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Protease Activity on an Immobilized Substrate Modified by Polymers: Subtilisin BPN′ Alan R. Esker,† Philip F. Brode III,*,‡ Donn N. Rubingh,‡ Deborah S. Rauch,‡ Hyuk Yu,§ Alice P. Gast,| Channing R. Robertson,| and Giuseppe Trigiante⊥ Miami Valley Laboratories, The Procter & Gamble Company, Cincinnati, Ohio 45253-8707, Department of Chemistry, Virginia Tech, Blacksburg, Virginia 24061-0212, Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, Department of Chemical Engineering, Stanford University, Stanford, California 94305, and IVEM, Oxford, Oxford OX1 3SR, U.K. Received April 20, 1999 We describe the adsorption and catalytic behavior of the serine protease subtilisin BPN′ on controlled pore glass (CPG) beads with a short (aminopropyl) or a long (aminoalkyl CH2 > 12) chain covalent link separating the reporter peptide succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (sAAPFpNA) from the surface. The propyl-linked sAAPFpNA modified glass surface (aminopropyl CPG:sAAPFpNA) showed a 2-fold increase in protease adsorption over an aminopropyl-glass surface. In contrast, the sAAPFpNA surface with the long chain connector showed a 2-fold drop in adsorption relative to an aminoalkyl surface. BPN′-catalyzed hydrolysis rates showed an inverse relationship to adsorption. Water-soluble polymers [poly(vinylpyrrolidone) (PVP), poly(ethylene oxide) (PEO), poly(4-vinylpyridine-N-oxide) (PVPO) and a copolymer of 1-vinyl-2-pyrrolidone and 1-vinylimidazole (PVPVI)] neutralize the 2-fold increase in BPN′ adsorption and provide more than a 3-fold increase in the initial rate of hydrolysis for BPN′-catalyzed cleavage of pNA. Another water-soluble polymer, poly(vinyl alcohol) (PVA), causes only a slight adsorption decrease and hydrolysis increase for the BPN′, aminopropyl CPG:sAAPFpNA system. None of the polymers causes a significant change in BPN′-catalyzed hydrolysis of, or adsorption on, aminoalkyl (CH2 > 12) CPG:sAAPFpNA. The apparent mechanism behind these effects is one in which the long alkyl chains and adsorbed polymers decrease the amount of adsorbed enzyme and increase the amount available for reaction in solution. A model is presented which describes the relationship between adsorption and surface hydrolysis.
Introduction The adsorption of proteins on solid surfaces is the result of many interacting factors including surface charge, protein charge, hydrophobic effects, pH, ionic strength, temperature, interfacial denaturation, and the presence of other surfactants.1-7 Enzymes, proteins which catalyze specific reactions, may alter the surface following adsorption, further complicating the problem.8-13 However, the technological and biomedical applications of these proteins require a better understanding of their surface properties. In a recent study, Brode and Rauch developed a method †
Virginia Tech. The Procter & Gamble Company. § University of Wisconsin. | Stanford University. ⊥ IVEM. ‡
(1) Andrade, J. D.; Hlady, V. Adv. Polym. Sci. 1986, 79, 1. (2) Norde, W. Adv. Colloid Interface Sci. 1986, 25, 267. (3) Lee, S. H.; Ruckenstein, E. J. Colloid Interface Sci. 1988, 125, 365. (4) Elwing, H.; Nilsson, B.; Svensson, K.-E.; Askendahl, A.; Nilsson, U. R.; Lundstro¨m, I. J. Colloid Interface Sci. 1988, 125, 139. (5) Lee, J.; Martic, P. A.; Tan, J. S. J. Colloid Interface Sci. 1989, 131, 252. (6) Thomann, J. M.; Mura, M. J.; Behr, S.; Aptel, J. D.; Schmitt, A.; Bres, E. F.; Voegel, J. C. Colloids Surf. 1989, 40, 293. (7) Lichtenbelt, J. W. Th.; Heuvelsland, W. J. M.; Oldenzeel, M. E.; Zsom, R. L. J. Colloids Surf., B: Biointerfaces 1993, 1, 75. (8) Verger, R.; Rietsch, J.; van Dam-Mieras, M. C. E.; de Haas, G. H. J. Biol. Chem. 1975, 251, 3128. (9) Muderhwa, J. M.; Brockman, H. L. J. Biol. Chem. 1992, 267, 1. (10) Blow, D. Nature 1991, 351, 444. (11) Converse, A. O.; Ooshima, H.; Burns, D. S. Appl. Biochem. Biotechnol. 1990 24/25, 67. (12) Brode, P. F., III.; Erwin, C. R.; Rauch, D. S.; Lucas, D. S.; Rubingh, D. N. J. Biol. Chem. 1994, 269, 23538. (13) Henis, Y. I.; Yaron, T.; Lamed, R.; Rishpon, J.; Sahar, E.; Katchalski-Katzir, E. Biopolymers 1988, 27, 123.
to determine adsorption on, and hydrolysis of, a model immobilized peptide substrate by a serine protease, subtilisin BPN′.14 This technique was used to study a series of subtilisin BPN′ variants produced through recombinant DNA techniques.15 Those variants, which led to decreased adsorption on the immobilized protease substrate relative to wild-type BPN′, also showed enhanced hydrolysis of the substrate. The authors speculated that this counterintuitive result was due to enhanced enzyme mobility which depends on interactions between the negatively charged surface and the electrostatic field near the active site of the enzyme. In the present study, the phenomenon of decreased enzyme adsorption resulting in enhanced hydrolysis of an immobilized substrate is further interpreted by means of a mathematical model which assumes parallel pathways for the reaction in solution and on the surface. The effect is mediated by the presence of water-soluble, surfaceactive polymers which alter the amount of surfaceadsorbed enzyme rather than through modifications of the native enzyme structure. The variables studied include the effect of polymer structural changes, polymer molecular weight, and direct modification of the surface. Materials and Methods Enzyme Solution. The steps used to isolate, purify, and prepare a stock solution of the serine protease used here, subtilisin BPN′ (MW ) 27 534 g/mol), from Bacillus amyloliquefaciens are outlined elsewhere.14 Prior to use, the enzyme stock solution is (14) Brode, P. F., III.; Rauch, D. S. Langmuir 1992, 8, 1325. (15) Brode, P. F., III; Erwin, C. R.; Rauch, D. S.; Barnett, B. L.; Armpriester, J. M.; Wang, E. S. F.; Rubingh, D. N. Biochemistry 1996, 35, 3162.
10.1021/la990472v CCC: $19.00 © 2000 American Chemical Society Published on Web 01/13/2000
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Table 1. Characteristic Properties of Aminopropyl and Aminoalkyl (CH2 > 12) CPG sizea
pore pore volumea particle size (diameter)a CPG surface areaa capacitya capacityb amine surface densityb CH2 surface densityb sAAPFpNA surface densityc sAAPFpNA surface densityc % of amines reactedb
aminopropyl CPG
aminoalkyl (CH2 > 12) CPG
486 Å ( 3.5% 1.04 cm3‚g-1 CPG 125-177 µm 50.0 m2‚g-1 231 µmol of NH2‚g-1 CPG 2.78 × 106 NH2‚µm-2 CPG 36.0 Å2 per amine 8.34 × 106 CH2‚µm-2 CPG 5.93 × 104 pNA‚µm-2 CPG 1690 Å2 per sAAPFpNA 2.13%
489 Å ( 6.71% 1.06 cm3‚g-1 CPG 125-177 µm 56.8 m2‚g-1 57.8 µmol of NH2‚g-1 CPG 6.13 × 105 NH2‚µm-2 CPG 163 Å2 per amine > 7.36 × 106 CH2‚µm-2 CPG 4.09 × 104 pNA‚µm-2 CPG 2440 Å2 per sAAPFpNA 6.67%
a Information from the supplier (Controlled Pore Glass, Inc.). b Calculated value. c Enzyme accessible surface concentration of sAAPFpNA determined experimentally.
passed through a TRIS buffer (0.1 M TRIS buffer with 10 mM CaCl2 at a pH of 8.6) equilibrated PD-10 column (Sephadex-G25) to remove the low molecular weight impurities and storage buffer. The purified enzyme is diluted in TRIS buffer to the appropriate active enzyme concentration. The active enzyme concentration is determined using a pNA assay reliant on color production from the enzyme-catalyzed hydrolysis of a soluble substrate, succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (sAAPFpNA) (Bachem, Inc.), at a wavelength of 410 nm using a Beckman DU-70 spectrophotometer.16 Polymers. All of the materials used in this study were used without further purification. Polydisperse poly(vinylpyrrolidone) (PVP) samples, with molecular weight 10k, 24k, 40k, and 360k, were obtained from Aldrich. Their corresponding monomer 1-vinyl-2-pyrrolidone was also obtained from Aldrich. A polydisperse 36k, 40:60 random copolymer of 1-vinyl-2-pyrrolidone and 1-vinylimidazole (PVPVI) was obtained from BASF. Polydisperse 10k poly(4-vinylpyridine-N-oxide) (4-PVNO) was obtained from K. Pramod of The Procter & Gamble Company. Polydisperse poly(ethylene oxide) (PEO) of molecular weight 300k was obtained from Aldrich, while monodisperse samples with Mn ) 31.1k, Mw/Mn ) 1.03 and Mn ) 710.9k, Mw/Mn ) 1.08 were obtained from Scientific Polymer Products, Inc. Poly(vinyl alcohol) (PVA), 99+% hydrolyzed, of molecular weights 85-146k and 124-186k were obtained from Aldrich. Solutions of the above polymers and the monomer (10 ppm) were made in TRIS buffer. All of the molecular weights are values provided by the manufacturer. Substrates. Aminopropyl and aminoalkyl (CH2 > 12) controlled-pore glass (CPG) were obtained from Controlled Pore Glass, Inc. The characteristics of these modified porous glass beads are found in Table 1. These surfaces were further modified by covalently coupling a model peptide substrate, sAAPFpNA, to the amine groups of the CPG surfaces with 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide hydrochloride (EDC) as described elsewhere.14 This process does not alter the overall surface area of the CPG. The characteristics of the substrateattached surfaces are also shown in Table 1. The enzyme accessible surface concentration of sAAPFpNA on the CPG was determined by removing the sAAPFpNA from the surface with a 20+ ppm active enzyme solution (BPN′) and quantifying the pNA absorbance at a wavelength of 410 nm with time (about 4 days) until no additional hydrolysis was detected. Procedure. Subtilisin BPN′ catalyzed hydrolysis of a model substrate (aminopropyl or aminoalkyl (CH2 > 12) CPG:sAAPFpNA) was carried out at 25.0 ( 0.5 °C in TRIS buffer for 90 min. Initially, sufficient CPG:sAAPFpNA was weighed into an annealed modified round-bottom flask to obtain a total surface area of 5.00 m2. TRIS or a 10 ppm polymer solution in TRIS was then added to the flask. The solution was degassed for 3 min to remove any air from the pores. After the flask was attached to a wristaction shaker, enzyme solution was added to the flask, providing a total solution volume of 30 mL. Then, the shaker and timer were started. Periodically, the shaker was stopped to take samples. The CPG:sAAPFpNA was allowed to settle for 30-45 s. A 600 µL sample of the clear supernatant was pipetted into (16) DelMar, E. G.; Largman, J. W.; Brodrick, J. W.; Geokas, M. C. Anal. Biochem. 1979, 99, 316.
an annealed 4 mL vial. This was stored on ice until adsorption and hydrolysis measurements were made. Adsorption Measurements. A standard pNA assay,16 initially performed on a blank (without CPG:sAAPFpNA), provided the initial active enzyme concentration. Subsequent pNA assays on each sample taken above provided the enzyme activity over the course of the reaction. The decrease in enzyme activity with time was attributed to adsorption such that a mass balance calculation converted active enzyme depletion from the solution to adsorption. Hydrolysis Measurements. Here, 600 µL of enzyme solution in TRIS was used to blank the spectrophotometer at a wavelength of 410 nm. Then 100 µL of sample was added to the cuvette, the resulting solution mixed, and the absorbance recorded after 100 s at 410 nm. A Beer’s law and mass balance calculation was applied to the absorbance measurement to determine the amount of pNA in solution. This value was corrected for base-catalyzed hydrolysis.14
Results and Discussion Subtilisin BPN′ Catalyzed Hydrolysis of, and Adsorption on, Aminopropyl and Aminoalkyl (CH2 > 12) CPG:sAAPFpNA. Figure 1 shows BPN′-catalyzed hydrolysis of, and adsorption on, the aminopropyl CPG: sAAPFpNA surface and compares this to the adsorption on the unreacted aminopropyl CPG surface. Figure 2 shows BPN′-catalyzed hydrolysis of, and adsorption on, the aminoalkyl (CH2 > 12) CPG:sAAPFpNA surface and compares this to the adsorption on the unreacted aminoalkyl CPG surface. Figure 1B shows enhanced adsorption to the substrate modified surface (>2× the unreacted surface), while Figure 2B shows a decrease in adsorption to the substrate-modified surface even though both CPG substrates show comparable initial hydrolysis rates of 373 ( 15 and 360 ( 80 molecules‚µm-2‚min-1 for the aminopropyl and aminoalkyl (CH2 > 12) CPG:sAAPFpNA surfaces, respectively. BPN′ adsorption to the unreacted aminopropyl surface is twice the adsorption seen on the aminoalkyl (CH2 > 12) surface, 14.9 ( 2.6 molecules‚µm-2 versus 7.3 ( 1.1 molecules‚µm-2. (Note that all of these values are much lower than the values of a closely packed BPN′ monolayer which would be on the order of 50 000 molecules‚µm-2.) The most plausible explanation for this effect lies in the surface charge of the CPG surface. Surface hydrophobicity does not appear to be the dominant factor governing BPN′ adsorption to these surfaces as the surface density of methylene groups for the two surfaces is roughly the same (see Table 1). Since a difference in hydrophobicity appears not to be the cause, it seems more likely that the long chain alkyl groups are more effective in shielding the enzyme from the surface charge of the CPG. This is consistent with the observation of decreased adsorption on the aminoalkyl (CH2 > 12) CPG following the attachment of sAAPFpNA, which would further separate the enzyme from the charged surface. That the opposite is
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Figure 1. BPN′ hydrolysis of, and adsorption on, 5.00 m2 of aminopropyl CPG:sAAPFpNA as a function of time for a 0.27 ppm BPN′ solution in TRIS at 25 °C. The solid line in B indicates BPN′ adsorption on 5.00 m2 of aminopropyl CPG without sAAPFpNA from a 0.28 ppm BPN′ solution in TRIS at 25 °C, while the dashed lines represent 95% confidence intervals on this value.
Figure 2. BPN′ hydrolysis of, and adsorption on, 5.00 m2 of aminoalkyl (CH2 > 12) CPG:sAAPFpNA as a function of time for a 0.28 ppm BPN′ solution in TRIS at 25 °C. The solid line in B indicates BPN′ adsorption on 5.00 m2 of aminoalkyl (CH2 > 12) CPG without sAAPFpNA from a 0.28 ppm BPN′ solution in TRIS at 25 °C, while the dashed lines represent 95% confidence intervals on this value.
seen for aminopropyl CPG:sAAPFpNA suggests a specific enzyme-substrate orientation effect optimizing the electrostatic interactions between the active site region of the enzyme and the negatively charged CPG surface. Further evidence indicating that charge is a key factor in BPN′ adsorption to these CPG surfaces is seen in the polymer effects below and the BPN′ variants discussed elsewhere.15 The Effect of Polymers. Several neutral polymers have been found to increase BPN′-catalyzed hydrolysis of the aminopropyl CPG:sAAPFpNA while simultaneously decreasing BPN′ adsorption to this surface. This trend is identical to one observed for variants of BPN′ in the 200220 loop, which adsorb less to the aminopropyl CPG: sAAPFpNA surface than native BPN′, and hydrolyze the surface-bound substrate with a greater initial rate.15 As will be seen later, the most straightforward explanation for this behavior implies a reduction in the catalytic efficiency of the enzyme consequent to its adsorption. Subtilisin BPN′ Catalyzed Hydrolysis of, and Adsorption on, Aminopropyl CPG:sAAPFpNA in the Presence of Polymers. PVP. Representative plots of the polymer effect for 10 ppm PVP on enzyme-catalyzed hydrolysis of, and adsorption on, aminopropyl CPG: sAAPFpNA are presented in Figure 3 for several molecular weights as well as for the monomer. The key features include a greater than 3× increase in the initial rate of hydrolysis, the lack of any effect in the presence of the monomer, the lack of any significant molecular weight dependence on hydrolysis, and reduced enzyme adsorption as compared to the unreacted aminopropyl CPG surface, as seen in Table 2 and Figure 3. The lack of any effect on
the part of the monomer indicates that the effect is not derived from a single specific polymer enzyme interaction which alters the enzyme conformation. It also indicates that this is a polymer effect. The absence of any observed molecular weight dependence among the polymers may simply reflect the polydisperse character of the samples in which higher molecular weight fractions obscure such effects. The enhanced hydrolysis at lower adsorption is similar to the general trend with BPN′ variants reported elsewhere.15 In this case, the effect is probably due to competitive adsorption of PVP through its amide bond dipole interaction with the negatively charged glass surface. PVP shows no effect on the aminoalkyl (CH2 > 12) surface indicating that its effect is a surface phenomenon rather than the result of a polymer-enzyme complex. A more detailed discussion of this appears later. PVPVI and 4-PVNO. Two other polymers with strong dipoles and similar structures were examined. These were a polydisperse random copolymer (PVPVI) consisting of 40% 1-vinyl-2-pyrrolidone and 60% 1-vinylimidazole and polydisperse 10k poly(4-vinylpyridine-N-oxide) (4-PVNO). Results of BPN′-catalyzed hydrolysis of, and adsorption on, aminopropyl CPG:sAAPFpNA in the presence of 10 ppm PVPVI and 4-PVNO are summarized in Table 2. The effect of PVPVI on both hydrolysis and adsorption is comparable to that seen for PVP. This is not too surprising given the 1-vinyl-2-pyrrolidone content of the copolymer. As for the 1-vinylimidazole content, it is expected to show some positive character at pH 8.6. The presence of 4-PVNO also caused a large increase in the initial hydrolysis rate and a maximum adsorption value (14.5 ( 1.2 molecules‚
Protease Hydrolysis of an Insoluble Substrate
Langmuir, Vol. 16, No. 5, 2000 2201 Table 2. BPN′-Catalyzed Initial Hydrolysis Rates and Maximum Adsorptions for Aminopropyl and Aminoalkyl (CH2 > 12) CPG:SAAPFpNA in the Presence and Absence of Polymerd
polymer none 85-146k PVA 124-186k PVA 31.1k PEO 300k PEO 710.9k PEO PVP monomer 10k PVP 24k PVP 40k PVP 360k PVP 36k PVPVI 10k 4-PVNO
m2
Figure 3. BPN′ hydrolysis of, and adsorption on, 5.00 of aminopropyl CPG:sAAPFpNA as a function of time for (b) 0.27 ppm BPN′, (9) 0.27 ppm BPN′ + 10 ppm 1-vinyl-2-pyrrolidone, (2) 0.27 ppm BPN′ + 10 ppm 10k PVP, (1) 0.28 ppm BPN′ + 10 ppm 24k PVP, (]) 0.27 ppm BPN′ + 10 ppm 40k PVP, and (") 0.28 ppm BPN′ + 10 ppm 360k PVP solutions in TRIS at 25 °C. The solid line on B indicates BPN′ adsorption on 5.00 m2 of aminopropyl CPG without sAAPFpNA from a 0.28 ppm BPN′ solution in TRIS at 25 °C, while the dashed lines represent 95% confidence intervals on this value.
µm2) similar to that observed for the unreacted aminopropyl CPG surface as shown in Figure 1. PVPVI and 4-PVNO at a concentration of 10 ppm had no effect on BPN′-catalyzed hydrolysis of the aminoalkyl (CH2 > 12) CPG:sAAPFpNA surface, just as PVP had no effect. Therefore, it is believed that PVPVI and 4-PVNO exert their effects through adsorption to the negatively charged CPG surface of the aminopropyl CPG:sAAPFpNA system. PEO. The three polymers discussed thus far all share a common structural motif, the presence of a fairly strong dipole. Another nonionic polymer, poly(ethylene oxide) (PEO), has a weak dipole, but its presence still causes increased BPN′-catalyzed hydrolysis of, and decreased adsorption on, the aminopropyl CPG:sAAPFpNA surface. This is presented in Table 2 for three different molecular weights of PEO present at concentrations of 10 ppm. The magnitude of the hydrolysis increase and adsorption decrease is comparable to that observed for PVP, PVPVI, and 4-PVNO. The key difference is that the 31.1k PEO sample shows a smaller increase in the initial hydrolysis rate and a smaller decrease in BPN′ adsorption on the aminopropyl CPG:sAAPFpNA as compared to the higher molecular weight samples. This observation is consistent with the phenomenon of increased polymer adsorption for larger molecular weights on solid surfaces. No molecular weight effect was seen for PVP, although this was probably due to the polydispersity of the samples as discussed above. PEO, just like PVP, PVPVI, and 4-PVNO,
substrate AP AP AA AP AA AP AA AP AP AA AP AA AP AA AP AA AP AA AP AA AP AA AP AA AP AA AP AA
initial enzyme concna
initial hydrolysis rateb
max BPN′ adsorptionc
0.27 0.27 0.28 0.27 0.29 0.28
398 ( 24 373 ( 15 360 ( 80 465 ( 25 320 ( 100 450 ( 40
31.2 ( 0.2 31.8 ( 0.4 4.8 ( 1.0 29.9 ( 0.2 2.5 ( 0.2 28.9 ( 0.3
0.30 0.30
1080 ( 60 1180 ( 70
16.9 ( 0.2 15.7 ( 0.8
0.27
1360 ( 100
12.9 ( 0.5
0.29 0.28 0.27
1400 ( 80 350 ( 70 378 ( 14
12.0 ( 0.5 1.5 ( 0.2 31.8 ( 0.3
0.27 0.28 0.28
1470 ( 190 350 ( 60 1360 ( 70
11.2 ( 0.7 1.3 ( 0.3 10.3 ( 0.6
0.27
1390 ( 140
9.2 ( 0.6
0.28
1440 ( 280
11.8 ( 0.4
0.27 0.28 0.28 0.26
1380 ( 100 350 ( 60 1680 ( 220 330 ( 70
9.7 ( 0.4 1.0 ( 0.3 14.5 ( 1.2 2.2 ( 0.6
a ppm. b Molecules‚µm-2‚min-1. c Molecules‚µm-2. d Key: AP ) aminopropyl CPG:sAAPFpNA; AA ) aminoalkyl (CH2 > 12) CPG: sAAPFpNA. The values for the initial hydrolysis rates were obtained from the least-squares slope in the linear part of the hydrolysis versus time plots. The maximum adsorption values were obtained by averaging the experimental values from a single experiment near the maximum in the adsorption versus time plots. The error bars represent 95% confidence intervals.
at a concentration of 10 ppm shows no effect on the BPN′catalyzed hydrolysis of the aminoalkyl (CH2 > 12) CPG: sAAPFpNA surface. PVA. Even though PVA possesses a strong dipole, the structure of PVA is different from the other polymers as it is the only one which is a hydrogen bond donor, while all the other polymers are potential hydrogen bond acceptors. It is probably the most interesting as its effect on BPN′-catalyzed hydrolysis of, and adsorption on, aminopropyl CPG:sAAPFpNA is different. This is seen in Table 2, where the most striking features are an initial rate of hydrolysis which is only slightly larger than, and an adsorption which is almost equal to, that of BPN′ in the absence of polymer. Whereas PVP, PVPVI, 4-PVNO, and PEO showed large changes in both BPN′ adsorption on, and hydrolysis of, aminopropyl CPG:sAAPFpNA despite structural differences, PVA with its own unique structure showed only small effects on both BPN′catalyzed hydrolysis and adsorption. One possible explanation is that PVA may weakly interact with the negatively charged glass surface (Si-O-). If PVA were to interact with the negatively charged glass surface through a hydrogen-bonding mechanism, rather than neutralizing the surface charge, PVA itself would take on a partial negative charge. This would alter the charge distribution rather than screening the charge, which means there would still be a negatively charged surface to interact with the enzyme, after it bound the peptide substrate. An alternative explanation is that PVA cannot compete with
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Figure 4. BPN′ hydrolysis of, and adsorption on, 5.00 m2 of aminoalkyl (CH2 > 12) CPG:sAAPFpNA as a function of time for (b) 0.28 ppm BPN′, (9) 0.29 ppm BPN′ + 10 ppm 85-146k PVA, (2) 0.28 ppm BPN′ + 10 ppm 10k PVP, (1) 0.28 ppm BPN′ + 10 ppm 36k PVPVI (40:60), ([) 0.26 ppm BPN′ + 10 ppm 10k 4-PVNO, and (`) 0.28 ppm BPN′ + 10 ppm 710.9k PEO solutions in TRIS at 25 °C. The solid line in B indicates BPN′ adsorption on 5.00 m2 of aminoalkyl (CH2 > 12) CPG without sAAPFpNA from a 0.28 ppm BPN′ solution in TRIS at 25 °C, while the dashed lines represent 95% confidence intervals on this value.
Figure 5. BPN′ hydrolysis of, and adsorption on, 5.00 m2 of aminopropyl CPG:sAAPFpNA as a function of time for (b) 0.27 ppm BPN′, (2) 0.27 ppm BPN′ + 10 ppm 10k PVP, and (4) 0.27 ppm BPN′ + 10 ppm 10k PVP (added after 24 min as indicated by the dashed vertical line) solutions in TRIS at 25 °C. The solid line in B indicates BPN′ adsorption on 5.00 m2 of aminopropyl CPG without sAAPFpNA from a 0.28 ppm BPN′ solution in TRIS at 25 °C, while the horizontal dashed lines represent 95% confidence intervals on this value.
BPN′ for adsorption to the surface. Despite the marked difference between PVA and the other polymers on the aminopropyl CPG:sAAPFpNA surfaces, 10 ppm PVA has no effect on BPN′-catalyzed hydrolysis of the aminoalkyl (CH2 > 12) CPG:sAAPFpNA surface, the same result seen for the other polymers. Comparison of Aminoalkyl (CH2 > 12) and Aminopropyl CPG:sAAPFpNA in the Presence of Polymers. In contrast to the large effects exhibited by the polymers on the aminopropyl surface, none of the polymers had any significant effect on BPN′-catalyzed hydrolysis of, or adsorption on, the aminoalkyl (CH2 > 12) CPG: sAAPFpNA surface. This is shown in Figure 4 and summarized in Table 2. For hydrolysis, all of the polymers fall on the curve for BPN′ without polymer. This is strong evidence that the polymer effect seen for aminopropyl CPG: sAAPFpNA is a surface effect. If the polymers were exerting their influence through a change in solvent properties, or through a polymer-enzyme complex, enhanced hydrolysis of the aminoalkyl (CH2 > 12) CPG: sAAPFpNA would be expected as the peptide surface presented to the enzyme is the same as that presented by the aminopropyl CPG:sAAPFpNA. The only difference between the two surfaces is the thickness of the linking layer between the negatively charged CPG and the peptide sAAPFpNA. Due to the low adsorption, there is more scatter in the adsorption data as seen in Figure 4. It appears that the polymers may cause a slight decrease in
enzyme adsorption which would not be inconsistent with polymer adsorption to the negatively charged glass surface. Polymer-Induced Desorption of BPN′ from the Aminopropyl CPG:sAAPFpNA Surface. In all of the experiments described above, the polymers were exposed to the CPG:sAAPFpNA surfaces for about 4 min prior to enzyme addition. A similar experiment was conducted by allowing the enzyme to adsorb on and start hydrolyzing the surface in the absence of polymer. Then part way through the experiment, a small volume of concentrated polymer solution was added to achieve a total polymer concentration of 10 ppm. Figure 5 shows the effect of adding 10k PVP, after 24 min, to achieve a final polymer concentration of 10 ppm. Both BPN′-catalyzed hydrolysis of, and adsorption on, the aminopropyl CPG:sAAPFpNA surface depart from the BPN′ without polymer limit to the BPN′ + 10k PVP limit by the end of the experiment. This result shows that the decrease in enzyme activity over the course of the experiment is not due to irreversible denaturation and is most likely due to adsorption. This is in accord with previous studies where BPN′ activity was recovered (enzyme desorbed) following the removal of substrate from aminopropyl CPG:sAAPFpNA14 and appreciable autolysis only occurred at higher bulk enzyme concentrations (>2×) and much longer time scales (t1/2 > 175 h).12 These observations appear to validate the original assumption that lost activity is primarily the result of reversible BPN′ adsorption. Moreover, the observation of
Protease Hydrolysis of an Insoluble Substrate
polymer-induced recovery of activity is consistent with the polymer displacing BPN′ from the surface. A Possible Mechanism. As BPN′ only shows enhanced adsorption to the aminopropyl CPG (versus no increase and maybe even a slight decrease on the aminoalkyl (CH2 > 12) CPG) after attachment of the sAAPFpNA substrate, it is clear that the enzyme peptide binding interactions alone do not explain enhanced adsorption to aminopropyl CPG:sAAPFpNA. This result suggests a mechanism in which electrostatic interactions with the CPG surface are responsible in part for BPN′ adsorption on the negatively charged substrate-coated CPG surface. If so, this effect can be altered by neutralizing the surface charge, changing the dielectric constant of the media between the enzyme and the surface, or by physically increasing the separation of the enzyme and the surface. Considering the chemistry of the system, the CPG:sAAPFpNA surfaces consist of the negatively charged CPG, an insulating layer consisting of the linking group with some dielectric constant, and the peptide sequence to which the enzyme binds. At pH 8.6, it is possible that the unreacted primary amines carry a positive charge which may neutralize some of the excess negative charge by looping back to the glass surface. The significant difference between the aminopropyl CPG: sAAPFpNA and the aminoalkyl (CH2 > 12) CPG:sAAPFpNA, however, is the length of the linking groups. It seems reasonable that the aminoalkyl (CH2 > 12) CPG forms a thicker insulating layer resulting in greater separation of the enzyme from the negatively charged surface and lower BPN′ adsorption. However, the two layers may also have different dielectric constants and no clear distinction between the two can be made. This mechanism also requires that the enzyme possess positively charged regions capable of interacting with the surface on adsorption. In this sense, the mechanism is consistent with the study of the effects of increasing the ratio of negative to positive residues in the 200-220 loop of the BPN′ molecule which results in decreased enzyme adsorption on, and increased hydrolysis of, the aminopropyl CPG:sAAPFpNA.15 As for the polymers, it seems likely that their effect results from adsorption to the surfaces. Table 2 indicates that the initial enzyme concentrations for identically prepared solutions are independent of the presence or absence of polymer showing the polymers have no significant effect on solution activity. Even though no direct measurements of polymer adsorption were made, it is clear from the literature that PEO, PVA, and PVP adsorb to a variety of solid interfaces from aqueous solution.5,7,17 Due to the large surface area used (5.00 m2) and the small polymer solution volume and concentration (30 mL of 10 ppm), any adsorption will be at submonolayer coverage (areas per monomer greater than 100 Å2 for all of the polymers if all of the polymer were to adsorb). The presence of polymer adsorption is supported by the fact that increased hydrolysis of the aminopropyl CPG: sAAPFpNA surface requires PVP instead of just the monomer. It is also supported by the smaller increase in hydrolysis seen for the lowest molecular weight of PEO which may adsorb less strongly to the CPG surface and thereby be less effective at increasing the hydrolysis of the aminopropyl CPG:sAAPFpNA surface. Both of these observations are consistent with increasing polymer adsorption with increasing molecular weight at solid surfaces due to entropic factors. It is unclear whether the polymer forms a looped structure which increases the (17) Molyneux, P. In Water Soluble Synthetic Polymers: Properties and Behavior; CRC Press: Boca Raton, FL, 1984; Vol. II, p 196.
Langmuir, Vol. 16, No. 5, 2000 2203 Scheme 1
thickness of the insulating layer providing increased charge separation, interpenetrates the insulating linking layer altering the dielectric constant of the layer, or both. Either of these effects could provide for the decreased adsorption. Some work to elucidate the relationship between adsorption and surface catalysis has been carried out;18-29 however, the technological and biomedical applications of these proteins still require a better understanding of their surface properties. Mathematical Model of the Reaction. To provide a quantitative model for our results, we need to propose a reaction scheme comprising both the enzyme adsorption process and the cleavage reaction. We assume that the enzyme partitions between the bulk solution and the CPG surface. This adsorption has been indirectly monitored for all the reactions. We also assume that the enzyme, whether adsorbed or not, is capable of cleaving the substrate albeit with two different kinetic routes. The hypothesized scheme for the reaction involves two parallel (surface and bulk) reaction channels with their relative rate constants leading to an intermediate (C or C′) which in turn leads to the product. The global chemical scheme is reported in Scheme 1,where the symbols are as follows: subtilisin total concentration, St; BPN′ total bulk concentration, Sbt ) rSt; BPN′ unbound bulk concentration, Sb; BPN′ total adsorbed concentration, Sat ) (1 - r)St ) Sa + C′; BPN′ uncomplexed adsorbed concentration, Sa; reactive peptide concentration, R; cleaved product concentration, P; enzyme-peptide complexes, C from bulk enzyme and C′ from surface enzyme; BPN′ surface partition constant, Kp ) Sbt/Sat ) r/(1 - r). The partition factor r is an index of the fraction of enzyme that does not adsorb to the CPG beads and ranges between 0 and 1. (18) Adam, G.; Delbru¨ck, M. In Structural Chemistry and Molecular Biology; Rich, A., Davidson, N., Eds.; Freeman: San Francisco, 1968; p 198. (19) Berg, O. G.; Winter, R. B.; von Hippel, P. H. Biochemistry 1981, 20, 6929. (20) Winter, R. B.; Berg, O. G.; von Hippel, P. H. Biochemistry 1981, 20, 6961. (21) Park, C. S.; Wu, F. Y.-H.; Wu, C.-W. J. Biol. Chem. 1982, 257, 6950. (22) Katchalski-Katzir, E.; Rishpon, J.; Sahar, E.; Lamed, R.; Henis, Y. I. Biopolymers 1985, 24, 257. (23) Henis, Y. I.; Yaron, T.; Lamed, R.; Rishpon, J.; Sahar, E.; Katchalski-Katzir, E. Biopolymers 1988, 27, 123. (24) Gaspers, P. B.; Robertson, C. R.; Gast, A. P. Langmuir 1994, 10, 2699. (25) Gaspers, P. B.; Gast, A. P.; Robertson, C. R J. Colloid Interface Sci. 1995, 172, 518. (26) Rubingh, D. N.; Bauer, M. D. In Polymer Solutions, Blends, and Interfaces; Noda, I., Rubingh, D. N., Eds.; Elsevier Science: Amsterdam, 1992; p 445. (27) Tilton, R. D.; Gast, A. P.; Robertson, C. R. Biophys. J. 1990, 58, 1321. (28) Tilton, R. D.; Robertson, C. R.; Gast, A. P. J. Colloid Interface Sci. 1990, 137, 192. (29) Kim, S.; Yu, H. J. Phys. Chem. 1992, 96, 4034.
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Approximations are needed to derive an analytical expression for the mathematical solution of the associated differential equation system. We assume that the chemical cleavage step is faster than the association one, as is commonly the case in enzyme kinetics, so that the concentration of the reaction intermediate C (and C′) is approximately constant. This approximation will be justified by the results in all but two cases. The two reaction channels are parallel and for either one (i.e. bulk)
dC/dt ) k1RSb - (k2 + k3)C ) 0 C)
k1RSb (k2 + k3)
)
1 RS K b
where K is the classical Michaelis-Menten constant, having the dimensions of concentration, and since Sb + C ) Sbt by mass conservation of enzyme, we have
C)
RSb R(Sbt - C) ) K K C)
RSbt K+R
and finally we obtain this expression for vb, the reaction rate in the bulk solution
νb ) -
k3SbtR dR ) k3C ) dt K+R
A similar relation holds for the surface reaction pathway. Rearranging the terms we have
dR(K/R + 1) ) -k3rStdt
( )
R + (R - R0) ) -k3rSt(t - t0) R0
(1)
This solution approximates first-order behavior if K . R, and linear (zeroth order) behavior if R . K. We shall henceforth omit the pedix for the k3 values for simplicity and refer to them as k and k′. The experimental data show that in the majority of cases the linear behavior is evident early in the reaction course. This allows us to simplify the general equation (including both reaction pathways)
ν ) ν b + νs )
measure adsorption. This plot is shown in Figure 6. If we divide the two initial rate values at r ) 0 and r ) 1 (which mathematically correspond to Stk′ and Stk, respectively) we obtain the ratio 100/2000 or 1/20, indicating that k′ is negligible compared to k and that the rate is mainly determined by the reaction of the bulk enzyme. The complete kinetic solution including bulk and surface reaction pathways reduces to
K ln
Integration gives
K ln
Figure 6. Initial hydrolysis rate as a function of bulk enzyme concentration of 5.00 m2 of aminopropyl CPG:sAAPFpNA for (b) 0.27 ppm BPN′, (9) 0.27 ppm BPN′ + 10 ppm 1-vinyl-2pyrrolidone, (2) 0.27 ppm BPN′ + 10 ppm 85-146k PVA, (4) 0.27 ppm BPN′ + 10 ppm 124-186k PVA, (1) 0.30 ppm BPN′ + 10 ppm 31.1k PEO, (3) 0.27 ppm BPN′ + 10 ppm 300k PEO, (white plus sign inside solid inverted triangle) 0.29 ppm BPN′ + 10 ppm 710.9k PEO, (]) 0.27 ppm BPN′ + 10 ppm 36k PVPVI (40:60), (`) 0.27 ppm BPN′ + 10 ppm 10k PVP, (white dot inside solid hexagon) 0.28 ppm BPN′ + 10 ppm 24k PVP, (white plus sign inside solid hexagon) 0.27 ppm BPN′ + 10 ppm 40k PVP, (") 0.28 ppm BPN′ + 10 ppm 360k PVP, and (0) 0.28 ppm BPN′ + 10 ppm 10k 4-PVNO solutions in TRIS at 25 °C. The error bars represent 95% confidence intervals, while a fit line is provided to highlight the trend. The dashed line marks the concentration for which r ) 1.
krStR k′(1 - r)StR + K+R K′ + R
to a globally zeroth order expression
ν ) St[kr + k′ (1 - r)] ) rSt(k - k′) + Stk′ This means that if we plot the initial rates of the reactions as a function of rSt (bulk enzyme concentration), we should obtain a straight line with intercept at rSt ) 0 of Stk′ and a slope of (k - k′). The parameter r can be obtained from the enzyme bulk depletion measurements performed to
R + (R - R0) ) -krSt(t - t0) R0
and one of the fit curves corresponding to this equation is reported as an example in Figure 7A. The agreement is good for all the reactions except two, corresponding to the lowest bulk enzyme concentrations (Figure 1, aminopropyl CPG:sAAPFpNA with no polymer added, and Figure 5, where a polymer is added after 24 min). In these situations the low enzyme concentration causes the stationary-state condition to break down, as the scarcity of one reactant makes the initial buildup of intermediate C slow enough to be observable. In these cases the general model k1
k3
} C 98 Sb + P Sb + R {\ k 2
still holds. This can be seen when the numerical fit, obtained using MATLAB sofware, is overlaid on one of the data sets (Figure 7B). The numerical fit is performed because the kinetic system of differential equations
dR/dt ) -k1SbR + k2C dC/dt ) k1SbR - (k2 + k3)C dP/dt ) k3C
Protease Hydrolysis of an Insoluble Substrate
Langmuir, Vol. 16, No. 5, 2000 2205
Table 3. Synoptic Table of the Kinetic Fit Parameters for the Subtilisin BPN′ Catalyzed Hydrolyses condition
BPN′ bulk (nM)
initial rate (molecules/(µm2min))
no polymer +VP monomer +PVP 10k + PVP 24k +PVP 40k +PVP 360k +PVPVI +PVNO +PVA 85k +PVA 124k +PVP 10k (24 min delay)
1.36 1.20 6.90 7.15 7.45 6.73 7.31 5.99 1.72 2.00 0.7 (before) 5.6 (after)
390 370 1470 1360 1390 1440 1380 1680 465 450
no polymer +PVP 10k +PVPVI +PVNO +PVA 85 +PEO 710
8.67 9.64 9.72 9.39 9.31 9.58
a
360 350 350 330 320 350
k3[E] (min-1)
Amino Propyl 3.4 × 102 5.0 × 102 2.0 × 103 2.0 × 103 2.0 × 103 2.0 × 103 N/A N/A N/A N/A 2.0 × 102 1.7 × 103 Amino Alkyl 2.7 × 103 2.6 × 103 2.7 × 103 2.8 × 103 2.6 × 103 2.8 × 103
K (molecules/µm2)
K (mM) 4.5 4.2 4.2 4.2 4.2 4.2
1.5a
1.6 × 104 a 1.5 × 104 1.5 × 104 1.5 × 104 1.5 × 104 1.5 × 104 N/A N/A N/A N/A 1.1 × 104 a
1.5 1.3 1.3 1.4 1.3 1.4
1.1 × 105 1.0 × 105 1.1 × 105 1.0 × 105 9.9 × 104 1.1 × 105
30.6 28.3 30.6 27.9 27.4 31.9
k3 (s-1) 1.4a 2.3 1.4 1.3 1.3 1.4
3.1
Data obtained from numerical fit using MATLAB software.
parameters (as derived from optimization of the analytical equation above or numerical MATLAB fit) are reported in Table 3. This allows us to comment on the reactions involved. It can be seen that k3 is reasonably constant and independent of the cross-linker chain length or polymers present, as would be expected if we assume that the chemical process of cleavage of the complex is unaffected by the surrounding environment. The value is however remarkably lower (about 50-fold) than the reported k3 in solution. A similar result has been reported (ref 14) and can be justified if we consider that this rate constant represents the turnover number of the enzyme, i.e., the number of substrate molecules hydrolyzed by one enzyme molecule per second, under conditions of substrate saturation (so that in solution all enzyme molecules are involved in the reaction at any given time). In a twodimensional case, though, a large fraction of the enzyme molecules is far removed from the surface and hence not available for reaction even if the substrate is saturating near the surface. This results in an apparently lower k3 that actually reflects a lower “effective” subtilisin concentration [S]eff. The value of K is constant only within each linker/ substrate series (aminopropyl and aminoalkyl) but is apparently greater (about 6-fold) in the aminoalkyl series. Given that k3 does not change and that k2 is small, this implies that k1 actually decreases in the aminoalkyl series. This would imply a slower association rate of the aminoalkyl substrate and the enzyme, but the reason for this is not obvious and needs to be further investigated. Figure 7. Plots of selected experimental data (b) with overlaid kinetic fit curves (dashed lines) as derived from the model described in the paper. The data show BPN′ hydrolysis of, and adsorption on, 5.00 m2 of aminopropyl CPG:sAAPFpNA as a function of time for a 0.27 ppm BPN′ solution in TRIS at 25 °C with (A) +10 ppm 360k PVP solutions (analytical solution) and (B) no polymer added (numerical fit).
with the side conditions
R(t)0) ) R0 Sb + C ) Sbt R + C + P ) R0 cannot be solved analytically. The values of the fit
Conclusions We have investigated the process of adsorption of the enzyme subtilisin BPN′ on derivatized controlled-pore glass (CPG) beads and the enzyme-catalyzed cleavage reaction of the surface-bound peptide CPG:sAAPFpNA under a variety of experimental conditions. The extent of enzyme adsorption showed a marked dependence on the presence of the substrate peptide on the surface. The addition of various polymers in solution affected enzyme adsorption only with an aminopropyl cross-linker and was not influential when a long chain aminoalkyl cross-linker was employed. All of the kinetic results obtained for enzymatic cleavage of the peptide could be satisfactorily described by a mathematical model that suggests the
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reaction is fundamentally driven by the unadsorbed enzyme, whereas the activity of surface-adsorbed enzyme is many times smaller under these conditions. This does not imply that the surface enzyme does not catalyze hydrolysis, as a nonzero intercept can be detected within experimental error, but rather that under the conditions studied it provides a far less important reaction pathway than enzyme in solution. We also discussed the effect of a number of different polymers and of the chain length of the cross-linking molecule on enzyme adsorption and reactivity and speculate that a more electrostatically screened surface disfavors enzyme binding, as does the
Esker et al.
presence of hydrogen bond acceptor polymers possibly competing for surface binding. Acknowledgment. We thank Mark Bauer, who purified the enzyme, and Bobby Barnett for useful discussions on the enzyme’s structure. A. Esker’s internship was supported in part by National Research Service Award 5 T32 GM08349 from the National Institute of General Medical Sciences. G. Trigiante acknowledges support from Genencor International, Inc. LA990472V
