In Situ Surface Plasmon Resonance Analysis of Dextran Monolayer

Richard A. Frazier, Martyn C. Davies*, Gert Matthijs, Clive J. Roberts, Etienne Schacht, Saul J. B. Tendler, and Philip M. Williams. Laboratory of Bio...
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Langmuir 1997, 13, 7115-7120

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In Situ Surface Plasmon Resonance Analysis of Dextran Monolayer Degradation by Dextranase Richard A. Frazier,† Martyn C. Davies,*,† Gert Matthijs,‡ Clive J. Roberts,† Etienne Schacht,‡ Saul J. B. Tendler,† and Philip M. Williams† Laboratory of Biophysics and Surface Analysis, Department of Pharmaceutical Sciences, The University of Nottingham, University Park, Nottingham NG7 2RD, U.K., and Biomaterial and Polymer Research Group, Laboratory of Organic Chemistry, University of Gent, Krijgslaan 281, B-9000 Gent, Belgium Received April 14, 1997. In Final Form: September 15, 1997X Surface plasmon resonance (SPR) has been employed to investigate the hydrolytic degradation of thiolated dextran monolayers on silver by the enzyme dextranase. The influence of pH upon the kinetics of this enzyme-catalyzed degradative reaction has been demonstrated, and SPR experiments reveal that dextranase does not completely remove the thiolated dextran monolayers, even at the enzyme’s most active pH. X-ray photoelectron spectroscopy and atomic force microscopy results support this observation, as do SPR measurements of protein adsorption to degraded samples, which show significant protein resistance after degradation. It is suggested that incomplete degradation occurs due to an inability of the enzyme to process, or complex with, the derivatized thiol bound portions of individual dextran macromolecules close to the silver surface.

Introduction The enzyme-catalyzed hydrolytic degradation of polymers is of wide commercial interest,1,2 particularly as enzymes can play an important role in the industrial processing of polymeric materials. Of equal importance is the study of the biodegradation of potential biomedical materials in vivo,3,4 as this has implications for applications such as implantation, or as a matrix for site-specific drug delivery systems.5 Dextrans are prone to enzymatic hydrolysis by dextranases via the endo mode of attack with subsequent cleavage at 1,6-R-glucosidic linkages.6 The endo mode is essentially a random attack along the length of the polysaccharide chain to produce a polydisperse mixture of lower molecular weight oligosaccharides.2 Such biodegradability of dextran has important consequences for its application in vivo, where degradable dextran hydrogels form a promising basis for site-specific drug delivery systems,7-9 particularly those targeted for release to the human colon,10,11 as dextranases are present in this region of the gastrointestinal tract. Dextranase metabolism of dextran is also of clinical importance in the prevention of dextran-based dental plaques.12,13 * To whom correspondence should be addressed at The University of Nottingham. † The University of Nottingham. ‡ University of Gent. X Abstract published in Advance ACS Abstracts, November 15, 1997. (1) Huang, J.-C.; Shetty, A. S.; Wang, M.-S. Adv. Polym. Technol. 1990, 10, 23-30. (2) Timmins, M. R.; Lenz, R. W. Trends Polym. Sci. 1994, 2, 15-19. (3) Stokes, K. Cardiovasc. Pathol. 1993, 2, 111S-119S. (4) Williams, D. F.; Zhong, S. P. Int. Biodeterior. Biodegrad. 1994, 34, 95-130. (5) Langer, R. Acc. Chem. Res. 1993, 26, 537-542. (6) Park, K.; Shalaby, W. S. W.; Park, H. Biodegradable Hydrogels for Drug Delivery; Technomic Press: Basel, 1993. (7) Nielsen, L. S.; Weibel, H.; Johansen, M.; Larsen, C. Acta Pharm. Nord. 1992, 4, 23-30. (8) Kamath, K. R.; Park, K. Polym. Gels Networks 1995, 3, 243-254. (9) Kurisawa, M.; Terano, M.; Yui, N. Macromol. Rapid Commun. 1995, 16, 663-666. (10) Brøndsted, H.; Hovgaard, L.; Simonsen, L. S. T. P. Pharma Sci. 1995, 5, 60-64. (11) Hovgaard, L.; Brøndsted, H. J. Controlled Release 1995, 36, 159166. (12) Safarı´k, I. Biomed. Biochim. Acta 1990, 49, 625-628.

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Little is known concerning the degradation of surfaceimmobilized dextrans, although modification of dextran is known to have an effect upon the rate and extent of degradation by dextranases.14-16 The presence of derivative functionalities appears to hinder the formation of the intermediary enzyme-substrate complex necessary to allow degradation to occur. It has been hypothesized that this hindrance may be largely a steric effect, though in some cases, as with the presence of substituted poly(ethylene glycol) chains, enzyme-substrate complexation may be prevented by a repulsive interaction between the substituted group and enzyme molecules.16 Surface plasmon resonance (SPR) has been recently demonstrated as an effective technique for the investigation of the extent of nonspecific macromolecular interactions at solid/liquid interfacial boundaries.17-19 SPR is particularly suited to studying systems under flow conditions, where association and dissociation kinetics can be derived from SPR data.20 To date, SPR has been used in combination with atomic force microscopy (AFM) to investigate the degradation of polymer films under various pH conditions.21-23 Here, SPR has been utilized to investigate the pH dependence (13) Willcox, M. D. P.; Patrikakis, M.; Knox, K. W. Aust. Dent. J. 1995, 40, 121-128. (14) Crepon, B.; Jozefonvicz, J.; Chytry, V.; Rihova´, B.; Kopecek, J. Biomaterials 1991, 12, 550-554. (15) Vercauteren, R.; Schacht, E.; Duncan, R. J. Bioact. Compat. Polym. 1992, 7, 346-357. (16) Chiu, H.-C.; Kona´k, C.; Kopeckova´, P.; Kopecek, J. J. Bioact. Compat. Polym. 1994, 9, 388-410. (17) Green, R. J.; Davies, J.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B. Biomaterials 1997, 18, 405-413. (18) Frazier, R. A.; Davies, M. C.; Matthijs, G.; Roberts, C. J.; Schacht, E.; Tasker, S.; Tendler, S. J. B. In Surface Modification of Polymeric Biomaterials; Ratner, B. D., Castner, D. G., Eds.; Plenum Press: New York, 1996; pp 117-128. (19) Frazier, R. A.; Matthijs, G.; Davies, M. C.; Roberts, C. J.; Schacht, E.; Tendler, S. J. B.; Williams, P. M. Biomaterials, submitted for publication. (20) Fa¨gerstam, L. G.; Frostell-Karlsson, A° .; Karlsson, R.; Persson, B.; Ro¨nnberg, I. J. Chromatogr. 1992, 597, 397-410. (21) Chen, X.; Shakesheff, K. M.; Davies, M. C.; Heller, J.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. J. Phys. Chem. 1995, 99, 1153711542. (22) Shakesheff, K. M.; Chen, X.; Davies, M. C.; Domb, A.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Langmuir 1995, 11, 3921-3927. (23) Chen, X.; Davies, M. C.; Roberts, C. J.; Shakesheff, K. M.; Tendler, S. J. B.; Williams, P. M. Anal. Chem. 1996, 68, 1451-1455.

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of the degradation of thiolated dextran monolayers by dextranase. Thiolated dextran monolayers are known to be protein resistant;18,19 therefore, we are also able to assess the extent of degradation by subsequent measurement of protein adsorption within an SPR experiment. Should removal of the thiolated dextran layer occur, then a corresponding decrease in protein resistance would be expected. AFM and X-ray photoelectron spectroscopy (XPS) analyses were also performed upon intact and degraded layers, in order to support the observations by SPR. Experimental Section Thiolated Dextran Samples. 2-Mercaptoethyl carbamoyl dextrans, or thiolated dextrans, were prepared as described elsewhere.19 Thiolated dextrans were prepared with varying degrees of thiol substitution and dextran molecular weights. SPR Analysis of Degradation. Dextranase from a penicillium species was received as a solid (Sigma, Poole, Dorset, U.K.). For SPR experiments, buffered dextranase solutions were prepared by dissolution in aqueous 10 mM sodium phosphate buffers of ranging pH. Each solution was of activity concentration of 6 units/mL, where 1 unit of dextranase liberates 1 µM isomaltose/min from a dextran substrate at pH 6 and 37 °C. The SPR instrument (Johnson & Johnson Clinical Diagnostics, Chalfont St. Giles, Buckinghamshire, U.K.), which has been described in detail elsewhere,17 essentially monitors interactions at, or above, a silver-coated glass sensor surface. Silver is employed as it provides a sharp resonance peak, allowing accurate determination of SPR angle shifts. A monochromatic laser light source at 780 nm wavelength is used. Sample solutions are flowed at a rate of 4 µL/s and are equilibrated within a thermoregulated block at a constant temperature of 34 °C prior to contact with the silver surface. Initially, 1 mg/mL of 4% thiolated 70 kDa dextran solution in 10 mM sodium phosphate buffer at pH 7.4 was passed over the silver surface, followed by two pH 7.4 buffer washes. Two further buffer washes were performed immediately in order to introduce the appropriate system pH for the degradation study. Buffered dextranase solution was then flowed over the surface and followed by two buffer washes at the same pH. Quoted degradation rates were calculated from the maximum initial slope of the SPR profiles by employing a 10 point linear regression function within the SPR instrument software. SPR Measurement of Protein Adsorption Following Dextranase Degradation. Dextranase degradation at pH 7.4 of thiolated dextran monolayers was performed as described above. Immediately following the buffer washes, a pH 7.4 buffered 0.5 mg/mL solution of bovine serum albumin (BSA; Sigma, Poole, Dorset, U.K.) was passed over the degraded surface. Some exchange of unbound dextran for BSA could occur during adsorption, possibly leading to misleading results. Therefore, to probe for the amount of adsorbed BSA, a 10 µL/mL anti-BSA antibody (Sigma, Poole, Dorset, U.K.) solution was flowed over the resulting surface. The anti-BSA antibody specifically binds to adsorbed BSA, the resulting SPR angle shift allowing a more reliable determination of the surface composition than would be achieved by quoting the SPR angle shift for BSA adsorption alone. AFM Analysis. A Rasterscope 3000 AFM (Danish Micro Engineering A/S, Herlev, Denmark) was utilized for contact mode imaging of silver-coated glass slides immediately following SPR experiments, in which a 4% thiolated 70 kDa dextran surface had been exposed to flow of either a blank pH 6 buffer or a dextranase solution at pH 6. XPS Analysis of Degradation. Typically, a 4% thiolated 70 kDa dextran monolayer prepared by incubating a silver-coated glass slide overnight in a 1 mg/mL aqueous solution was analyzed by XPS. Following the XPS analysis from this layer, the sample was removed from the XPS instrument and its surface was then exposed to a 6 units/mL dextranase solution at pH 6 for 5 min. These conditions were chosen for maximum activity of the enzyme, and to mimic the exposure time encountered within SPR experiments. Dextranase solution was then drawn off the surface by capillary action using filter paper and the surface rinsed with deionized water and then dried before acquisition of further XPS spectra.

Figure 1. SPR profiles showing the chemisorption of 4% thiolated 70 kDa dextran to silver (ca. 0-350 s), followed by the flow of blank 10 mM sodium phosphate buffers at pH 3.5 and pH 9.8 over the dextran monolayers (ca. 350-700 s). The spikes in the profiles correspond to buffer washes. Little degradative effect was observed in either extreme case of the pH range studied. XPS spectra were recorded with a VG ESCALAB Mk2 instrument (VG Scientific Ltd., East Grinstead, Sussex, U.K.) employing Mg KR X-rays (hυ ) 1253.6 eV) at two-electron emission angles relative to the sample plane, namely 25° and 75°. The X-ray gun was operated at 10 kV and 20 mA, corresponding to a power of 200 W.

Results and Discussion Experiments were performed by SPR analysis to investigate the pH dependence of degradation, and then the comparative extent of degradation of a range of thiolated dextrans. In the latter case, the extent of protein adsorption to the nondegraded and degraded surfaces was also determined. Initially, experiments employed a 4% thiolated 70 kDa dextran monolayer, as this had been previously shown to form the most stable and highcoverage monolayer.19 pH Dependence of Dextranase Degradation. Dextranase degradation of monolayers of 4% thiolated 70 kDa dextran was monitored at a range of pH values. So that the thiolated dextran monolayer was consistent between experiments, 4% thiolated 70 kDa dextran was always chemisorbed from a pH 7.4 buffered solution. To ensure that the pH environment did not effect the formation and subsequent stability of the 4% thiolated 70 kDa dextran monolayers, control experiments were performed during which monolayers were exposed to a flow of blank buffer at each pH. As can be observed in Figure 1, little effect due to buffer pH at pH 9.8 was observed. A slight negative SPR angle shift was measured during the flow of acidic pH 3.5 buffer; however, the magnitude and rate of the drift were not of a significant level compared to later shifts recorded during dextranase degradation experiments at the same buffer pH. When dextranase was flowed over the 4% thiolated 70 kDa dextran monolayers, a degradative effect was observed at all pH values apart from pH 9.8, where no degradation was detected. SPR profiles are displayed in Figure 2, where in most cases the value of the SPR angle can be observed to sharply decrease upon exposure of the 4% thiolated 70 kDa dextran surface to dextranase. For degradation at pH 6, 7 and 8.2, the value of SPR angle reaches a steady state following this initial decrease, indicating that no further surface degradation has occurred. At pH 3.5 and 4.9, a steady state is not observed, and there appears to be a secondary adsorption stage immediately following the initial surface degradation. This

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Figure 2. SPR profiles showing the degradation of 4% thiolated 70 kDa dextran monolayers by dextranase at pH 3.5, 4.9, 6.0, 7.0, 8.2, and 9.8. A variation in the extent and rate of degradation is detected. Spikes in the profiles correspond to buffer washes.

may occur because the system pH was near to the expected isoelectric point for dextranase (pH 4),24 and hence the enzyme was prone to interact with the degraded surface. In general, however, the magnitude of the negative SPR angle shift and the initial rate of decrease in the value of the SPR angle varied considerably depending upon the pH environment. To visualize the change in the surface due to the degradation process, 4% thiolated 70 kDa dextran layers on silver-coated slides were imaged by AFM following exposure to either a blank pH 6 buffer solution or dextranase at pH 6 within an SPR experiment. Typical AFM images are displayed as 1 µm square scans in Figure 3. Figure 3a shows a dextran layer after exposure to the blank buffer alone; a coverage of smooth globular features was evident, as expected from previous studies.19,25 After exposure to dextranase, however, the layer exhibited evidence of degradation, as in Figure 3b, where rough surface features consistent with either residual material and/or possibly the rough underlying silver surface were revealed.17 Extent of Degradation. Figure 4a displays a graph in which the magnitude of dextranase degradation is plotted against pH. At the most acidic pH 3.5, the average SPR angle shift observed was -50 ( 16 mDA (n ) 30). As the pH was increased, the SPR angle shift for degradation was observed to increase, through -80 ( 15 mDA (n ) 30) at pH 4.9, to a maximum between pH 6 and pH 7, where the shifts were -110 ( 19 mDA (n ) 30) and -107 ( 14 mDA (n ) 30), respectively. This activity peak may be broader than that suggested in Figure 4a, because at pH 4.9 the degradation shift taken before the secondary adsorption stage was in the region of -100 mDA (Figure 2). This would agree more with the expected behavior from solution studies, where the maximum would be between pH 5 and pH 6.24 At further basic pH, the degree of degradation declined sharply until at pH 9.8 no degradation of the surface was detected. In all cases it was noted that degradation of the 4% thiolated 70 kDa dextran monolayer did not proceed to completion, as would have been indicated by a return to (24) Janson, J.-C.; Porath, J. In Methods in Enzymology 8, Complex Carbohydrates; Neufeld, E. F., Ginsburg, V., Eds.; Academic Press: New York, 1966; pp 615-621. (25) Tasker, S.; Matthijs, G.; Davies, M. C.; Roberts, C. J.; Schacht, E.; Tendler, S. J. B. Langmuir 1996, 12, 6436-6442.

Figure 3. The 1 µm square AFM images showing the topography of (a) a 4% thiolated 70 kDa dextran monolayer on a silver SPR slide and (b) an image recorded following degradation by dextranase at pH 6.

an SPR angle value indicative of bare silver. The SPR angle shift for chemisorption of 4% thiolated 70 kDa dextran was 172 ( 18 mDA (n ) 24),19 whereas the maximum negative SPR angle shift of -110 ( 19 mDA at pH 6 indicated incomplete degradation. This value corresponded to a loss of approximately 65% of the original layer, which implied that a significant dextran layer remained at the surface. Indeed, experimental evidence from XPS suggested that dextran remained at the surface following incubation of 4% thiolated 70 kDa dextran with dextranase at pH 6, the most active pH. Figure 5a shows a composite of typical wide scans in the range of 250-550 eV from a nondegraded dextran layer, degraded dextran, and blank silver. These indicate a decrease in the magnitude of the C 1s and O 1s peaks relative to the Ag 3d peaks following degradation, implying a loss of dextran from the surface. However, when the degraded dextran wide scan is compared with that of silver, it is clear that the degradation was incomplete. XPS peak area ratios of Ag 3d5/2:C 1s

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Figure 4. (a) Plot of SPR angle shift (mDA) against pH for the degradation of 4% thiolated 70 kDa dextran monolayers by dextranase. (b) Plot of degradation rate (mDA/s) against pH for the degradation of 4% thiolated 70 kDa dextran monolayers by dextranase.

determined from spectra recorded at 25° and 75° emission angles indicated a decrease in the dextran layer coverage/ thickness after degradation, and further implied that this corresponded only to a partial loss of dextran from the surface. At an emission angle of 25°, the Ag 3d5/2:C 1s peak area ratio was 0.046 from an intact dextran layer and 0.141 following incubation with the enzyme, thereby showing in the latter case a greater contribution from the silver substrate. Similarly at 75° emission, the Ag 3d5/2:C 1s peak area ratio was seen to increase from a prior value of 0.189 up to a value of 0.276 following degradation. The Ag 3d5/2:C 1s peak area ratio for blank silver was 4.216, so while a reduction in the level of carbon at the surface was observed, nevertheless a significant dextran-like layer was still detected following degradation. Closer examination of the C1s region spectra for the intact and degraded dextran layers, shown in Figure 5b,c, supports the presence of a dextran-like layer following degradation. Two dominant carbon species are expected from dextran, being single-bonded C-O and O-C-O in a 5:1 ratio. Minor contributions from the 2-mercaptoethyl carbamoyl tethers of aliphatic (C-N and C-S) and carboxyl species in a 2:1 ratio are also expected. However, these would contribute in the order of only 1-2% of the total C1s intensity, since they are present in only 4% of the glucose subunits of the thiolated dextran. Indeed, in the two C1s spectra shown, three major peaks may be resolved at 285.0, 286.7, and 288.3 eV, respectively. The

Figure 5. (a) Overlay of wide scan XPS spectra (250-550 eV) for blank silver, a degraded dextran layer, and an intact thiolated dextran layer, showing the C 1s, Ag 3d and O 1s peaks. (b) Fitted single-region C 1s spectrum from a 4% thiolated 70kDa dextran monolayer before degradation. (c) Fitted single-region C 1s spectrum from the same sample following degradation with dextranase at pH 6 for 5 min.

aliphatic carbon peak at 285.0 eV comprises a significant amount of residual surface contamination, in the order of 25% of the overall C1s intensity before degradation, rising to nearer 38% following degradation. In contrast, the intensities of the C-O peak at 286.7 eV and O-C-O peak at 288.3 eV were seen to decrease in relation to the aliphatic peak after degradation, which would tally with a loss of some of the dextran layer. Indeed, the remaining presence of the hydrolysable single-bonded O-C-O at 288.3 eV strongly suggests the presence of at least an oligosaccharide layer at the surface. The observation of incomplete activity may be due to inhibition of the formation of an enzyme-substrate complex by the presence of substituted 2-mercaptoethyl carbamoyl functionality.14-16 This could be ascribed to either a steric effect, or an inability of the enzyme to recognize, or process, the substituted dextran. An alter-

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Table 1. SPR Angle Shifts and Degradation Rates Calculated from SPR Profiles pH

SPR angle shift for degradation (mDA, n ) 30)

degradation rate (mDA/s, n ) 30)

3.5 4.9 6 7 8.2 9.8

-50 ( 16 -80 ( 15 -110 ( 19 -107 ( 14 -37 ( 6 3(4

-6.9 ( 1.4 -8.8 ( 1.8 -8.6 ( 1.4 -5.3 ( 1.1 -0.5 ( 0.1 0.1 ( 0.2

native explanation may be that the individual dextran molecules indeed suffered degradation and depolymerization, leaving thiol-anchored oligosaccharides bound at the surface. The enzyme may not be conformationally able to bind and cleave these final oligosaccharides from their 2-mercaptoethyl carbamoyl tether, as steric hinderance will play a role near the silver surface. Either explanation is feasible, though in the first instance the thiol species are likely to be oriented away from the accessible dextran surface at the silver-dextran interface; hence enzyme-substrate complexation should not have been hindered by its presence. Rate of Degradation. A dependence upon pH of the observed maximum rate of degradation, seen by the decrease in SPR angle, was also observed. In these experiments, the degradation kinetics include a ratelimiting contribution from diffusion, as the enzyme must complex with the substrate before hydrolytic degradation may begin.6 Therefore, intrinsic rate constants cannot be directly quantified,26,27 as would be possible for dissociation or desorption with no solute present in the flowing solution.20,26 However, a qualitative comparison of degradation rates may be made, since each SPR experiment was subject to the same diffusion limits, as identical enzyme concentrations, flow rates, and flow cell geometries were employed. Figure 4b shows a plot of the degradation rate, measured from the steepest SPR degradation profile gradients, against system pH. These data are also summarized in Table 1 and indicate a clear change in the rate of degradation as pH is varied. The pattern of variation observed was similar to that for the extent of degradation, although the peak rate occurred at a slightly lower pH than at which maximum activity was observed in Figure 4a. Indeed, between pH 4.9 and pH 6, the degradation rate increased to a maximum, while more acidic or basic pH saw a decline in the rate of degradation. The observation of a lower peak in the degradation rate compared to Figure 4a may be easily rationalized, particularly with reference to Figure 2, as the measurement of the degradation rate was not subject to the same isoelectric effects as were the final SPR angle shifts observed. Dependence of Thiolated Dextran Degradation and Subsequent Protein Adsorption upon Macromolecular Properties. To further investigate the degradation of thiolated dextrans, experiments were undertaken in which 1% thiolated 5 kDa dextran, 2% thiolated 70 kDa dextran, 4% thiolated 70 kDa dextran, and 4% thiolated 500 kDa dextran were each studied for their dextranase degradation. For simplicity, and to allow subsequent investigation of protein adsorption properties, these experiments were performed employing pH 7.4 sodium phosphate buffer. Dextranase Degradation of Thiolated Dextran Monolayers. Figure 6a and Table 2 display SPR angle shift (26) Karlsson, R.; Michaelsson, A.; Mattsson, L. J. Immunol. Methods 1991, 145, 229-240. (27) Davies, J. Nanobiology 1994, 3, 5-16.

Figure 6. (a) Bar graph displaying the SPR angle shift data arising from the degradation of (A) 1% thiolated 5 kDa dextran, (B) 2% thiolated 70 kDa dextran, (C) 4% thiolated 70 kDa dextran, and (D) 4% thiolated 500 kDa dextran by dextranase at pH 7.4. (b) Bar graph comparing the SPR angle shifts arising from anti-BSA antibody detection of BSA protein adsorption to intact and degraded (A) 1% thiolated 5 kDa dextran, (B) 2% thiolated 70 kDa dextran, (C) 4% thiolated 70 kDa dextran, and (D) 4% thiolated 500 kDa dextran layers. Table 2. SPR Angle Shift Data for the Degradation of Thiolated Dextran Monolayers and Their Subsequent Adsorption of BSA

thiolated dextran sample (A) 1% thiolated 5 kDa dextran (B) 2% thiolated 70 kDa dextran (C) 4% thiolated 70 kDa dextran (D) 4% thiolated 500 kDa dextran

SPR angle shift for dextranase degradation (mDA, n ) 12)

SPR angle shift for anti-BSA antibody binding to adsorbed BSA (mDA, n ) 12)

-25 ( 5

117 ( 20

-52 ( 5

221 ( 27

-40 ( 5

149 ( 14

-54 ( 5

160 ( 21

data from the degradation stage of each experiment, from which it is apparent that each thiolated dextran of differing molecular weight degraded to a different extent. The greatest degradation shifts were for 2% thiolated 70 kDa dextran and 4% thiolated 500 kDa dextran. A lower SPR angle shift was measured for degradation of 4% thiolated 70 kDa dextran, while 1% thiolated 5 kDa dextran exhibited the least degradation and lowest SPR angle shift. Relative to the SPR angle shifts for the chemisorption of each thiolated dextran,19 approximately 20% of the 1% thiolated 5 kDa dextran layer was lost, compared to nearer 25% of the 4% thiolated 70 kDa dextran layer. Both the

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2% thiolated 70 kDa and 4% thiolated 500 kDa dextran layers lost approximately 35% of the original layer through degradation. From inspection of the trend between individual thiolated dextrans, it was of interest to observe that the relative degradation of each surface could be related to their previously determined protein adsorption properties.18,19 Therefore, we suggest that the thiolated dextrans with the greatest resistance to nonspecific protein adsorption additionally exhibit the most resilience to enzymatic hydrolysis and vice versa. This trend may be as a result of the relative efficiency of the thiol-binding reaction with the silver substrate. Thiol binding to the silver substrate is hindered less for a lower molecular weight thiolated dextran;25 hence 1% thiolated 5 kDa dextran forms the greatest density coverage of thiolate bonds at the silver surface. This presents a lower proportion of the bound dextran layer that may dissociate from the surface during degradation. At the other extreme, thiol access to the silver surface is likely to be considerably hindered during chemisorption of the high molecular weight 4% thiolated 500 kDa dextran. Therefore, a less efficiently bound layer results with a more sparse distribution of silver-thiolate bonding, which consequently allows a greater fraction of the dextran layer to dissociate during degradation. The behavior of the two 70 kDa thiolated dextrans would seem to agree with this argument, as the SPR angle shift for degradation was less for the 4% thiolated 70 kDa dextran, which should produce a more densely bound layer, than for the 2% thiolated 70 kDa dextran. Protein Adsorption to Degraded Thiolated Dextran Layers. It has been argued above that a significant thiol bound oligosaccharide layer remains following degradation by dextranase. If this is indeed the case, then a hydrophilic layer should remain that is resistant to protein adsorption. To investigate this possibility, protein adsorption was monitored following dextranase degradation of the thiolated dextran monolayers, and the results are summarized in Table 2. Figure 6b shows a histogram comparing the SPR angle shifts resulting from anti-BSA antibody detection of BSA adsorption to intact thiolated dextran layers19 and to degraded layers (Table 2). It was immediately apparent in all cases that degradation resulted in a 2- to 3-fold increase in the level of antibody adsorption. This indicated a significant loss of dextran from the surface. However, it did not suggest a complete absence of a protein resistant layer, as a protein monolayer was not detected, the presence of which would result in an SPR angle shift for anti-BSA antibody of 332 ( 32 mDA (n ) 18). A comparison of the antibody adsorption shifts to each layer revealed the same trend for protein adsorption to both intact and degraded layers. This common trend suggests that the relative surface coverages of bound material are consistent both prior to and after degradation. Therefore, these results would support the inference that surface coverage is the primary factor that determines protein resistance.

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Conclusions The application of SPR analysis has enabled the in situ investigation of the enzymatic hydrolysis of thiolated dextrans by dextranase. The versatility of the SPR technique has allowed the observation of a pH dependence for both the rate and extent of surface degradation within the same experiment. By inspection of the rate of degradation measured from SPR profiles, an optimum activity of dextranase has been observed to reside in the region from pH 4.9 to pH 6, which agrees well with expectations from the literature of degradation in solution (maximum in region of pH 5-6).24 However, our results suggested that complete removal of thiolated dextran layers did not occur, even at the optimum activity pH values. This was shown by the magnitude of the SPR angle shifts for degradation and by XPS and AFM analysis of monolayers prior to and after degradation. Incomplete degradation was proposed to occur either due to a steric effect of the thiol binding at the silver surface, preventing access and/or recognition, necessary for the formation of a dextranase-dextran complex or due to depolymerized portions of dextran remaining bound at the surface via thiol tethers. Further SPR experiments suggested that the extent of removal of thiolated dextran layers from the silver substrate depended upon the same factors as did their previously studied protein resistance.18,19 It is supposed that by increasing the degree of thiol substitution at a constant dextran molecular weight, a more stable layer was presented, which was less susceptible to enzymatic hydrolysis. However, increasing the dextran molecular weight may have a more pronounced effect, as the lowest molecular weight 1% thiolated 5 kDa dextran exhibited the least degradation, despite a low proportion of thiol substituents. Conversely, the highest molecular weight 4% thiolated 500 kDa dextran degraded to the greatest magnitude, despite a high degree of thiol substitution. This effect could be attributed to hinderance of enzyme access to near the surface of a tightly bound layer as opposed to that of a more diffuse higher molecular weight monolayer, where greater accessibility may be allowed. Measurement of the protein resistance of degraded layers inferred that a hydrophilic and strongly bound surface layer remained following degradation. This agreed with earlier assumptions from the magnitude of negative shifts following degradation, which consistently fell short of those expected for loss of a complete layer. Considering the results from XPS, it appeared that this layer was most likely of a mono- or oligosaccharide nature, which is presumed to offer a partial hydrophilic steric barrier to protein adsorption. Acknowledgment. The authors thank the European Community Brite Euram Programme for support. R.A.F. thanks Courtaulds PLC for funding his Ph.D. Research Studentship. S.J.B.T. is a Nuffield Foundation Science Research Fellow. LA970382V