Anal. Chem. 2002, 74, 2943-2949
On-Line Trypsin-Encapsulated Enzyme Reactor by the Sol-Gel Method Integrated into Capillary Electrophoresis Kumiko Sakai-Kato, Masaru Kato, and Toshimasa Toyo’oka*
Department of Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada Shizuoka, Shizuoka, 422-8526, Japan
A novel trypsin-encapsulation technique using the solgel method was developed for the preparation of an online enzyme reactor integrated into capillary electrophoresis. Trypsin was encapsulated in tetramethoxysilane-based hydrogel, and its enzymatic activity was evaluated using r-N-benzoyl-L-arginine ethyl ester and two peptides (bradykinin and [Tyr8]-bradykinin). The enzyme encapsulation was carried out in a single step under mild conditions within a capillary, and 1.5-cm gel was formed at the inlet of the capillary. The resultant monolithic reactor showed excellent enzymatic activity, which was ∼700 times higher than that in free solution, without stopping the flow. Separation of the unreacted substrates and products in the same capillary also showed high selectivity, and sample size in this system decreased 3 orders of magnitude from conventional tryptic reaction schemes. The encapsulated trypsin maintains its substrate specificity even in a sol-gel matrix. Furthermore, the encapsulated trypsin exhibits increased stability even after continuous use compared to that in free solution. An enzyme is a biological catalyst, which has high efficiency under mild conditions and is highly selective. In the concept of utilizing the natural catalysts for the manufacture of various products, such as foods or pharmaceuticals, the technique for immobilizing enzyme was born.1 The advantages of an immobilized enzyme are numerous. Enzyme reactors offer the convenience of repeated use of the enzyme without much loss of its activity. The ability to easily separate immobilized enzymes from the reaction mixture offers an additional advantage of multiple use of the enzyme resulting in reduced cost and time to remove the enzyme from the measurement samples. In some cases, there have been speculation that the immobilized enzymes are more stable than their soluble forms. It would be particularly useful if immobilization enabled the use of enzymes in organic solvents or at temperatures above those that would ordinarily cause a loss of activity. Since the 1960s, various methods have been developed to immobilize enzymes.2-6 * Corresponding author. E-mail:
[email protected]. Fax: +8154-264-5593. Tel: +81-54-264-5656. (1) Michaelis, K.; Ehrenreich, M. Biochem. J. 1908, 10, 283-290. (2) Chibata, I. Immobilized Enzymes; Kodansha: Tokyo, 1978. (3) Pechet, M.; Goldstein, L.; Katchalski, E. Biochemistry 1964, 3, 1905-1913. (4) Weetall, H. H. Biochim. Biophys. Acta 1970, 212, 1-7. 10.1021/ac0200421 CCC: $22.00 Published on Web 05/23/2002
© 2002 American Chemical Society
The immobilization of enzymes is important in many applications,2 including industrial and biomedical, and is, potentially, of practical consequence in continuous-flow substrate conversion systems, combined with a separation system. This combined reactor-separator system is well-suited for automation and highthroughput systems. Most of the immobilized enzyme reactors, however, have been used off-line from the separation system used to analyze the reaction products. To take advantage of the effectiveness of an enzyme reactor for a high-throughput system, the enzyme reaction in the flow-through system is very desirable. Some techniques have been reported to immobilize enzymes into the flow-through system.7-11 For example, a chromatographic support, which is commercially available, was used for the noncovalent immobilization of an enzyme, followed by the online separation using another column.9 The covalent attachment of the enzyme through glutaraldehyde onto the inner wall of a fused-silica capillary was also developed for use in tandem with capillary electrophoresis (CE) directed toward microseparation.10 An immobilized enzyme is also used in pre- or postcolumn derivatization devices for high-performance liquid chromatography (HPLC).11 More recently, Wainer’s group described a coupledcolumn enzyme reactor based upon dopamine β-hydroxylase, and two different stationary phases were used to immobilize the enzyme in different manners.12 Another recent report by Jiang and Lee described an on-line coupling of a microenzyme reactor with micromembrane chromatography.13 Recently, the sol-gel encapsulation method has attracted much attention for the preparation of a desirable protein-doped matrix for biosensors. The silicate matrix is formed by hydrolysis of an alkoxide precursor followed by condensation to yield polymeric oxo-bridged SiO2 networks. Conventional sol-gel methods involve the use of a high concentration of methanol as (5) Glassmeyer, C. K.; Ogle, J. D. Biochemistry 1971, 10, 786-792. (6) Brown, H. D.; Patel, A. B.; Chattopadhyay, S. K. J. Biomed. Mater. Res. 1968, 2, 231-235. (7) Petro, M.; Svec, F.; Fre´chet, M. J. Biotechnol. Bioeng. 1996, 49, 355-363. (8) Amankwa, L. N.; Kuhr, W. G. Anal. Chem. 1992, 64, 1610-1613. (9) Alebic´- Kolbah, T.; Wainer, I. W. Chromatographia 1993, 37, 608-612. (10) Nashabeh, W.; El Rassi, Z. J. Chromatogr. 1992, 596, 251-264. (11) Marka-Varga, G.; Johansson, K.; Gordon, L. J. Chromatogr., A 1994, 660, 153-167. (12) Markoglou, N.; Wainer, I. W. J. Chromatogr., B 2001, 766, 145-151. (13) Jiang, Y.; Lee, C. S. J. Chromatogr., A 2001, 924, 315-322.
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a cosolvent for the precursors, often with high acidity.14 However, Ellerby et al. developed a modified version of the process that removes the need for the addition of methanol. This version does not expose the biomolecules to the damaging effects of a low-pH environment.15 These improvements enabled encapsulated proteins to retain their structure15,16 and biological activity for a prolonged period17 and to enhance their utility as a new type of matrix for biomolecules. Until now, however, the utility of the revised version has been limited to static formats, including silica gel film18 or monoliths in cuvettes for optical biosensors15,17,19,20 and it has not been applied to the flow-through supports for proteins, except for the immunoaffinity chromatography in which IgG-encapsulated sol-gel glasses were ground and packed into a column.21 The sol-gel method has been noted as the monolithic packing method for HPLC22 and capillary electrochromatography (CEC).23-26 The specific advantage of the sol-gel method is the ease in which capillary columns can be prepared in a single step.24-26 An additional advantage of these monolithic methods arises from the fritless design, which was a serious problem for conventional columns packed with silica particles. Recently, we developed a novel protein-encapsulation technique using the sol-gel methods for the preparation of monolithic capillary columns for CEC.27 Two proteins, bovine serum albumin (BSA) and ovomucoid from chicken egg white, were encapsulated in tetramethoxysilane (TMOS)-based silica matrix and their enantioselectivity was evaluated for the separation of some selected enantiomers. The protein encapsulation was carried out within a capillary in a single step under mild conditions. The resultant monolithic columns showed adequate chromatographic performance, including mechanical strength, penetration of pressurized flow, and chiral separation. These results show the possibility of this technique as a powerful tool for biomolecule encapsulation. To extend our original concept of monolithic molded materials to the area of catalysis with nanoscale samples, this report deals with the encapsulated trypsin in the sol-gel matrix for the on(14) Brinker, C. J.; Scherer, G. W. Sol-Gel Science: The Physics and Chemistry of Sol-Gel Processing; Academic Press: New York, 1990. (15) Ellerby, L. M.; Nishida, C. R.; Nishida, F.; Yamanaka, S. A.; Dunn, B.; Valentine, J. S.; Zink, J. I. Sceince 1992, 255, 1113-1115. (16) Edmiston, P. L.; Wambolt, C. L.; Smith, M. K.; Saavedra, S. S. J. Colloid Interface Sci. 1994, 163, 395-406. (17) Braun, S.; Rappoport, S.; Zusman, R.; Avnir, D.; Ottolenghi, M. Mater. Lett. 1990, 10, 1-5. (18) Yao, T.; Harada, I.; Nakahara, T. Bunseki Kagaku 1995, 44, 927-932. (19) Flora, K.; Brennan, J. D. Anal. Chem. 1998, 70, 4505-4513. (20) Dave, B. C.; Dunn, B.; Valentine, J. S.; Zink, J. I. Anal. Chem. 1994, 66, 1120A-1127A. (21) Cichna, M.; Knopp, D.; Niessner, R. Anal. Chim. Acta 1997, 339, 241250. (22) Minakuchi, H.; Nakanishi, K.; Soga, N.; Ishizuka, N.; Tanaka, N. Anal. Chem. 1996, 68, 3498-3501. (23) Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Soga, N.; Nagayama, H.; Hosoya, K.; Tanaka, N. Anal. Chem. 2000, 72, 1275-1280. (24) Dulay, M. T.; Kulkarni, R. P.; Zare, R. N. Anal. Chem. 1998, 70, 51035107. (25) Kato, M.; Dulay, M. T.; Bennett, B.; Chen, J.-R.; Zare, R. N. Electrophoresis 2000, 21, 3145-3151. (26) Dulay, M. T.; Quirino, J. P.; Bennett, B. D.; Kato, M.; Zare, R. N. Anal. Chem. 2001, 73, 3921-3926. (27) Kato, M.; Sakai-Kato, K.; Matsumoto, N.; Toyo’oka, T. Anal. Chem. 2002, 74, 1915-1921.
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line enzyme reactor, in which the enzyme reaction and microseparations could be simultaneously performed in a single capillary. EXPERIMENTAL SECTION Materials and Chemicals. Fused-silica capillary (75-µm i.d.) was obtained from Polymicro Technologies Inc. (Phoenix, AZ). TMOS, methacryloxypropyltrimethoxysilane (MPTMS), and R-Nbenzoyl-L-arginine ethyl ester (BAEE) hydrochloride were purchased from Tokyo Kasei (Tokyo, Japan). Trypsin from porcine pancreas and DL-norleucine methyl ester hydrochloride were purchased from Sigma-Aldrich (Milwaukee, WI). R-N-Benzoyl-Larginine (BA) was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Bradykinin and [Tyr8]-bradykinin were purchased from Peptide Institute, Inc. (Osaka, Japan). Capillary Pretreatment. A capillary column (40 cm) was pretreated with MPTMS, which covalently anchors the capillary wall to the silanol groups of the silicate matrix.28 In the preliminary experiment, this pretreatment proved to be very effective in preventing the gel from leaking out of the capillary. The polyimide coating of the treated capillary is burned with fuming sulfuric acid to make a detection window. Monolithic Capillary Column Preparation. The sol-gel reaction basically followed the procedures developed by Ellerby et al.15 and described in our previous report.27 The monomer solution was obtained by mixing the following reagents just prior to use: (1) 761 µL of TMOS, (2) 169 µL of water, and (3) 11 µL of 0.04 N HCl. This monomer solution was stirred for 20 min so that hydrolysis proceeds to form a fully or partially hydrolyzed silane, SiOH4-n(OMe)n. A total of 120 µL of trypsin solution (10% (w/v)) in 50 mM Tris-HCl (pH 7.0) containing 20 mM CaCl2 was added to 20 µL of the hydrolyzed solution. After mixing and ultrasonication for 5 s, the mixture solution was carefully aspirated with a 1.0-mL disposable syringe from the inlet of the capillary, which was in advance filled with 50 mM Tris-HCl (pH 7.5), until the sol plug was 1.5 cm long under microscopic observation. Introducing the running buffer in advance is very useful in order to shorten the conditioning time. Both ends of the capillary were sealed and placed at 4 °C for more than 4 days. Once fabricated, the capillary was cut to an adequate length for the instruments. The total length and effective length were 34 and 25 cm, respectively. The capillary was carefully installed in a CE cartridge and conditioned electrokinetically (-2 kV) with 50 mM Tris-HCl (pH 7.5) for 1-2 h to eliminate the resultant methanol or proteins, which were not encapsulated in the solgel matrix. Assay of Trypsin Activity. Activity of the free trypsin in aqueous solution was determined by a UV-visible recording spectrophotometer UV-2100 (Shimadzu, Kyoto, Japan) with 1 or 2 mM BAEE as the substrate using the standard procedure.29 BAEE is hydrolyzed to BA. Also, 0.5 mg/mL [Tyr8]-bradykinine was hydrolyzed at the peptide bond of N-terminal Arg by free trypsin (0.3 mg/mL) in the water bath kept at 25 °C for various periods. The reaction was stopped by adding methanol, followed by centrifugation (3000 rpm, 5 min), and filtration through a 0.22(28) Hje´ten, S. J. Chromatogr. 1985, 347, 191-198. (29) Schwert, G. W.; Takenaka, Y. Biochim. Biophys. Acta 1955, 16, 570-575.
µm membrane. The filtrate was injected into the capillary without trypsin-encapsulated gel, and hydrolysis activity was determined by the quantification of the resultant peptide, which was lost Arg, using norleucine methyl ester as an internal standard. The enzymatic activity of the encapsulated trypsin was determined using BAEE and [Tyr8]-bradykinine as substrates. The substrates were introduced electrokinetically (4 kV, 10 s) from the inlet of the capillary. The enzymatic reactions were carried out on-line by allowing a thin plug of the substrates to flow through a trypsin-encapsulated matrix using electroosmotic flow (EOF). The substrates were converted to products at the trypsinencapsulated gel, followed by separation and determination at the part of the capillary downstream of the gel. Determination of Trypsin Content in Gel. Protein content was determined in order to evaluate the amount of encapsulated trypsin in the gel. A glass tube (6-mm i.d.) was filled with the gel containing trypsin, and both ends of the tube were soaked in buffer bottles filled with 50 mM Tris-HCl (pH 7.5). After applying the same voltage as the capillary for 2 h, the encapsulated trypsin in the sol-gel matrix was dissolved in 1 N NaOH for 30 min and protein contents were measured by the method of Lowry et al.30 using BSA as standard. Equipment. CE experiments were carried out on a HewlettPackard 3DCE system (Palo Alto, CA) equipped with a diode array detector. Substrates were introduced electrokinetically at the anodic side (4 kV, 10 s). In most experiments, a voltage of 4 kV was applied. The temperature was kept at 25 °C in all experiments. The mobile phase was 50 mM Tris-HCl (pH 7.5). Substrates were diluted in the mobile phase. Before use, all solutions were filtered through a 0.22-µm membrane (Millipore, Bedford, MA) and degassed by ultrasonication. Water was purified by MilliQ apparatus (Millipore). RESULTS AND DISCUSSION The goal of this research was to develop a new on-line enzyme reactor that encapsulates trypsin in the sol-gel matrix and is integrated into a CE system. The sol-gel reaction proceeds by the following steps:14 (1) the hydrolysis of alkoxysilane; (2) the condensation of hydrated silica to form siloxane bonding (tSi-O-Sit); and (3) the polycondensation by linkage of additional silanol group to form the cyclic oligomers. While the silicate network grows, it traps the protein molecules.
hydrolysis: Si(OR)4 + H2O f (RO)3SiOH + ROH condensation: 2(RO)3SiOH f (RO)3Si-O-Si(OR)3 + H2O (RO)4Si + (RO)3SiOH f (RO)3Si-O-Si(OR)3 + ROH
1. Design of the Capillary Enzyme Reactor. Figure 1 is a schematic illustration of the capillary enzyme reactor. At the inlet of the capillary, trypsin is encapsulated in the gel matrix. The substrates are introduced electrokinetically from the inlet of the capillary (Figure 1a). The substrates are converted into products by encapsulated trypsin (Figure 1b). Finally, the unreacted (30) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265-275.
Figure 1. Schematic illustration of an on-line enzyme reactor integrated into CE. (a) Substrates are introduced electrokinetically into the trypsin-encapsulated reactor. (b) Substrates are catalyzed into products while they flow through the trypsin-encapsulated gel by electrophoresis and EOF. (c) The products and unreacted substrates are separated at the separation section of the capillary by electrophoresis.
substrates and products are separated by electrophoresis (Figure 1c). 2. Preparatory Condition for the Trypsin-Encapsulated Capillary. In the previous report,27 we investigated the optimum gelation condition for protein encapsulation in the sol-gel matrix, such as buffer condition and protein content. In this study, the trypsin concentration (10% (w/v)) and the ratio of the protein solution volume to the hydrolyzed solution (6:1) was followed by the basic procedures developed in the previous reports.27 After gelation for more than 4 days, encapsulation of trypsin into the gel matrix was evaluated by measuring the protein content in the gel. About 80% of the protein was not incorporated into the matrix and was washed out of the column by EOF during the first conditioning. The average amount of trypsin in the capillary was ∼0.90 µg/cm gel. Buffer conditions, such as concentration and pH, are very critical to the enzymatic activity. Because the enzymatic reaction and the separation were performed in the same capillary column, the buffer condition was considered from both standpoints. According to the standard procedure,29 50 mM Tris-HCl (pH 7.5) was selected for the enzymatic reaction and separation. With this buffer, the tryptic activity of BAEE in free solution was ∆ 3.9 min-1 (mg of protein)-1, which was close to the labeled value, ∆ 3.1 min-1 (mg of protein)-1. ∆ means the increase in absorbance at 253 nm. The separation of BAEE and its product BA was examined within a capillary without gel using 50 mM Tris-HCl (pH 7.5) as the running buffer. As illustrated in Figure 2, both peaks were completely separated. As shown in Figure 3, [Tyr8]-bradykinin and its digest were also completely separated using the same buffer. The digest, which lost N-terminal Arg, migrated behind [Tyr8]-bradykinin and EOF time was ∼20.2 min. These results show the feasibility of using this buffer for both the enzymatic reaction and separation. To prevent autolysis of trypsin during the gelation, 20 mM CaCl2 was added to 50 mM Tris-HCl (pH 7.0) as the initial buffer during the gelation. Analytical Chemistry, Vol. 74, No. 13, July 1, 2002
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Figure 2. Electropherogram of BAEE and BA separated within a capillary without gel. Conditions: sample, 2 mM BAEE and BA; injection, 4 kV, 10 s; fused-silica capillary, 34-cm total length; mobile phase, 50 mM Tris-HCl (pH 7.5); field strength, 114 V/cm; detection, 214 nm.
Figure 3. Electropherogram of [Tyr8]-bradykinin and its digest separated within a capillary without gel. Conditions: sample, the reactant of 0.5 mM [Tyr8]-bradykinin hydrolyzed by free trypsin (0.31 mg/mL); injection, 4 kV, 10 s. Other conditions are as in Figure 2.
Next, the substrates were introduced electrokinetically from the inlet of the capillary, which is filled with trypsin-encapsulated sol-gel matrix. This enzyme reactor system was evaluated by the production of digests. The gel length was changed from 10 to 1.5 cm to examine reaction activity. Total length was fixed to 34 cm. The capillary containing 10- and 6-cm gel took a long time to become stable in terms of current, migration time of the analytes, and peak shapes. These results are probably derived from the fact noted in our previous report that the protein-containing gel decreases EOF.27 Another cause may be that the longer gels might have a higher degree of nonspecific interaction with the substrates, causing the undesirable effects on separation. On the other hand, the capillary with a matrix less than 3 cm stabilized more quickly and had better repeatability of migration time. Moreover, enzyme activity was maintained even with the shorter gels as well as good separation beween substrates and products. Therefore, the data were obtained using the capillary containing the 1.5-cm gel, which is the shortest gel tested (total length, 34 cm). Figure 4 illustrates the typical electropherogram of BAEE and BA, when 200 mM BAEE was introduced into the capillary as 2946 Analytical Chemistry, Vol. 74, No. 13, July 1, 2002
Figure 4. Electropherogram of an on-line tryptic reaction using BAEE as a substrate. Conditions: sample, 200 mM BAEE; injection, 4 kV, 10 s; trypsin-encapsulated gel length, 1.5 cm; total length, 34 cm; mobile phase, 50 mM Tris-HCl (pH 7.5); field strength, 114 V/cm; detection, 214 nm.
Figure 5. Electropherogram of an on-line tryptic reaction using [Tyr8]-bradykinin as a substrate. Conditions: sample, 0.5 mg/mL [Tyr8]-bradykinin; internal standard (I.S.), DL-norleucine methyl ester. Other conditions are as in Figure 4.
substrate. Encapsulated trypsin successfully hydrolyzed BAEE to BA without stopping the flow. The BAEE and BA peaks were completely separated just as the peaks were separated within a capillary without trypsin-encapsulated gel (Figure 2). The increase in peak elution time is due to a decrease in EOF as observed in our previous report.27 When the amount of BA was large, the peak shape tended to be fronting. The sample injection method was the same (4 kV for 10 s) in all cases. BAEE less than 50 mM was completely hydrolyzed to BA as it flowed through the gel matrix, and no BAEE peak was observed. However, when the activity of encapsulated trypsin decreased, a BAEE peak as well as a BA peak was also observed. Figure 5 illustrates the electropherogram of [Tyr8]-bradykinin and its digest. The Arg peak that is also produced by the hydrolysis of [Tyr8]-bradykinin was detected at 200 nm with the retention time of 11.1 min, but not at 214 nm. The peak shapes of the peptides deteriorated compared to Figure 3, and the retention times increased. Adachi et al. studied the elution profiles of substrate and product using immobilized invertase in an acryl-
Table 1. Property of Trypsin Encapsulated in the Sol-Gel or in Free Solutiona
protein (mg/mL) Vmax (mM min-1 mg-1) Km (mM) sample size (mol)
encapsulatedb
freec
encapsulated/free
20.2 983 ( 158 0.923 ( 0.158 4.91 × 10-12
0.314 1.42 ( 0.101 0.312 ( 0.049 2.50 × 10-8
64.3 692 2.96 1.96 × 10-4
a V 8 max and Km values are expressed as mean ( standard error of the mean (SEM); n ) 3. The enzymatic activity was determined using [Tyr ]bradykinin as a substrate. Other conditions are as in Figure 2. b Reaction conditions of the encapsulated trypsin are as in Figure 4. c Various concentrations of [Tyr8]-bradykinin were hydrolyzed in free trypsin solution for 5 min at 25 °C and the reactants were analyzed in a capillary without gel.
Figure 6. (a) Velocity of the encapsulated trypsin depending on substrate concentrations. (b) Double-reciprocal plot of the enzymatic activity of encapsulated trypsin. Conditions: substrates, [Tyr8]-bradykinin. Other conditions are as in Figure 4.
Figure 7. (a) Velocity of the free trypsin depending on substrate concentrations. (b) Double-reciprocal plot of the enzymatic activity of free trypsin. Conditions: substrates, [Tyr8]-bradykinin. Various concentrations of [Tyr8]-bradykinin were hydrolyzed in trypsin solution (0.31 mg/mL) for 5 min at 25 °C. The reactant was analyzed within a capillary without gel. Other conditions are as in Figure 2.
amide gel column and reported that asymmetrical elution profiles having long tails may reflect the influence of the diffusional resistance in the gel.31 Therefore, the peak broadening of [Tyr8]bradykinin may be caused by the diffusional resistance in the gel matrix. Nonspecific interaction between [Tyr8]-bradykinin and gel matrix would be also the cause of the peak deterioration. 3. Tryptic Activity to Peptides. We investigated the availability of this system as a real bioreactor using [Tyr8]-bradykinin as substrate. DL-Norleucine methyl ester was used as an internal standard for the quantitative determination of the digests (Figure 5). (31) Adachi, S.; Hashimoto, K.; Matsuno, R.; Nakanishi, K.; Kamikubo, T. Biotechnol. Bioeng. 1980, 22, 779-797.
Table 1 shows the tryptic activity on the encapsulated column and in the free trypsin using [Tyr8]-bradykinin as substrate. The reaction time in the gel was calculated from the retention time of the unreacted [Tyr8]-bradykinin and the gel length. By encapsulating trypsin into the gel matrix, the concentration of trypsin was increased by 64 times. Figure 6a and Figure 7a depict the change of the initial velocity depending on the substrate concentration. Both systems show a similar increase in the initial velocity at the lower substrate concentration, followed by gradual slope saturation. On the encapsulated column, inhibition occurred at the higher concentration, which was not observed in the free solution. Double-reciprocal plots of the tryptic activity (Lineweaver-Burk plot) are shown in Figures 6b and 7b. The Km and Vmax values Analytical Chemistry, Vol. 74, No. 13, July 1, 2002
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Table 2. Enzymatic Activity of Encapsulated and Free Trypsin
native-bradykinin [Tyr8]-bradykinin ratio ([Tyr8]-/native-)
encapsulateda (mM/column)
freeb (mM/min)
0.143 0.744 5.21
0.021 0.154 7.33
a The activity of encapsulated trypsin was determined using 1.0 mM bradykinin and [Tyr8]-bradykinin. The activity of encapsulated trypsin for each substrate was compared using the concentration of each substrate digested in one analysis. b The activity of free trypsin was determined using 0.5 mM bradykinin and [Tyr8]-bradykinin.
Figure 8. Stability of encapsulated and free trypsin at 25 °C during the repeated usage. Conditions: stability of the encapsulated trypsin was determined using 200 mM BAEE as substrate. Other conditions are as in Figure 4. Stability of free trypsin was determined using 1 mM BAEE as a substrate. The trypsin solution (20 mg/mL) was placed in 50 mM Tris-HCl buffer (pH 7.5) at 25 °C. Aliquots were withdrawn at the indicated times, diluted 100 times, and assayed. Other conditions are as with the standard procedures.29 The initial velocity was measured in each format.
were obtained from these double-reciprocal plots and the following eq 1,
1/v ) (Km/Vmax)(1/[S]) + 1/Vmax
(1)
where v is the velocity of the enzymatic activity, Km is the Michaelis constant, Vmax is the maximum velocity, and [S] is the concentration of substrates. The Km value of [Tyr8]-bradykinin for the encapsulated column was 0.923 on average (Table 1), which is ∼3 times higher than that of free trypsin. On the other hand, Vmax for the encapsulated trypsin was 983 mM min-1 (mg of protein)-1 on average, which is ∼700 times higher than that for free trypsin. This remarkable increase in Vmax is mainly attributable to the high concentration of trypsin in a very narrow space of a capillary. The enhanced mass transfer may also make it possible to increase the frequency of interaction between trypsin and substrate. As mentioned in section 4, the increase in the stability of trypsin by encapsulation could be another reason. These results show the effectiveness of this trypsin-encapsulated technique for the assay of enzyme activity in combination with CE. It is also notable that sample size in this system decreased 3 orders of magnitude from conventional tryptic reaction schemes (Table 1). 4. Stability. Figure 8 shows the operational stability of trypsin encapsulated or in free solution, during its use. The encapsulated trypsin was used continuously during the tested period. The free trypsin was stored in the same mobile phase of 50 mM Tris-HCl buffer (pH 7.5) at 25 °C. The concentration of free trypsin was the same as that of the encapsulated trypsin in a capillary (20 mg/mL). Aliquots were withdrawn at the indicated intervals, diluted 100 times, and assayed using 2 mM BAEE as substrate. In 1 day, the activity of the encapsulated trypsin decreased 20% of its initial value. On the other hand, free trypsin almost completely lost its activity even in 1 day. After 50 repeated analyses 2948 Analytical Chemistry, Vol. 74, No. 13, July 1, 2002
for 4 days, the activity of the encapsulated trypsin decreased by 75% of its initial value. This increase in the lifetime of the encapsulated trypsin is thought to be caused by the prevention of autolysis in the sol-gel matrix. Furthermore, the encapsulated trypsin held the enzymatic activity after more than 3 months of storage at 4 °C. 5. Substrate Specificity. Physically confining an enzyme into the matrix has various effects on the characteristics of the enzymatic catalysis,32 such as enzymatic activity or substrate specificity. These effects may occur as a result of a specific interaction between the matrix and the enzyme, or the environmental differences between the bulk property of the solutions and the microenvironment in the sol-gel matrix. A structural change of enzyme would also affect the characteristics. In fact, there are many cases where substrate specificity changed dramatically in immobilized protease.2 To investigate the effect of encapsulation, the substrate specificity was investigated both on encapsulated and with free trypsin using bradykinin and [Tyr8]-bradykinin as substrates. As shown in Table 2, [Tyr8]-bradykinin was preferentially hydrolyzed over bradykinin in both formats. Because Phe of bradykinin is substituted by Tyr in [Tyr8]-bradykinin, they have different structure and electrical charge. Consequently, our result indicates that the manner of structural or electrical interaction between trypsin and each substrate is presumably highly maintained even in the sol-gel matrix. CONCLUSIONS We developed a novel on-line trypsin-encapsulated enzyme reactor by the sol-gel method integrated into CE. This simple in situ encapsulation procedure, which is carried out under mild conditions, was shown to be suitable for the immobilization of trypsin while still maintaining its catalytic activity. When integrated into a CE system, a very sensitive analytical technique is achieved where the activity of the encapsulated trypsin toward [Tyr8]bradykinin increased ∼700 times that of free trypsin, and the sample size in this system decreased 3 orders of magnitude from conventional tryptic reaction schemes. Furthermore, the encapsulated trypsin exhibited increased stability during continuous use compared to that in free solution. On the other hand, the diffusional resistance of [Tyr8]-bradykinin in the gel phase or (32) Bowers, L. D. Anal. Chem. 1986, 58, 513A-530A.
nonspecific interaction between the gel matrix and the peptide, which results in the deterioration of the peak profiles, needs to be overcome to achieve more efficient analysis. It is, however, noteworthy that both the biomolecule encapsulation in a nanometer environment and sample analysis were performed in a single capillary with very little sample handling. This opens a new pathway for the application to automated and high-throughput analytical schemes.
ACKNOWLEDGMENT This work was supported by a grant from Japan Health Sciences Foundation. The authors acknowledge Prof. R. N. Zare and Dr. M. T. Dulay for the useful comments on this work. Received for review January 23, 2002. Accepted March 28, 2002. AC0200421
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