Kinetics and Mechanism of Substitution Reactions between Myoglobin

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Kinetics and Mechanism of Substitution Reactions between Myoglobin and Activated Self-Assembled Monolayers Investigated Using Surface Plasmon Resonance Alice A. Deckert,* Jennifer Lesko, Stephanie Todaro, Margaret Doyle, and Christine Delaney Department of Chemistry, Allegheny College, Meadville, Pennsylvania 16335 Received March 21, 2002. In Final Form: May 30, 2002 The rate of the reaction between solution-phase myoglobin and SAMs fabricated from ω-X-hexadecylthiomethylbenzenes where X was a mesylate, chloride, or bromide was investigated using surface plasmon resonance (spr). The data were fit to a model in which nonspecific adsorption competes with a second-order substitution reaction between myoglobin and the surface species. The second-order reaction rate constants obtained from the regression analysis were 440 ( 140 M-1s-1 for the bromide, 56 ( 10 M-1s-1 for the mesylate, and 38 ( 12 M-1s-1 for the chloride. The experimental rate law fit to the data and the increase in rate with decreased basicity of the leaving group were consistent with an SN2 mechanism.

Introduction Investigations of the immobilization of proteins and other biologically relevant molecules to solid substrates have increased dramatically during the past decade.1-4 Specifically, the possibility of forming biosensors from biomolecules covalently bound to suitable organic thin films is of increasing interest, and numerous studies have addressed some of the major issues inherent in covalent immobilization of large molecules to surfaces.4-15 This interest is sparked, in part, by the inherent stability of covalent bonds.5,6 Each biosensing application requires a unique recognition system. Therefore, researchers must find novel methods for protein immobilization each time a new assay is developed. Thus, the development of a simple and general method for the fabrication of robust devices * To whom correspondence should be addressed. E-mail: [email protected]. (1) Scouten, W. H.; Luong, J. H. T.; Brown, S. R. Tibtech 1995, 13, 178. (2) Ostuni, E.; Yan, L.; Whitesides, G. M. Colloids Surf., B 1999, 15, 3. (3) Ferretti, S.; Paynter, S.; Russell, D. A.; Sapsford, K. E.; Richardson, D. J. Trends Anal. Chem. 2000, 19, 530. (4) Chechik, V.; Crooks, R. M.; Stirling, C. J. M. Adv. Mater. 2000, 16, 1161. (5) Okuso, H.; Kurihara, K.; Kunitake, T. Langmuir 1994, 10, 3577. (6) Lee, S.; Anzai, J.; Osa, T. Bull. Chem. Soc. Jpn. 1991, 64, 2019. (7) Fryxell, G. E.; Rieke, P. C.; Wood, L. L.; Englehard, M. H.; Williford, R. E.; Graff, G. L.; Campbell, A. A.; Wiacek, R. J.; Lee, L.; Halverson, A. Langmuir 1996, 12, 5064. (8) Templeton, A. C.; Hostetler, M. J.; Kraft, C. T.; Royce, M. W. J. Am. Chem. Soc. 1998, 120, 1906. (9) Van Ryswyk, H.; Turtle, E. D.; Watson-Clark, R.; Tanzer, T. A.; Herman, T. K.; Chong, P. Y.; Waller, P. J.; Taurog, A. L.; Wagner, C. E. Langmuir 1996, 12, 6143. (10) Caruso, F.; Rodda, E.; Furlong, D. N. J. Colloid Interface Sci. 1996, 178, 104. (11) Guiomar, A. J.; Guthrie, J. T.; Evans, S. D. Langmuir 1999, 15, 1198. (12) Willner, I.; Lapidot, N.; Riklin, A.; Kasher, R.; Zahavy, E.; Katz, E. J. Am. Chem. Soc. 1994, 116, 1428. (13) Tatsuma, T.; Tsuzuki, H.; Okawa, Y.; Yoshida, S.; Wtatnabe, T. Thin Solid Films 1991, 202, 145. (14) Anzai, J.; Lee, S.; Osa, T. Chem. Pharm. Bull. 1989, 37, 3320. (15) Karymov, M. A.; Kruchinin, A. A.; Tarantov, Y. A.; Balova, I. A.; Remisova, L. A.; Sukhodolov, N. G.; Yanklovich, A. I.; Yorkin, A. M. Sens. Actuators, B 1992, 6, 208.

employing a wide range of biological recognition systems is needed. All general methods that have been developed to date have their own advantages and disadvantages. Some covalent immobilization techniques provide strong bonds for robust devices but require multiple fabrication or synthesis steps.13-17 Physical adsorption techniques are general and simple but do not always allow for control of protein coverage or orientation and result in less robust devices because of protein leaching.18-20 Several strategies have been developed that employ strong interactions between a protein and a surface bound species that result in robust devices and allow for control of protein coverage but require protein engineering.21-23 Strong ligand-acceptor interactions can be employed to fabricate robust devices but can often only be applied to a limited set of recognition systems.24-26 Unfortunately, both the development of general methods and the application of existing methods for protein immobilization are hampered by a lack of basic understanding of the reactions and interactions that occur at thin-film and matrix surfaces. To date, very few comprehensive studies of the kinetics and mechanism of covalent reactions between proteins and thin films have been carried out.7-9 The work detailed in this paper will provide fundamental knowledge required for the rational design of more sensitive and selective biosensors. (16) Gregorius, K.; Mouritsen, S.; Elsner, H. I. J. Immuological Methods 1995, 181, 65. (17) Willner, I.; Riklin, A. Anal. Chem. 1994, 66, 1535. (18) Hamachi, I; Noda, S.; Kunitake, T. J. Am. Chem. Soc. 1990, 112, 6744. (19) Decher, G. Science 1997, 277, 1232 (20) Lvov, Y. M.; Lu, Z.; Schenkman, J. B.; Zu, X.; Rusling, J. F. J. Am. Chem. Soc. 1998, 120, 4073. (21) Firestone, M. A.; Shank, M. L.; Sligar, S. G.; Bohn, P. W. J. Am. Chem. Soc. 1996, 118, 9033. (22) Samuelson, L. A.; Kaplan, D. L.; Lim, J. O.; Kamath, M.; Marx, K. A.; Tripathy, S. K. Thin Solid Films 1994, 242, 50. (23) Sigal, G. B.; Bamdad, C.; Barberis, A.; Strominger, J.; Whitesides, G. M. Anal. Chem. 1996, 68, 490. (24) Owaku, K.; Goto, M.; Ikariyama, Y.; Aizawa, M. Anal. Chem. 1995, 67, 1613. (25) Lahiri, J.; Isaacs, L.; Grzybowski, B.; Carbeck, J. D.; Whiteside, G. M. Langmuir 1999, 15, 7186. (26) Reichert, A.; Nagy, J. O.; Spevak, W.; Charych, D. J. Am. Chem. Soc. 1995, 117, 829.

10.1021/la025767e CCC: $22.00 © 2002 American Chemical Society Published on Web 09/17/2002

Myoglobin and Activated Self-Assembled Monolayers

Our approach is to employ well-known nucleophilic substitution chemistry on a thin film that contains a good leaving group such as mesylate, bromide, or chloride. This film is susceptible to attachment of nucleophilic substrates, such as amino acids with thiol groups in their side chains.7 Since several thiol groups are likely to be accessible on the surface of any given protein or biological recognition system, we can, in principle, attach nearly any polypeptide with this approach. We have chosen to start our kinetic and mechanistic investigation with myoglobin because we have experience with this protein and have already demonstrated a specific interaction with mesylate surface groups.27 Experiments performed as a function of film composition showed that the coverage of myoglobin on the film was dependent on the percentage of docosylmesylate (CH3(CH2)11OSO2(CH3)) contained in the film. We achieved optimal myoglobin attachment with film compositions between 22% and 30% docosylmesylate. We hypothesized that steric hindrance of the surface-bound active sites at higher site densities accounted for the observed maximum myoglobin coverage for the films containing between 22% and 30% docosylmesylate.27 We have also shown that myoglobin retains redox activity when immobilized on films consisting of 25% docosylmesylate.28 These initial studies have shown that substitution reactions can be used as a potentially general method for the attachment of proteins to surfaces without loss of protein activity. However, our preliminary results could not answer outstanding questions concerning the kinetics and mechanism of the reaction between myoglobin and the thin film that are addressed in this work. Extension of the preliminary study to measure the rate constants for reaction and to include other primary functional groups has allowed us to elucidate some details of the mechanism for binding of myoglobin to these thin films. A leaving group that is a weaker base than mesylate should result in a faster substitution reaction. Our studies using primary alkyl halides (R-Cl, R-Br) and a primary mesylate (R-OSO2CH3) show that the reaction rate increases with decreasing basicity of the leaving group. In addition, the integrated rate law fit to the data is consistent with a second-order reaction step. These results indicate that the reaction occurs through an SN2 reaction mechanism. Experimental Section spr Response Curves. All spr response curves were obtained using a Nomadics miniature integrated spr sensor with temperature compensation and flow cell. The gold surface was cleaned by successive applications of 0.5% Triton 100-X solution in 0.10 M aqueous NaOH solution prior to monolayer formation. When successive rinses with deionized water (resistivity better than 17.5 MΩ-cm) showed an spr angle within 0.005% of each other, the gold surface was considered to be clean. A 2 mM solution of the desired monolayer molecule in ethanol was then flowed over the surface for between 60 and 120 s. A final rinse with deionized water produced a consistent change in the spr angle of about 0.23 ( 0.07 rad. This angle change was consistent with a layer thickness of 2.2 nm which is approximately equal to the expected thickness (2.1 nm) of a monolayer assembled from 16-bromohexadecylthiomethylbenzene (BHTB), 16-mesylhexadecylthiomethylbenzene (MHTB), or 16-chlorohexadecylthiomethylbenzene (CHTB) assuming a tilt angle of about 25° off vertical.3 After successful formation of the monolayer on the gold surface, the monolayer was exposed to a solution of crystallized and lyopholized horse skeletal muscle myoglobin from Sigma (95(27) Deckert, A. A.; Farrell, C.; Roos, J.; Waddell, R.; Stubna, A. Langmuir 1999, 15, 5578. (28) Janesko, B. G.; Deckert, A. A. In preparation.

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Figure 1. A representative spr response curve for one experiment is shown. A monolayer of CHTB had been previously assembled on the gold surface of the spreeta sensor. At the time indicated on the figure, the TRIS buffer (pH 7.0) solution was replaced with a solution containing 162 µM myoglobin in TRIS buffer. Once the spr response leveled out, TRIS buffer (pH 7.0) was again introduced into the flow cell at the time indicated. The difference in the spr angle (∆θnet) indicated on the figure was proportional to the final surface coverage of myoglobin. 100%) in TRIS buffer at pH 7.2 at room temperature (17-20° C). The spr response curves were obtained for a 100-fold variation in myoglobin concentrations (7.2 µM and 750 µM). The spr response was monitored as a function of time until a constant spr angle was obtained. Finally, the resulting monolayers were exposed to buffer solution and a final deionized water rinse. The net change in the spr angle was observed to be fairly constant for all reactions at about 0.075 rad. A clean gold surface on the spr sensors was regenerated by sonication of the gold surface in 30% peroxide for approximately 1 min. After treatment in peroxide, the spr angle for the clean surface under deionized water was within 0.005% of the angle of the clean surface under water before exposure to the monolayer or myoglobin solutions. Each sensor could be reused up to 3 times before failing to give a consistent spr angle for the clean surface under water. Synthesis of Monolayer Molecules. The ω-X-hexadecylthiomethylbenzenes used to form the monolayers investigated were all synthesized using well-known techniques from the 16mercapto-hexadecanoic acid precursor. The methyl ester of the acid was formed and subsequently reduced to the alcohol with LAH. To make the primary mesylate, bromide, and chloride, the mercapto functionality was protected by benzylation with benzyl chloride. Finally, the alcohol functionality was reacted with either mesyl chloride, thionyl chloride, or thionylbromide to form the mesyl, chloro, or bromo derivatives, respectively. All products were verified by proton NMR and recrystallized prior to use. Details of the syntheses are provided as Supporting Information.

Results Figure 1 shows a representative spr response curve for the entire process from initial buffer response to final buffer response. The time when the myoglobin is introduced, the time when buffer is again introduced, and ∆θnet are indicated in Figure 1. Figure 2a-c shows the spr response to four different bulk concentrations of myoglobin for sensors coated with (a) BHTB, (b) MHTB, and (c) CHTB. The reaction rate clearly increases as a function of the bulk myoglobin concentration for each surface species. The solid lines shown in Figure 2 are least-squares fits to a biexponential function. The three spr response curves shown in Figure 3 are for a bulk myoglobin concentration of 384 µL for different surface-bound species. Figure 3 clearly shows that the

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linear regression Figure 4a SAM bromide mesylate chloride

ka+kr

(M-1s-1)

477 ( 38 191 ( 14 117 ( 19

kd

(x10-2s-1)

3.3 ( 1.1 5.4 ( 0.4 5.2 ( 0.6

quadratic regression Figure 4b kakr

(x103

M-2s-2)

15 ( 4 7.4 ( 0.8 2.9 ( 0.9

kdkr

(M-1s-2)

6.8 ( 2.6 3.0 ( 0.5 2.0( 0.6

calculated values ka

(M-1s-1)

34 ( 10 140 ( 17 80 ( 20

kr (M-1s-1) 440 ( 140 56 ( 10 38 ( 12

Figure 3. The data for a myoglobin concentration of 384 µM are compared for the three surfaces studied. The open circles are the data for the BHTB, the open squares are the data for the MHTB, and the open triangles are the data for the CHTB SAM. The solid lines are the least-squares fits to a biexponential function. The bromo-terminated surface clearly results in a faster response than the mesyl-terminated surface which is faster than the chloro-terminated surface.

Figure 2. The spr response curves for four concentrations of myoglobin are shown for each of the activated SAMs studied. In each plot, the open circles show data for a myoglobin concentration of 600 µM, the open squares show the data for a myoglobin concentration of 384 µM, the open triangles show the data for a myoglobin concentration of 288 µM, and the open diamonds show the data for a myoglobin concentration of 79.4 µM. The solid lines through each data set are the least-squares fit to a biexponential function. The spr response is clearly dependent on myoglobin concentration for all surfaces. (a) BHTB (b) MHBT (c) CHTB.

reaction is fastest with a bromine-terminated surface and slowest with a chlorine-terminated surface. These observations are consistent with the better leaving group (Br) undergoing substitution more rapidly. The solid lines in Figure 3 are the least-squares fits to the integrated rate equation. The kinetic model for competing adsorption/desorption and reaction steps developed below predicts that the sum of the decay constants (R+ + R-)for a biexponential fit should be linear with myoglobin concentration. In addition, (R+ + R-)2 - (R+ - R-)2 is predicted to be quadratic in myoglobin concentration. Figure 4a shows the values of (R+ + R-) extracted from the biexponential fits to the spr response curves graphed as a function of myoglobin concentration. These data are clearly linear with myoglobin concentration for each surface species investigated.

Figure 4b shows (1/4)(R+ + R-)2 - (R+ - R-)2 as a function of myoglobin concentration. These data are clearly not linear and fit well to a quadratic function for each surface species investigated. Table 1 shows the results of the regression analyses presented in Figure 4. The adsorption, desorption, and reaction rate constants are calculated from the regression data. The uncertainties reported reflect the standard error for each coefficient from the linear regression analysis. Table 2 shows the average of the net change (∆θ) in the spr response for each myoglobin concentration studied. The net change in the spr response was determined as the difference between the spr response to buffer solution before introduction of myoglobin and the final, constant spr response to buffer after reaction with myoglobin was complete as shown in Figure 1. The averages are over two or three trials for each reactive SAM surface for a total of six to eight trials for each concentration. Table 2 shows that ∆θnet did not depend significantly on the myoglobin concentration. This indicates that although the reaction rate depended on the myoglobin concentration, as shown in Figure 2, the total amount of tightly bound myoglobin did not. As a control, a nonreactive SAM consisting of 16-ol, hexadecylthiomethylbenzene (AHTB) was investigated over most of the myoglobin concentration range. The average total change in spr response (∆θnet) for two or three trials is also shown in Table 2 for this SAM. The response was much lower with an average of 0.034 ( 0.006. In addition, the sum of the decay constants for the biexponential fits to the data did not show a linear correlation with myoglobin concentration.

Myoglobin and Activated Self-Assembled Monolayers

Langmuir, Vol. 18, No. 21, 2002 8159 Scheme 1

Figure 4. The decay constants extracted from the biexponential fits are plotted according to the model discussed in the text. The closed circles show the data for the BHTB SAM, the open squares are for the MHTB SAM, and the closed triangles are the data for the CHTB SAM. (a) The sum of the decay constants, (R+ + R-), is plotted as a function of the myoglobin concentration. The solid lines are linear least-squares fits to the data. The slopes provide the sum of the adsorption and reaction rate constants, (ka + kr), and the y-intercepts give the desorption rate constant, kd. (b) (1/4)(R+ + R-)2 - (R+ - R-)2 is plotted as a function of the myoglobin concentration. The solid lines are quadratic least-squares fits to the data. The quadratic coefficient gives the product of the adsorption and reaction rate constants, (kakr); the linear coefficient provides the product of the reaction and the desorption rate constants, (kdkr). Table 2 [Mb] (µM)

〈∆θnet〉 reactive SAMs

〈∆θnet〉 alcohol SAM

7.20 12.0 20.0 33.3 55.6 79.4 113 162 288 384 600 averages

0.055 0.100 0.058 0.034 0.044 0.063 0.095 0.082 0.050 0.071 0.130 0.071 ( 0.009

0.049050 0.017850 0.061350 0.070600 0.012400 0.014400 0.037400 0.023300 0.032700 0.034 ( 0.006

Discussion The change in spr response at any time is representative of all changes occurring at the surface. This includes changes in the bulk solution index of refraction, the amount of myoglobin adsorbed to the surface, and the

amount of myoglobin covalently attached to the surface. Initially, when the solution is changed from buffer to myoglobin in the flow cell, the spr signal increases rapidly because of the increased index of refraction of the myoglobin solution. However, the instrument response time is typically very fast (less than a second) and any changes after this initial fast response are due to adsorption or reaction at the surface since the bulk solution properties are not changing. The time constants extracted from the biexponential fits are all much longer than one second and thus correspond to reaction and adsorption changes at the surface. The results obtained are consistent with a kinetic scheme in which a chemical reaction competes with nonspecific adsorption and desorption. The spr response curves shown in Figures 2 and 3 do not fit well to a simple exponential association. This indicates that more than one kinetic process is being monitored by the spr signal. An adsorption/desorption step must be included to account for the results shown in Figure 1 which indicates that some myoglobin becomes loosely and nonspecifically adsorbed since the spr response diminishes upon washing with buffer solution. Three observations are consistent with an additional chemical reaction step that covalently binds the myoglobin to the surface. First, the amount of tightly bound myoglobin that does not wash off the surface shows no trend with the identity of the leaving group or with myoglobin concentration. This is the expected result for a covalent interaction since the density of active sites on each surface is expected to be approximately the same. Second, the reaction rate constants extracted from the fits to the data increase with decreasing basicity of the leaving group on the surface as expected for a substitution reaction. Third, a nonreactive -OH terminated surface resulted in a significantly smaller change in the spr angle after washing, and the data for this surface do not fit to the same integrated rate law as the reactive surfaces. Scheme 1 is qualitatively consistent with these observations. The first step in Scheme 1 is an equilibrium binding step where ka denotes the rate constant for adsorption and kd denotes the rate constant for desorption of nonspecifically bound myoglobin. The second step in Scheme 1 is a substitution reaction in which the leaving group (X) is replaced by the myoglobin. For the second step, kr denotes the rate constant for reaction. Scheme 1 predicts two types of surface coverage: a loosely bound species that can be desorbed (Mb‚X-S ) θ1) and a covalently bonded species that cannot be washed off the surface (Mb-+S ) θ2). The rate equations for each type of fractional coverage (θ1 and θ2) are shown as eqs 1 and 2.

dθ1 ) ka[Mb](1 - θ1 - θ2) - kdθ1 dt

(1)

dθ2 ) kr[Mb](1 - θ1 - θ2) dt

(2)

These coupled differential equations can be solved for θ1

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and θ2 using the method of elimination. The solutions are biexponential and are shown as eqs 3 and 4.

θ1 ) c1e-R+t + c2e-R-t

(3)

θ2 ) c3e-R+t + c4e-R-t + 1

(4)

Where:

predicted to be linear in myoglobin concentration whereas the difference between the square of the sum and the square of the difference should be quadratic in myoglobin concentration. These relationships are shown as eqs 6 and 7 and plotted in Figure 4.

R+ + R- ) (ka + kr)[Mb] + kd (R+ + R-)2 - (R+ - R-)2 ) 4kr[Mb](ka[Mb] + kd)

(6) (7)

R+ ) ((ka + kr)[Mb] + kd) + x((ka + kr)[Mb] + kd)2 - 4kr[Mb](ka[Mb] + kd) 2 R- ) ((ka + kr)[Mb] + kd) - x((ka + kr)[Mb] + kd)2 - 4kr[Mb](ka[Mb] + kd) 2

The spr response is due to the total coverage of myoglobin on the surface or θ ) θ1 + θ2. Equation 5 shows the integrated rate equation for the total fractional coverage (θ).

θ ) 1 - C1e -R+t - C2e-R-t

(5)

In eq 5, C1 ) -(c1 + c3) and C2 ) -(c2 + c4). The initial conditions of the problem result in negative coefficients; thus, the final integrated rate equation is defined so that C1 and C2 are positive numbers. Multiplying eq 5 through by the proportionality constant between the spr response and the fractional coverage gives the final equation used to fit the data and shown as solid lines in Figures 2 and 3. The values of the decay constants extracted from the biexponential fits to the data can be used to calculate the adsorption, desorption, and reaction rate constants shown in Table 1. The sum of the decay constants (R+ + R-) is

These data are able to distinguish between several models. If Scheme 1 were modified so that the two steps were sequential rather than parallel or if the reaction sequence were SN1 rather than SN2 as proposed, eq 6 would still be linear in myoglobin concentration, but eq 7 would be linear rather than quadratic. The data shown in Figure 4b are clearly not linear which rules out a sequential adsorption/reaction scheme or an SN1 reaction sequence. Conclusions The substitution reaction between bulk myoglobin and SAMs terminated by good leaving groups most likely proceeds by a one-step, SN2 mechanism. The rate law determined from the experimental data supports this conclusion as does the increasing reaction rate with decreasing basicity of the leaving group attached to the surface. Acknowledgment. The authors acknowledge Allegheny College and the National Science Foundation for support of this research through NSF grant CHE-0094392. Supporting Information Available: Experimental details. This material is available free of charge via the Internet at http://pubs.acs.org. LA025767E