alabama 35899 Received August 22,1990. I n Final Form: February 6, 1991 Time-de ndent transitions in an adsorbed layer of fibrinogen (-300 ng/cm2) on two different poly(ether ure&e ureas) were studied both by use of an attenuated total reflection infrared flow cell and by measurement of the fraction of protein removable by exposure to a 1 % sodium dodecyl sulfate (SDS) solution. The fraction of the adsorbed fibrinogen removed by exposure to SDS decreased with residence time of the adsorbed protein on the surface. The infrared spectral changes are consistent with timedependent conformationalchanges in the adsorbed fibrinogen, particularly a gain in &structures (sheets, turns, and bends). A correlation between the center-of-gravity frequency shift of the amide I1 band of adsorbed fibrinogen and the amount of fibrinogen retained after SDS exposure is also shown. Observed discrepancies between the two types of experiments can be explained by postulating a distribution of bound states for the adsorbed protein. Introduction There have been a number of attempts to characterize changes in surface-adsorbed proteins using methods such as infrared s p e c ~ o s c o p ysurfactant ,~~ elution,'$ fluorescence techniques? and circular dichroism.10 Some evidence of time-dependent conformational changes in adsorbed protein layers has been reported.lt4 An apparent change in protein-surface interactions was shown by Bohnert and Horbett,' who demonstrated that the resistance of adsorbed protein to removal by detergent increased with increasing residence time of the protein on the surface. They proposed that the increased residence time resulted in strengthening of protein-surface interactions, possibly accompanied by conformational changes in the adsorbed protein. However, little work correlating changes in the structure of adsorbed proteins with the attachment of these proteins to polymer surfaces has been performed. In this investigation, we describe the adsorption of fibrinogen to polyurethanes, with changes monitored by both an attenuated total reflection (ATR)infrared
* Author to whom correspondence should be addressed.
+ Present address: IBM Research Division, Almaden Research Center, K93/801,660Harry Rd., San Jose, CA 95120. t Department of Chemical Engineering. 8 Center for Bioenmneerinn. (1) Pitt, W. 0.;Fabkiue-Himan, D. J.; Mosher, D. F.; Cooper, S. L. J . Colloid Interface Sei. 1989, 1!29 ( I ) ,231. (2)LenkT. J.;Ratner.B.D.:Gendreau,R.M.;Chittur,K.K. J.Biomed. Mater. Rea. 1989,N (61, 649. (3)Pitt, W.;Cooper, 5.L.ACS Symp. Ser. 1987, No. 343, 324. (4)Catillo, E.J.; Koenig, J. L.;Andereon, J. M. Biomateriala 1986, 7., 89. -~ (5) M o h y , B. W.; Stromberg, R.R.J . Colloid Interface Sci. 1974, 46 ( 1 ) . 162. ~-~ (6) Morrirwy,B. W.;Fensbder,C.A. Trane.Am.Soc.Artif.Zntern. Organr 1976, XXZZ, 278. (7)Bohnert, J. L.;Horbett, T. A. J . Colloid Interface Sci. 1986,111 (2). ,-,.sB9. (8) Rapou, R. J.; Horbett, T. A. J. Biomater. Sci., Polym. Ed. 1989, -I (1). .-,,89. - -. (9) Rainbow,M.R.; Atherton, S.;Eberhart, R. C. J. Biomed. Mater. Rea. 1987,21 (a), 639. (10)Soderquirt, M.E.;Walton, A. C. J . Colloid Interface Sci. 1980, ,
~
I
.
75 (21, 386.
technique and elution with sodium dodecyl sulfate (SDS) solutions. The objective was to test the hypothesis of Bohnert and Horbett by comparing changes in the infrared spectra of adsorbed fibrinogen to the increased resistance to removal by SDS with residence time. Materials and Methods Buffers. Citrate-phosphatebuffered saline (CPBS)was wed for all of the infrared experimentsrequiring use of a buffer. This buffer contains0.01 M sodium citrate,0.01M sodium phosphate, and 0.12 M NaCl and has a pH of 7.4. For experiments with 'Wradiolabeled protein, CPBS containing0.01 M sodium iodide was used (CPBSI). The nonradioactive iodideis presentto ensure that any free l=I is only a small fraction of the free iodide in solution. This decreases the amount of free iodide that can be picked up by a surface,reducing spurious countsthat may result from surface affinity for free iodide. Proteins. Baboon fibrinogen was prepared from citrated baboon blood by a polyethylene glycol, &alanine fractional precipitation as described previously.'l Trasylol (Mobay Chemical Corp.) was added to plasma prior to fibrinogen purification to inhibit proteolyticdigestion. The fibrinogenwas greater than 90% clottable. Aliquots were stored at -70 "C at concentrations greater than 1 mg/mL, thawed as needed, and diluted to the desired concentration in CPBS. Samples for use in infrared experimentswere dialyzed either before or after freezing toremove 8-alanineremaining from the preparation procedure. 19-radiolabeled fibrinogen was prepared by the iodine monochloride (ICl)method of MacFarlane12as modified by Helmkamptset al. and Horbett." The labeled fibrinogen was added to a stock solution of baboon fibrinogen to give a specific activity of approximately 108 (counts/min)/mg of fibrinogen. Polymers. The two polyurethanes tested were synthesized by endcapping a diol with 4,4'-diphenylmethane diisocyanate (MDI) and chain extending with ethylenediamine (ED). The MD1:EDdiol ratio was 2:l:l. Poly(tetramethy1eneoxide)of molecular weight 2000 was used to prepare one polymer, designated as PTMG2000-PEU. This polymer is typical of block poly(ether ( 1 1 ) Weathersby, P. K.;Horbett, T. A.; Hoffman, A. S. Thromb. Rea. 1977,10, 245. (12) MacFarlane, A. Nature 1969,182,53. (13) Helmkamp, €2. W.; Goodland, €2. L.; Bale, W. F.;Spar, I. L.; Mutschler, L. E. Cancer Res. 1960,20, 1496. (14) Horbett, T. A. J . Biomed. Mater. Res. 1981, 16, 673.
0743-7463/91/2407-1755$02.50/0 0 1991 American Chemical Society
Lenk et al.
1756 Langmuir, Vol. 7, No. 8, 1991 Table I. ESCA Analysis of the Polyurethanes (Surface Composition in Atomic Percent) surface carbon oxygen nitrogen HS-PEU bulk (elemental analysis) 74 14 12 2% on glass, 55O angle 75 14 11 2% on glass, 800 angle 76 14 10 PTMG2000-PEU bulk (elemental analysis) 79 18 3.0 2% on glaas, 55O angle 79 19 1.5 2% on glass, 80° angle 80 19 0.8 0.2% on Ge, 8 0 O angle 80 19 0.9 Table 11. ESCA Analysis of the Polyurethanes: Resolution of the Area of the C 1s Spectrum into Constituent Peaks surface hydrocarbon ether carbonyl HS-PEU bulk (elemental analvsisl 63 26 10 2% on glass, 550 angie 63 11 26 2% on glass, 80° angle 26 9 64 PTMG2000-PEU bulk (elemental analysis) 54 44 2.5 2% on glass, 55O angle 44 1.7 54 2% on glass, 80° angle 44 0.4 56 0.2% on Ge, 80° angle 51 48 0.1
Y)
c
3
u
292
290
(15) Lenk, T . J.i Rapoza, R. J.; Slack, S. M.; Horbett, T.A.; Ratner, B. D. Manuscript in preparation.
286
284
282
280
Binding Energy (eV)
'
urethane ureas) used in biomedical applicaLans. The seconl polymer, a hard segment model analogue, was prepared with dipropylene glycol as the diol and is designated HS-PEU. Molecular weight distributions (determined by GPC against polystyrene in dimethylacetamide) are as follows: PTMG2000-PEU, M, = 1.6 X 106, M n= 2.4 X 1% HS-PEU, M, 2.9 X 106,M, = 3.0 X 104. Surfaces for the radiolabeled experiments were prepared by centrifugally coating 30 pL of a 2% solution of the polymer in 1,1,1,3,3,3-hexafluoro-2-propanol(HFIP) onto silane-treated 9-mm glass borosilicatecoverslips.16 Surfaces for infrared studies were prepared by centrifugally coating 400 rL of a 0.2 % solution of the polymer in HFIP onto one surface of a 102 X 10 X 2 mm germanium ATR crystal using a special holder for the c r y ~ t a l . ~ Solutions were filtered through a 0.5-rm Teflon fiiter before coating. A lower polymer concentration was used for the germanium crystals than for the glaes coverslipsbecause thinner polymer films increase the sensitivity of the ATR technique to protein adsorbed onto the polymer. The fibs were determined by ellipsometry to be approximately 110-160 A thick. Grazing angle electron spectroscopy for chemical analysis (ESCA) of polymer films on germanium was conducted by using coatings on 5 X 5 mm poliihed germanium squares to allow mounting on samples to the variable angle stage. ESCA was performed on a Surface Science Instruments SSX-100 spectrometer using a monochromatized aluminum K, X-ray source. A low-energy electron flood gun and a wire grid mounted 1 mm above the samples aided in charge neutralization. ESCA analysisof the polymersused in this study is summarized in Tables I and 11. The PTMG2000-PEU shows depletion of hard segment near the surface (based upon a low concentration of nitrogen), as is common for phase-separated polymers of this type. The HS-PEU yields a surface compositionnearly identical with the bulk. Because PMTG2000-PEU undergoes phase separation, and because different coating procedures were used for the infrared and elution experiments, the surfaces of the two types of coatings were compared. Figure 1 shows C 1s ESCA spectra at an 80' takeoff angle (the uppermost surface, -10 A sampling depth) for coatings of PTMG2000-PEU from a 2% solution in HFIP onto borosilicateglassand from a 0.2 % solution onto germanium. Little difference is observed between the two surfaces in the "as cast" state. This is further verified by examination of the ESCA results in Tables I and 11. Transmission infrared spectra of the two polyurethanes are shown in Figure 2. The spectrum of PTMG2000-PEU, which is mostly polyether, is dominated by the symmetric C-O-C ether
280
Figure 1. Comparative ESCA C 1sspectra of PTMG2000-PEU coatings collected at an 80° takeoff angle. Top, coating from 0.2% solution in HFIP onto germanium; bottom, coating from 2% solution in HFIP onto borosilicate glass.
I
I
PTMG2000-PEU
3500
3000
2500
2000
1500
1000
Waven umbers
$500
3000
2500
2000
Wavenumbers
1500
1000
Figure 2. Transmission infrared spectra of PTMG2000-PEU and HS-PEU. stretch at 1113 cm-I and the C-H stretch of the poly(tetramethylene oxide) (the two sharp bands at 2846 and 2941 cm-9. HS-PEU contains a much higher percentage of ureaand urethane linkages, and this is reflected in the intensities of the urea amide I1 and amide I11 vibrations (1524 and 1226 cm-I) and the N-H stretch at 3318 cm-l. Water contact anglesin air were measured for the two polymers in the as cast state and after 17 h of hydration using a h e - H a r t Model 100-00 115goniometer. The results of measurements on three different samples of each polymer are shown in Table I11 and are comparable to values observed previously for similar materials.'eJ' The contact angles are slightly smaller on HS(16) Tingey, K.G.; kdrade, J. D.; Zdrahala, R. J.; Chittur, K.K.; Gendreau,R. M. h o g . Btomed. Engr. (Surf.Charoct.Biomot.) 1988,255. (17) G r a d , T.G.; Cooper,S.L.Biomoteriols 1986, 7, 315.
Langmuir, Vol. 7, No. 8, 1991 1757
IR Studies of Adsorbed Fibrinogen Table 111. Water Contact Angles on the Polyurethanes PTMG2000-PEU
as cast 17-h hydration
advancing 74+3 70& 1
receding 44*2 40t 1
HS-PEU
advancing 6411 6111
receding 38f1 36i 1
PEU, probably due to the enrichment of poly(tetramethy1ene oxide)at the surface of PTMG2000-PEU,althoughneither surface is especially"hydrophilic". There is considerable hysteresis (2530°) on both surfaces. This might be ascribed to surface rearrangement and surface heterogeneity for PTMG2000-PEU, but the existence of nearly as much hysteresis on HS-PEU, which should not be subject to those concerns, indicates that surface roughness probably plays a large role. A 17-h hydration also appears to have little effect on the measured contact angles. Infrared Experiments. Polymer coatings on germanium ATR elementa were prepared as described above. The crystal was then assembled into an infrared flow cell1*using an ethylene/ propylene rubber gasket (60% ethylene) as a spacer to create a flow channel. After assembly, the cell was left for 10-12 h to allow for gasket creep before beginning spectral collection. The polymer coatings were hydrated for 5-20 h in CPBS, at which time fresh buffer was introduced and a single-beam spectrum of the hydrated polymer in the presence of buffer was collected. After hydration, 50 mL of fibrinogen solution (0.05 mg/mL) was introduced into the flow cell a t 60 mL/min (shear rate -700 5-1) using a syringe pump and a 50-mL syringe. This was approximately 10times the volume of the flow cell and tubingcombined. After the appropriate adsorption time (3 min on HS-PEU, 5 min on PTMG2000-PEU) the protein solution was displaced by 50 mL of fresh buffer. At this point, the adsorbed protein was considered to have begun its "residence time" in buffer. These adsorption times gave surface fibrinogen concentrations of N 300 ng/cm* in adsorption experiments with radiolabeledprotein. This concentration was chosen to be low enough for significantchanges in elutability to occur over reasonable times and high enough to produce a strong infrared absorption. Because previous work has revealed possible spectral changes in some proteins with adsorbed concentration,B the surface concentration was kept as similar as possible on both polymers to avoid extraneous effects. Except for the actual pumping to introduce and displace the protein solution (about 45-50 s in each case), adsorption and buffer residence occurred in the presence of static solution. This kept the conditions as close as possible to those of the radiolabel experiments, although the initial protein addition was quicker for the radiolabel experiments (requiring only a few seconds of pipetting). Spectra were collected every 3-5 s during the initial cell filling and adsorption, at 15,30, and 60 min, and every 1-2 h for the remainder of the experiment. All spectra collected after 6 min of residence time in buffer consisted of 1000 coadded scans (5-6min collectiontime). Polarized spectra were collected by using a 2-pm grating polarizer. Spectra of adsorbed protein were created by subtracting the last spectrum of hydrated polymer in buffer before introduction of protein into the cell from the spectra of the buffer-polymerprotein system. Hydrated spectra with polarized light were used as the references for the polarized spectra. Although the appearance and consistency of the spectra can sometimes be improved by adjusting the subtraction factor u ~ e d , l *work * ~ in our laboratory has shown the amide I1 band of adsorbed protein to be insensitive to small changes in the subtraction factor, so a subtraction factor of 1 was used for all spectra in these experiments. Absorption frequencies and intensities were measured by using a five-point center-of-gravity and the program WIDPLT~~ with a linear baseline from 1720 to 1480 cm-l for the amide I and I1 peaks. The pertinent characteristics of the different flow runs are summarized in Table IV. (18) Leininger,R. I.; Fink, D. J.; Gendreau, R. M.;Hutaon, T. B.; Jakobeen, R. J. Trans. Am. SOC.Artij. Intern. Organs 1983,29, 152. (19) Powell, J. R.; Wasacz, F. M.; Jakobsen, R. J. Appl. Spectrosc.
1986, 40 (3,339. (20) Douseeau, F.; Therrien, M.; Pezolet, M. Appl. Spectrosc. 1989,43 13). . .. 538. (21) Cameron, D. G.; Kauppinen, J. K.; Moffat, D. J.; Mantech, H. H. Appl. Spectrosc. 1982, 36 (3), 246.
Radiolabel Experiments. Samples were hydrated 12-16 h at room temperature in CPBSI, which was replaced by fresh buffer before beginning the experiments. Samples were equilibrated at 25 O C for at least 2 h before adding protein. Protein was allowed to adsorb from a 0.05 mg/mL solution for the appropriate time to obtain a surface concentration of about 300 ng/cm2, after which the protein solution was displaced by fresh CPBSI. The samples were then left in the displacing buffer for the desired residence time (up to 48 h), rinsed, and placed in counting tubes with 1% SDS solutions in Tris electrophoresis sample buffer. These samples were counted, rinsed after approximately 24 h, and then recounted to determine the amount of protein removed by exposure to SDS. Peak-Fitting. Component peaks were fit to the amide I and I1 regions of nondeconvoluted infrared spectra using a leastsquares routine described by Fraser and SUzuki.22The program was modified to run on the IBM PC and accept spectral files in the J-CAMP format. Each spectrum was initially modeled by nine Gaussian peaks, as listed in Table V. The use of a Lorentzian shape function resulted in long tails at both ends of the baseline and did not accurately reflect the shape of the spectra. Approximate band positions were determined from second derivative spectra, and the initial intensities and widths were chosen after trial fitting to several spectra. These peaks were drawn above a baseline from 1720 to 1480 cm-l. It was possible to constrain any combination of peak widths, intensities, or positions during fitting. Initially, widths and positions were constrained, and the peak heights varied to give the best fit for each spectrum. Once the initial intensities were obtained, the widths, positions and intensities of all peaks were allowed to vary simultaneously. Fitting continued until further iteration resulted in a decrease in the least-squares error of less than 5 % from the previous iteration. The two spectra nearest 1,20, and 40 h of residence time from each of three runs on PTMG2000PEU and two runs on HS-PEU were used in fitting studies. A typical fit spectrum is shown in Figure 3.
Rssults Comparable amounts of protein were adsorbed in all experiments, as indicated by the amide I1 intensity after 1 h of surface residence (Table IV). These values were scaled to a water band intensity of 700 mAU to correct for experiment-to-experiment variations in alignment and flow cell assembly.23 When compared with values of fibrinogen adsorption in the radiolabel experiments, a value of 16.6 f 2.1 (ng/cm2)/mAU for protein quantitation is obtained, comparable to values obtained in other work with the same flow cell.24 The time-dependent changes in percentage of adsorbed fibrinogen retained on t h e polymer surfaces after exposure to SDS are presented in Figure 4. Both residence time and t h e adsorbing surface affect the ability of SDS to remove the adsorbed protein. Time-dependent changes in the amide I1 band are shown in the deconvoluted spectra in Figure 5. These intensity changes in individual components of the amide I1 band can be more conveniently described by the change in the amide I1 center-of-gravity frequency (Figure 6). Despite some variation, there is a consistentshift in t h e center-of-gravity frequency to higher wavenumber with residence time on the surface. The reason for the offset of run 2 is not clear, but the shift is similar to that of the other three runs. A comparison of t h e elutability and center-of-gravity frequency results is shown in Figure 7. The infrared average values were obtained by combining points from all three runs so that (22) Fraser, R. D. B.; Suzuki,E.In Physical Principlesand Techniques of Protern Chemistry; Leach, S. J., Ed.; Academic Prese: New York, 1973; Part C, p 301. (23) Chittur, K. K.; Fink, D. J.; Leininger,R. I.; Hutaon, T. B. J. Colloid Interface Sci. 1986,111 (21,419. (24) Fink, D. J.; Hutaon, T. B.; Chittur, K. K.; Gendreau,R.M. A d . Biochem. 1987, 166, 147.
1758 Langmuir, Vol. 7, No. 8,1991
Lenk et al.
Table IV. Characteristicr of Individual Infrared Experiments (All Experiments Used Baboon Fibrinogen at a Concentration of 0.06 mg/mL) no. surface hydration, h ads time, min:s run time, h Am I1 int at 1 ha COG at 1 h resolution, cm-1 1 PTMG2000-PEU 5 529 45 22.7 1548.25 8 2 PTMG2000-PEU 9 6:Ol 40 20.1 1549.33 8 1548.42 4 3 PTMC2000-PEU 21 516 40 20.9 4 PTMG2000-PEU 1548.30 4 8 5:05 51 19.0 5 HS-PEU 1547.92 8 4.8 3:03 56 17.0 1547.66 8 6 HS-PEU 7.5 300 50 18.8 18.3 1548.71 4 7' HS-PEU 13 258 a Baseline from 1480 to 1720 cm-', scaled to water band intensity of 700 mAU. * Run developed problems after several hours, could not be used for long-term studies.
Table V. Initial Peaks for Fitting position, cm-1 inteneity, mAU width, cm-1 1680 5 30 1650 13 30 1630 4 30 1620 4 30 2 30 1590 1576 4 30 1550 13 30 4 30 1630 1515 5 30 I
a
h
3
a
E
v
I
2.0 2
1.6
g
1.2
-E P
B 0.8
5 minutes
0.4
1600
I
I
1500
Wavenumber 0.0 1720
1680
1640 1600 1560 Wavenumber (cm-'I
1520
1480
A
Figure 3. A bandfit spectrum of approximately 300 ng/cm2 of
baboon fibrinogen that has resided on PTMG2000-PEU for 1h.
I
b
u
80
e
a
-
i
30
a,
0
20
0 0
10
20
Residence Time
30
40
50
(hours)
10
Figure 4. Comparison of the amount of fibrinogen retained on HS-PEU and PTMG2000-PEU after exposure to a 1% SDS
solution: open circles, PTMG2000-PEU;closed circles, HS-PEU.
I
I
1600
each time interval would contain at least three points. Since measurement times were not identical for all of the runs, error bars are shown in both x and y directions. The y axes were scaled by equating the initial and final values for each type of data. Figure 8shows the amide I1center-of-gravity shift during initial adsorption of fibrinogen to PTMG2000-PEU and HS-PEU. Protein adsorbs to HS-PEU with a high initial center-of-gravity frequency, but the value then drops as more protein is adsorbed. PTMG2000-PEU, on the other hand, shows little change in the amide I1center-of-gravity frequency during the initial adsorption period. Bandfitting of the infrared spectra was used in an
I
1500
Wavenumber
Figure 5. Time-dependent changes in the amide I1 band of fibrinogenon polyurethanes: (a) PTMG2000-PEU;(b) HS-PEU. The spectra were deconvoluted using a half-width at half-height of 13 cm-I and k = 1.5.
attempt to understand how the amide I and 11bands were changing. Average peak parameters from bandfitting results for all spectra are shown in Table VI. In examining changes with time, the values at 40 h were compared with those from 1h. The significance of the differences was determined by using Student's t test with a95% confidence level. Individual band areas are presented in Table VI1
Langmuir, Vol. 7, No. 8,1991 1759
IR Studies of Adsorbed Fibrinogen
-
1551
1560
c
c
€
I
14.10
'
I
'
.
I
10
0
20
I
8
30
40
.
0
50
0
1545
t
I
1
I
I
O
15501
1549
4
0
90
Exposure Time (seconds)
c
=
60
30
0
Residence Time (hours) 1552
HS-PEU
I
Figure 8. The shift in the amide I1 center-of-gravityfrequency during initial adsorption of fibrinogen onto PTMG2000-PEU (open circles) and HS-PEU (filled circles) from a 0.05 mg/mL solution. The bulk solution was displaced after approximately 5 min on PTMG2000-PEU and 3 min on HS-PEU. Table VI. Average Band Parametem for Fitted Spectra of Fibrinogen on PEUs peak, cm-1 weition, cm-' width, cm-1 % of band area
0
~
0
10
20
30
40
50
60
Residence Time (Hours)
Figure 6. (a) Time-dependentshift in the amide I1 center-ofgravity frequency for fibrinogen adsorbed to PTMG2000-PEU. C l o d squares, run 1;open squares, run 2; closed circles, run 3; open circles, run 4. Surface concentration was approximately 300 ng/cm*. (b) Time-dependent shift in the amide I1 centerof-gravityfrequency for fibrinogen adsorbed to HS-PEU closed circles, run 5; open circles, run 6. Surface concentration was approximately 300 ng/cm*.
I
0
10
20
30
40
22.4 f 7.4 27.9 f 2.4 29.8 f 1.2 28.4 f 2.8 30.1 f 2.8
4.3 f 3.3 16.6 f 2.7 47.6 f 4.3 17.3 f 2.2 14.2 f 1.9
Table VII. Band Areas for Fibrinogen Adrorbed to PEUs
0.0
2.5
1680 1650 1630 1620
0.5 0.0 40
1593.3 f 3.2 1574.0 f 0.9 1550.0 f 0.4 1529.9 i 1.4 1515.5f 1.5
peak, cm-l
50
1 .o
30
1590 1575 1550 1530 1515
3.6 f 0.9 16.4 f 0.6 46.0 f 1.1 19.6 f 1.1 14.4 f 1.0
1.5
20
25.6 f 3.8 50.6 f 6.3 6.0 f 2.6 17.8 f 4.8
1590 1575 1550 1530 1515
2.0
10
32.0 f 3.0 30.3 f 1.4 20.0 f 2.9 29.3 f 2.0
1680 1650 1630 1620
Residence Time (hours)
0
1677.4 f 1.1 1649.4 f 0.7 1630.0 f 0.9 1619.4 f 1.0
20 h PTMG2000-PEU 27.3 f 4.3 24.5 f 2.1 50.7 f 0.8 46.7 f 2.1 5.2 f 2.0 3.9 f 1.7 20.8 f 1.8 20.8 f 1.7
U.Z
Amide Shift
1680 1650 1630 1620
50
Residence Time (hours)
Figure 7. Comparison of the amide I1 center-of-gravityshift and percent of protein retained on the surface after SDS elution for fibrinogen on the polyurethanes: (a) PTMG2000-PEU, (b) HS-PEU. The endpoints were pinned to establish the similarity of the time course of the changes. as a percentage of total amide I area (1680, 1650, 1630, and 1620 cm-l bands) or total amide I1 area (1590,1575, 1550,1530, and 1515 cm-1 bands).
Discussion Long-Term Changes. The time-dependent elutability studies reveal an increasing amount of fibrinogen
1590 1575 1550 1530 1515
l h
5.7 f 3.6 17.3 f 1.8 44.9 f 0.9 17.6 f 1.2 14.5 f 1.6 HS-PEU 21.9 i 0.6 23.5 f 2.4 56.1 f 1.3 58.5 f 1.4 5.4 f 1.4 7.8 f 1.5 10.2 f 2.1 16.6 f 0.7 2.3 f 2.4 5.7 f 1.2 15.4 0.3 15.2 f 2.3 51.5 f 3.2 46.2 f 0.6 18.1f 0.3 17.9 i 2.2 12.9 f 1.2 14.7 i 2.3
40 h 28.7 f 2.0 44.4 f 1.1 5.6 f 2.5 21.3 f 2.0 6.6 f 3.6 18.2 f 2.4 45.1 f 1.3 15.2 i 1.8 14.8 f 1.3 26.0 f 5.0 52.4 f 4.5 9.5 f 1.9 12.1 f 3.3 0.5 f 0.6 15.3 f 5.0 55.4 f 4.3 15.2 f 2.0 13.6 f 3.2
change
+ +
-
no
+ + -
-
no
+ + -
-
no
+
-
no
a Sign of any significant difference in paired values from 1to 40 h using the t distribution (a= 0.05).
retained on the surface with increasing residence time (Figure 4), an indicator of stronger attachment of individual proteins to the surface. Previous investigators have hypothesized that such attachment may be facilitated by a conformational change in the adsorbed protein.' The observed decrease in protein elutability in these experiments is greater on HS-PEU than on PTMG2000-PEU. A time-dependence is also seen in the changes in the amide I1 peak of the infrared spectrum of fibrinogen residing under similar conditions. The deconvoluted spectra show changes in peak shape with time, and these changes are also greater on HS-PEU than on PTMG2000PEU (Figure 5). The most prominent feature of the changes is what appears to be a shoulder near 1520 cm-l. An attempt to determine the magnitude of this change by
1760 Langmuir, Vol. 7, No. 8,1991 peak fitting revealed that it was probably not so much an increase at 1520 cm-l as a decrease near 1530 cm-l that created this feature (Table VII). Not much is known about the conformational significance of the amide I1 region, as most studies of conformational assignments have been conducted by using D20 (deuteration shifts the amide I1 frequencyby about 100cm-1) or using Raman spectroscopy (in which the amide I1 vibration is not active). Because of this lack of specific assignments, it was decided to represent the changes in the amide I1 band as a change in the center-of-gravity frequency. It may be significant that the changes on PTMG2000-PEU are small, but consistent, while the changes on HS-PEU are larger, but with more scatter in the data. Four scenarios could explain changes in the infrared spectrum of adsorbed fibrinogen: changes in the internal protein bonding, such as changes in secondary structure; changes in protein~urfaceinteractions; changes in protein/ water bonding, or the hydration state of the protein; and interfering spectral changes resulting from continued hydration of the polymer films. The spectral changes in the polymer films are greatest during the initial few hours of hydration.25 They should be negligible during the time period in question and will not be discussed further here. It is also possible that the adsorbed protein may reorient the polyurethane surface in some manner, although this effect should not be significant more than a few angstroms into the surface. The three other scenarios seem equally plausible, and all may be occurring simultaneously. The amide I1 band has been assigned to a combination of C-N stretchingand N-H bending modes.% A change in hydrogen bonding should affect primarily the N-H bend, with a shift to higher frequency associated with increased hydrogen bonding.2’ If the change in frequency is treated strictly as a change in hydrogen bonding, a plausible conclusion is that some hydrogen bonds within the adsorbed protein are converted to stronger bonds with residence time on the surface. This could be due to breaking of internal protein bonds and formation of specific protein/surface bonds or simply loss of water of hydration (replacing protein/water bonds with protein/surface bonds). Of course, it must be kept in mind that there is already a distribution of hydrogen-bonded states present in the protein and that these may be distinguished by various descriptive frequencies. A shift in the frequency of the amide I1 band could indicate a conformational change in the adsorbed protein through changes in intramolecular hydrogen bonding, and it is impossible to completely separate internal changes in the adsorbed protein from changes in protein-surface interactions. The amide I vibration offers more insight into possible conformational changes. The band assignmentsare better understood, and band-fitting can be profitably used to determine relative changes of the constituent bands with time. For fibrinogen adsorbed to PTMG2000-PEU, both the 1680- and 1630-cm-l bands increase in area with time at the expense of the 1650-cm-’ band (TableVII). A study of globular proteins in DzO assigned the high and low wavenumber components of the amide I band to @-structures (sheets, turns, and bends), with the lowest wavenumber bands assigned to @-sheet.28The combination of band (25) Tingey, K. G.;Lenk,T.J.; Chittur, K. K.; Ratner,B.D.; Andrade, J. D. Manuscript in preparation. (26) Miyazawa, T.; Shimanouchi, T.; Mizuehima, T. J. Chem. Phys.
1968, 29 (31, 611. (27) Bellamy,L. J. The Infrared Spectra of Complex Molecules; Chapman and Hak New York, 1980. (28) Byler, D. M.; Susi, H. Biopolymers 1986, 25, 469.
Lenk et al. Table VIII. Amide I and I1 Area and Intensity Measurements quantity amide 1/11 intensity ratio amide 1/11 area ratio relative amide I area relative amide I1 area
Ih 20 h PTMG2000-PEU
40h
*
+
1.15 0.14 1.33 0.09 1.50 h 0.06 1.00 f 0.02 1.25 f 0.11 1.40 f 0.24 1.00 h 0.02 1.08 h 0.08 1.06 h 0.09
+
1.16 h 0.17 1.31 0.13 1.41 h 0.14
*
*
HS-PEU amide 1/11 intensity ratio amide 1/11 area ratio relative amide I area relative amide I1 area
chanae~
1.38 h 0.21 1.39 f 0.31 1.60 h 0.34 1.26 f 0.16 1.33 1.00 h 0.03 0.88 1.00 h 0.00 0.84
* 0.21 * 0.10 * 0.06
1.62 h 0.28 0.99 f 0.12 0.77 h 0.02
+
no
+ +
no
-
Sign of any significant difference in paired values from 1 to 40 h using the t distribution (a = 0.05). 0
area changes in the adsorbed protein is consistent with an increase in the percentage of turns and bends with residence time. There appears to be little change in the @-sheet content and a loss of helical and/or random structures. The results on HS-PEU are less clear, a consequence of the lower resolution and the availability of only two runs. There are no statistically significant band position changes. Both the 1680-and 1630-cm-’ bands increase in area with residence time on the surface (Table VII), again indicating an increase in 8-structures with time in the adsorbed fibrinogen. In this case, the 1620-cm-l band decreases in area along with the 1650-cm-I band, possibly indicating a loss of regular @-sheetstructure along with the loss of helical or random structure. The fractional areaaccounted for by the “random and helix” peak at 1650 cm-l is also larger for fibrinogen on HS-PEU than on PTMG2000PEU. This and the increased width of the 1630-cm-l peak, which partially overlaps the 1650-cm-’ peak, seem unlikely to be due to additionally formed helical regions and probably represent additional random structure. Increased random structure could indicate increased denaturation upon adsorption, which might account for the much lower fraction of fibrinogen that can be removed from HS-PEU even at very short times. In Table VIII, overall amide 1/11intensity and integrated band area ratios are displayed. The area and intensity ratios compare favorably in average values, although the intensity ratios are much more scattered, a consequence of small variations in peak width. Both surfaces show an increase in amide 1/11 area ratio with time. Since fibrinogen on HS-PEU becomes resistant to elution more quickly than on PTMG2000-PEU,it appears that the ratio may correlate with the amount of protein “tightly” bound with respect to SDS elution. For BSA adsorption to PEUs, adsorbed protein (which also appeared to be somewhat denatured) showed an increase in the amide 1/11intensity ratio relative to bulk protein.2 Relating these results to the fibrinogen results, the data appear consistent with the idea of increasing conformational changes in adsorbed fibrinogen corresponding to increased amounts of fibrinogen “tightly bound” to the surface. However, the amide 1/11 ratio changes by different mechanisms on the two surfaces, as shown by comparison of relative peak areas (normalized to the amide I1 area at 1h). On PTMG2000PEU, the amide I1 area is relatively unchanged with residence time, while the amide I area increases dramatically. On HS-PEU, the amide I area is relatively unchanged over 40 h, while the amide I1 area declines by over 20%. No significant amount of protein is lost from either surface during this time according to the experiments with radiolabeled fibrinogen. Whether these con-
IR Studies of Adsorbed Fibrinogen trasting types of change indicate different protein-surface interactions is unclear. Short-Term Changes. It is clear from Figure 4 that important short-term changes are occurring in fibrinogen on HS-PEU. While the amount of fibrinogen removable from PTMG2000-PEU remains near 100% for up to an hour, the amount of fibrinogen retained on HS-PEU is about 30% after only 1 mi; of surface exposure to the fibrinogen s0lution.~3A decrease in elutability occurs soon after adsorDtion on HS-PEU. and we would exDect this rapid change to be reflected in the infrared dah. In Figure 8 we see that the center-of-gravityfrequency for fibrinogen adsorbed to PTMG2000-PEU does not change appreciably over the initial minutes of adsorption and in fact is nearly the same as the value at 1 h (see Figure 6). On HS-PEU, by contrast, there is a large decrease in center-of-gravity frequency with continued adsorption during the first minute. The long-terminfrared changes on HS-PEU, however, are in the opposite direction, and that increase in center-of-gravityfrequency was attributed to increased protein-surface interactions. To be consistent with the long-term results, we propose that the higher amide I1 frequency for fibrinogen initially adsorbed to HS-PEU indicates that the initially adsorbed protein is also attached tightly. Then, however, the next adsorbing protein attaches less tightly (lower center-ofgravity frequency), and so on, until the average centerof-gravityfrequency drops to the normal value for "loosely bound" protein. These changes occur in less than 1min, consistentwith our observationthat retention of fibrinogen on HS-PEU after SDS elution has already risen to the initial "plateau" by 1min of exposuretime. Due to physical constraints on protein addition and sample handling, elutabilities were not measured for exposure times less than 1 min. It should be noted that the use of a dilute protein solution for adsorption essentially eliminates the effect of bulk protein on the protein spectra. Even though the bulk protein solution is displaced from the cell at 3 min, no perceptible change in amide I1 intensity or center-ofgravity frequency occurs, indicating that the bulk protein contribution to the overall signal is negligible (i.e., the initial change is not due to switchingfrom a predominantly "bulk protein" signal to an "adsorbed protein" signal as more protein adsorbs). This is also supported by the fact that no initial change is observed for PTMG2000-PEU, where bulk protein solution is also present in the cell. Only adsorbed protein is monitored in the first minute, and the observed spectral changes are a direct reflection of changes in the adsorbed protein. Evidence for Protein Orientation. With the spectral region and experimental conditions used in this work, the s- and p-polarized component of the incident beam will have penetration depths that differ by about a factor of two (p > s).m Because of this, it is possible for orientation changes to appear as spectral changes when using radiation of uncontrolled polarization. It therefore becomes necessary to determine whether observed spectral changes are due to physical changes in the adsorbed protein or to changes in orientation of the protein on the surface. In an effort to look for orientation in the adsorbed fibrinogen layers, some spectra were collected by using polarized radiation. These spectra were collected in sets of four with the radiation polarized as follows: (1) unpolarized (Ul),(2) s-polarized (s), (3) p-polarized (p), and (4) unpolarized (U2). Each set of four spectra required about (29)Mirabelln,F. M.;Harrick,N. J. ZnternalReflection Spectroscopy: Review and Supplement; Harrick Scientific Corp.: Oesining,NY, 1986.
Langmuir, Vol. 7, No. 8, 1991 1761 Table IX. Dichroic Ratios of the Amide I and Amide I1 Peaks for Fibrinogen Adsorbed to Polyurethanes polarized (P/d amide 11 intensity PTMG2000-PEU 1.10 0.07 HS-PEU 1.13 k 0.26 amide I intensity PTMG2000-PEU 0.94 0.14 HS-PEU 1.01 0.27
* *
unpolarized (Ul/U4)
(a= 0.01)
1.01 0.02 1.00 0.02
yea no (a> 0.10)
* 0.02 0.06
no (a > 0.10) no (a> 0.10)
1.01 0.95
different
30 min of collection time. The s-polarized radiation is oriented perpendicular to the plane of beam propagation and will interact more strongly with dipoles oriented parallel to the surface of the ATRcrystal. The p-polarized light is oriented in the plane of propagation and will have components both parallel and perpendicular to the surface of the ATR crystal. The unpolarized spectra were collected as controls to ensure that neither random error or timedependent changes over the course of collecting the polarized spectra would be interpreted as changes due to polarization. No significantdifferencesbetween measured parameters in the pairs of unpolarized spectra were found. All changes observed with polarized light were therefore attributed to the orientation effects measurable with polarized light. Statistical comparisons were made by using Student's t test. There were six sets of spectra (from three different runs) for each surface. The first area to be investigated was the dichroic ratios of the amide I and I1 bands. A small difference was seen in the amide I1band of fibrinogen adsorbed to PTMG2000PEU, with the intensity greater for p-polarized light (Table IX). Since the dipole momenta of the C-N stretch and N-H bend (amide 11)in an amide linkage are thought to be nearly perpendicular to that of the C=O stretch (amide I),m a corresponding decrease in the amide I intensity is expected, but cannot be clearly established above the noise. Still, since the amide I1 vibration is perpendicular to the axis of a helix, the amide I1 dichroism could indicate a general orientation of adsorbed fibrinogen with helices predominantly parallel to the adsorbing surface. A common model of fibrinogen is that of three balls on a string. The balls are globular domains, while the string consists of two superhelical (a helix of three helices) connecting regions.31 A plausible scenario would have fibrinogen lying flat on the surface of PTMG2000-PEU, with the large superhelices parallel to the surface. On HS-PEU, the faster rate of attachment upon contact could result in the adsorption of "bent" molecules of fibrinogen, with helices oriented in many directions relative to the surface, and thus no consistent orientation. Another concern in these experiments is whether the amide I1 center-of-gravity shifts and the amide 1/11 intensity ratio changes can be ascribed to orientation effects or whether they are indicative of physical changes in the adsorbed fibrinogen. The amide I1 center-of-gravity results are shown in Table X. The center-of-gravity frequency is higher for spectra using p-polarized light. This means that increased orientation perpendicular to the surface could cause an increase in the amide I1 centerof-gravity. However, the largest difference that was observed between spectra at the two polarizations (0.67 cm-1 on PTMG2000-PEU and 1.38 cm-l on HS-PEU) is small compared to the center-of-gravity shifts over the course of each run ( 1.5 cm-l on PTMG2000-PEU and 3 cm-1 on HS-PEU). There is too little data to establish
-
N
(30) Nevskaya, N. A.; Chirgadze, Y. N. Biopolymers 1976, I 4 637. (31) Doolittle, R. F. In Huemostasis and Thrombosis; Bloom, A. L., Thomas, D. P.,Ede.; Churchill-Livingaton:New York, 1987; Chapter 11.
1762 Langmuir,
VoZ. 7, No.8, 1991
Lenk et aZ.
Table X. Differen- in Amide I1 Centersf-Gravity Frequenciem for Spectra of Fibrinogen on Polyurethanes Using Polarized Light
I
Elutable
I
Non-elutable
diff (a=
p-s
PTMG2000-PEU 0.35f0.24 HS-PEU 0.70f 0.46
0.01) yes yes
unpolarized different (Ul-U4) (ar0.01) -0.04f0.09 no 0.02 f 0.22 no AC
whether or not fibrinogen changes orientation with residence time on the surface, but it is also clear that the protein is not completely oriented in any one direction even at long times. Part of the amide I1 center-of-gravity shift can be ascribed to orientation changes, but the bulk of the change is clearly due to other causes. The amide 1/11ratio on PTMG2000-PEU is 1.26 i 0.15 for p-polarized light and 1.48 f 0.16 for s-polarized light (a significant difference at a = 0.025). This is consistent with the amide I1 dichroism observed. However, it provides further evidence against high levels of orientation of the adsorbed protein at long times. The overall amide 1/11 ratio increases with residence time, the opposite of what would be expected for increasing orientation and stronger interaction with p-polarized radiation. A similar difference in amide 1/11ratio is seen on HS-PEU (1.40f 0.18 (p) to 1.59 f 0.30 (s)), but it is not significant with the standard deviations recorded. The amide 1/11ratio for both polarizations is higher on HS-PEU than on PTMG2000-PEU, consistent with the overall values. There does appear to be some orientation of fibrinogen on PTMG2000-PEU, as indicated by dichroic differences in amide I1 intensity, center-of-gravity frequency, and amide 1/11intensity ratio. Similar trends are seen on HSPEU, but the data are much more scattered. There is too little data to indicate clearly whether orientation of the adsorbed fibrinogen increases with time after the first measurements. The SDS studies indicate that there is a period of about 1 h where nearly all of the adsorbed fibrinogenon PTMG2000-PEU exists in a "loosely bound" state, which could provide an opportunity for rearrangement of protein on the surface. Since the first polarized measurements were made after 3 h of residence time, it is impossible to tell whether fibrinogen adsorbs onto PTMG2000-PEU in an oriented manner or whether it rearranges during this initial period of "loose" attachment to produce a more oriented layer. Distribution of Adsorbed States. The relationship of the observed center-of-gravity frequency shift to the amount of protein retained on the surface after SDS exposure is different for the two polyurethanes (Figure 7). On PTMG2000-PEU, the time course of the two events shows excellent correspondence. On HS-PEU, the time course of the amide I1 peak shift lags considerably behind the observed change in fibrinogen retention. This inconsistency appears to be due to the differences in the measurement techniques used. The percentage of protein retained after SDS exposure measures changes only in specific proteins-those that have gone from a state in which they were removable to a state in which they are not removable. Infrared analysis, on the other hand, measures an average change for all of the molecules present on the surface. Keeping these differences in mind, the apparent discrepancies can be explained by viewing the protein layer on the surface as composed of a continuous distribution of adsorbed states. The concept of multiple adsorbed states for proteins on surfaceswas proposed by Soderquist and Walton in 1980,lO yet most models of protein adsorption continue to make use of just two states, "reversibly" and "irreversibly" ad-
Adsorbed State
Figure 9. Illustration of a continuous distribution of binding states for adsorbed protein. The gray area represents protein that would be retained after exposure to a removing agent.
sorbed. This is probably a great oversimplification of the real situation, as it has been shown that the concept of reversible adsorption depends on the means of perturbation of the adsorbed protein. For example, proteins that appear irreversibly adsorbed when exposed to buffer solution can be readily displaced by protein in solution32 or removed by exposure to surfactant solution^.^*^ However, because most experiments involve only one method of measuring protein changes and because most methods determine only if protein is or is not "changed" (on/off tests), the two-state model (an on/off model) continues to be widely used. A hypothetical normal distribution of adsorbed states on a surface is shown in Figure 9. The distribution is characterizedby a particular mean and standard deviation, and ranges from zero (completely removable protein, even by simpleexposure to buffer) to infinity (the protein cannot be removed from the surface). At some point, depending on the method of removal, there exists a critical adsorbed state (4).Proteins in a binding state below 4 will be removed by a given perturbation, and those in an adsorbed stateabove 4 will remain on the surface. Increases in the amount of "irreversibly bound" protein could come about by various types of changes in the distribution with time, including broadening, shape changes, and shifts in the entire distribution with respect to &. As a first approximation, we will examine broadening of the distribution to illustrate how this idea affects the interpretation of different types of data. Since infrared spectroscopy measures an average value of the state of all molecules on a surface, an infrared parameter that represents the binding of protein to a surface would correspond to the mean of this distribution. Measurement of removal of protein by a specific agent, on the other hand, would providea measurement of how much protein was located above and below the "critical adsorbed state" corresponding to the particular method of removal. Because we are assuming that one limit of the distribution always corresponds to completely removable protein (the distribution changes only by broadening), the mean and standard deviation of the distribution are related. By assuming a shape for the distribution, one can predict changes in either the mean of the distribution or the fraction of area above the critical adsorbed state by specifying the other. Because of the apparent strong relationship between the shift in amide I1center-of-gravity frequency for fibrinogen on polyurethanes and the elutability of the fibrinogen by SDS, we will use the elution results for fibrinogen on PTMG2000-PEU and HS-PEU to illustrate the expected behavior of the center-of-gravity shift on those same surfaces for three different distributions. (32)Brash, J. L.;Samak, Q. L. J. Colloid Interface Sci. 1978,e (3), 495.
IR Studies of Adsorbed Fibrinogen
Langmuir, Vol. 7, No. 8, 1991 1763 T e u)
6
-
zb
5. C
a 0
10
20
30
40
50
0
10
Time (hours)
20
30
40
50
Time (hours)
c : e u)
6
E 5
-1 2
-10
* % Retained
C
m
; 0
10
20
30
40
50
0
Time (hours)
IC
1 .o
C
- 0.6
20
a
d
e
.-0
e
Q)
* %Retained
10
+
-0.4
Mean
10
20
30
C
0
a w
Y
C
m
0 0
Q)
40
50
f
Time (hours)
20
30
40
-08 -06
50
Time (hours) 90
.--m
30 0
e
10
-
40
.-m
+ Mean
80
v Q)
.-m
C
70 60 -45
e
a 50 40
U --t
30
% Relained
-2
Mean
20
0
10
20
30
40
50
Time (hours)
Figure 10. Correspondence of chan es in the mean and "tightly bound" fraction for models of adsor%edfibrinogen distribution on PTMG2000-PEU (a) mean from normal distribution; (b) mean from y distribution;(c)mean fromexponentialdistribution. The relative shapes of the "mean" curves can be compared to the amide I1 center-of-gravityshift in Figure 7a.
For demonstration purposes, normal, exponential, and y distributions of the adsorbed states were assumed. In Figure 10, the shift in the mean (corresponding to the
infrared measurements) is compared to the change in the area beyond an arbitrarily set critical adsorbed state (the "tightly bound" fraction measured by elutability) over the range of elutability values observed in the actual experiments with PTMG2000-PEU. There is good correspondence between the time rate-of-change of the mean and bound fraction for all three of the distributions, much like the actual results for the center-of-gravity and bound fraction on PTMG2000-PEU (Figure "a). In Figure 11, a similar comparison is made for HS-PEU. A lag in the time behavior of the mean with respect to the bound fraction is observed with all three distributions, although none of them has the same magnitude as the actual results on HS-PEU (Figure 7b). The explanation for this lag is most easily understood by looking a t the normal distribution. In the early stages of change (as the tail of the distribution begins to creep past &) small shifts in the mean of the distribution are accompanied by small changes in the area beyond &. This leads to an approximate correspondence of the time rate of change of the twovalues, the result seen with PTMG2000-PEU. However, at intermediate values of area beyond &, when the mean (the maximum of the normal distribution) is near 4,a
Figure 11. Correspondence of chan e8 in the mean and "tightly bound" fraction for models of adsor%edfibrinogen distribution
on HS-PEU:(a)mean from normal distribution; (b) mean from y distribution; (c) mean from exponential distribution. The relative shapes of the "mean" curves can be compared to the amide I1 center-of-gravityshift in Figure 7b. small change in the mean will result in a large change in the area above 4. This causes a lag in the time change behavior of the mean relative to the area beyond &. Finally, at high values of area beyond &there will be very little additional area that can extend beyond &,while the mean can still increase indefinitely. This leads to a lag in the time change behavior of the area beyond & relative to changes in the mean and results graphically in the separation of the lines describing the changes in the two quantities when the endpoints are pinned. This is the effect seen on HS-PEU. A similar argument applies to the other distributions. The concept of a distribution of binding states for adsorbed protein on a surface offers a reasonable explanation of the differences in the correspondence of the elution and infrared results for fibrinogen on PTMG2000-PEU and HS-PEU. There are some difficulties in applying this concept. The most notable of these is measuring the shape of the actual distribution of adsorbed states on a surface. There are also no firm grounds for expecting a unimodal distribution, and the distribution may have more than one maximum at which proteins are in semistable states. Also, although the adsorbed state will have some relationship to the energy of interaction of the protein and the surface, the use of chemical removal agents may introduce steric as well as energetic factors.
1764 Langmuir, Vol. 7, No. 8, 1991
Summary and Conclusions On both HS-PEU and PTMG2000-PEU, the fraction of adsorbed fibrinogen resistant to removal by SDS increases with residence time in buffer. Infrared experiments show a shift in center-of-gravity of the amide I1 band of adsorbed fibrinogen with residence time. A comparison of fitted bands in the amide I region at 1 and 40 h on both surfaces is indicative of a loss of helical and random structure with time on both surfaces, and an increase in &structures, lending support to the hypothesis that the observed increases in protein attachment are accompanied by, and perhaps facilitated by, changes in secondary structure. Since both polymers would be expected to provide a somewhat hydrophilic surface upon hydration, the differences in initial elutability could be due to hydrogen bonding of the protein to N-H and carbonyl groups on the polymer surface. HS-PEU, which cannot phase-separate, would be expected to have high urethane and urea levels a t the outermost surface, providing multiple sites for hydrogen bonding. The PTMG2000-PEUI on the other hand, has been shown to have a surface dominated by the polyether soft segment under vacuum (Tables I and 11), although the resence of some hard segment in the outermost 15 on similar polymers has been shown by matrix secondary ion mass s p e ~ t r o m e t r y .A~plasticized ~ of hard and soft segment may exist on the surface of the polymer in the hydrated state, with the hard segment domains not as accessible as on the entirely hard segment polymer. This would lead to lower amounts of protein being tightly attached upon initial adsorption, until the
1
(33) Hearn, M. J.; Ratner, B. D.; Briggs, D. Macromolecules 1988,21, 2960.
Lenk et al.
polymer surfacecould rearrange in response to the presence of protein. Using both the infrared and elutability results, it is possible to construct a model for protein adsorption on the two polyurethanes tested. On HS-PEU, the first protein contacting the surface would attach tightly, probably bonding at multiple sites, perhaps replacing part of the hydration shell with bonds to the polymer surface. Later protein adsorbed on top of this would be attached more loosely but, over time, would also undergo structural changes and be able to bind more tightly. Little of the initial protein adsorbed on PTMG2000-PEU is adsorbed tightly. It either adsorbs in aspecific orientation (perhaps it is only held tightly enough in one specific orientation), or it rearranges to a more ordered form after adsorption. Such rearrangement could be aided by the lack of strong interactions with the surface. Over time, the protein undergoes structural rearrangement and attaches more tightly, perhaps aided by rearrangement of the polymer to expose more urea and urethane linkages for multiple hydrogen bonds. As the hard segment components come into contact with the protein, they could be “locked”into place through favorable interactions with the protein, eventually forming a surface that could hold much of the adsorbed fibrinogen very strongly.
Acknowledgment. The authors gratefully acknowledge Dr. Steven Slack for preparation of the fibrinogen used in this work and Dr.Ron Rahman and Ms. Barbara Pederson for synthesis of the polyurethanes. The assistance of Ms. Deborah Leach-Scampavia in the ESCA analysis of the polymer films is also appreciated. The authors acknowledge the support of NHLBI Grants RR01296, HL25951, HL19419, RR01367, and HL38936.
