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Excimer Fluorescence of Pyrene
Analytical Biochemistry 306, 247–251 (2002) doi:10.1006/abio.2002.5717
Development of Peptide Substrates for Trypsin Based on Monomer/Excimer Fluorescence of Pyrene 1 Taeho Ahn,* ,2 Joon-Sik Kim,† Ho-Il Choi,‡ and Chul-Ho Yun§ *Research Institute of Natural Science, †AngioLab, and §Department of Genetic Engineering, Pai-Chai University, Taejon 302-735, Korea; and ‡PepTron, Inc., Daeduk Bio-Community, Taejon 305-390, Korea
Received December 6, 2001; published online June 21, 2002
An assay using fluorogenic peptides based on the monomer/excimer fluorescence features of pyrene was developed to measure the proteolytic activity of trypsin, a serine protease. Two pyrene moieties were incorporated into the respective N- and C-terminus of the peptides as (pyrene)-C-Xaa-C-(pyrene), where Xaa represents amino acid residues of 5-, 6-, 7-, or 8-mer containing the cleavage site of trypsin. The proteolytic cleavage of the substrates led to an increase in monomer fluorescence and a decrease in excimer fluorescence of pyrene. Kinetic parameters (k cat and K m) for the enzymatic hydrolysis of the substrates were successfully determined. The parameters are dependent on the chain length of the substrate and optimal catalytic activity was obtained with substrates that consisted of 9 or 10 amino acid residues. The present assay system is sensitive and the preparation of the substrate is very simple. We suggest that this method may be suitable for high-throughput screening and also applicable to the characterization of other proteases. © 2002 Elsevier Science (USA)
The activity measurement of serine proteases usually utilizes peptide substrates containing a colorimetric or fluorescent leaving group such as 4-nitroaniline or 7-amino-4-methylcoumarin at their C-terminus (1, 2). The enzymatic hydrolysis of these synthetic peptides produces spectral features that enable the quantification of cleavage. These methods are known to be simple in preparation of the substrates and practical for studying the characterization of the protease. How1
This work was supported by the Korea Research Foundation, MOE-KRF Research Professor Grant D00029. 2 To whom correspondence should be addressed at the Research Institute of Natural Science, Pai-Chai University, Taejon 302-735, Korea. Fax: ⫹82-42-520-5385. E-mail: [email protected]. 0003-2697/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
ever, in these substrates the specificity of the amino acid residues at the P⬘ position (counterpart of the S⬘ subsite of enzyme), which is important for the substrate discrimination and catalytic efficiency (3–5), cannot be reflected. Alternatively, other methods utilizing fluorescence resonance energy transfer (FRET) 3, fluorescence polarizations, or radioactive peptides have been suggested (6 –9). In the FRET assay, a pair consisting of energy donor and acceptor, such as 5-(2-aminoethylamino)naphthalene-1-sulfonic acid and 4-(4-dimethylaminophenylazo)benzoic acid, is incorporated into the same peptide. The cleavage of substrate leads to an increase in fluorescence intensity as the acceptor, which quenches the emission energy of donor, becomes separated from the donor. FRET systems are very sensitive methods for the detection of proteolytic activity and have been successfully applied to assays for several proteases (10 –12). However, the syntheses of FRET substrates are usually complex and often result in a decrease in yield depending on the peptide sequences (6). In the present paper, we describe an assay for proteolytic activity of trypsin utilizing monomer and excimer fluorescence of a pyrene moiety incorporated into the N- and C-terminus of the peptide substrate. The proteolytic cleavage of substrates labeled with pyrene produces a dual emission character: an increase in monomer fluorescence and a decrease in excimer emission intensity of pyrene. This assay is sensitive and will be applicable to other proteases. Furthermore, the preparation of substrate is very simple. Very similarly, Packard et al. (13) reported the design of new profluorescent protease substrates utilizing spectral characteristics of xanthene dyes. In that system, the proteo3
Abbreviations used: TAME, p-toluenesulfonyl-L-arginine methyl ester; Me 2SO, dimethyl sulfoxide; FRET, fluorescence resonance energy transfer; E/M ratio, excimer to monomer fluorescence ratio. 247
248
AHN ET AL.
lytic cleavage of substrates which are labeled with dyes on each side of cleavage site produces an increase in the fluorescence intensity on the basis of exciton theory (14). Furthermore, substrate cleavage was also accompanied by spectral changes. But the procedure for peptide derivatization with fluorophores seems to be more complex compared with the present system. MATERIALS AND METHODS
Materials All amino acid derivatives and resins were obtained from Bachem Feinchemikalien (Bubendorf, Switzerland). N-[3-Pyrenyl]maleimide was purchased from Molecular Probes (Eugene, OR). Bovine trypsin (EC 3.4.21.4) was obtained from Calbiochem–Novabiochem AG (La¨ ufelfingen, Switzerland). p-Toluenesulfonyl-Larginine methyl ester (TAME) was from Sigma (St. Louis, MO). The purity of trypsin was checked with SDS–PAGE and used without further purification. All other chemicals were of highest grade commercially available. Design of Peptide Substrates The amino acid sequences used in the present study were as follows: CPARLAC (7-mer, PS-1), CPARLAIC (8-mer, PS-2), CGPARLAIC (9-mer, PS-3), and CGPARLAIGC (10-mer, PS-4). To incorporate a pyrene moiety into the peptide substrates with N-[3-pyrenyl]maleimide, a thiol-reactive probe, all synthesized peptides contained Cys residues at both the N- and the C-terminus. Peptide Synthesis The peptide substrates for trypsin were synthesized on an Applied Biosystems peptide synthesizer (Model 430A) using a standard 9-fluorenylmethyloxycarbonyl protocol (15). Crude peptides were purified by reversephase HPLC using a Phenomenex W-Porex C 18 column, elution being made with an acetonitrile/water gradient (10 –50% of acetonitrile) containing 0.1% trifluoroacetic acid. The sequences of peptides were confirmed by a MilliGen/Biosearch 6600 Prosequencer.
FIG. 1.
Structure of pyrene-labeled substrate (PS-3) for trypsin.
Enzyme Reaction and Fluorescence Measurement The fluorescence was recorded with a Shimadzu RF5301 PC spectrofluorometer equipped with a thermostated cuvette and rapid-mixing apparatus. All experiments were performed at 30°C. The enzyme reaction was started by adding protease to a buffer containing 25 mM Tris–HCl, pH 7.6, 100 mM NaCl, 5 mM CaCl 2, and diluted substrate. The final concentration of Me 2SO in the reaction mixture was less than 1% (v/v). As a control experiment, the catalytic activity of trypsin was measured using TAME in the presence of up to 3% Me 2SO. When excimer (E) and monomer (M) fluorescence of pyrene was measured at equilibrium-state, the excitation wavelength was 342 nm and emission was in the range of 360 –500 nm. Fluorescence intensities at 375 nm (for monomer) and 458 nm (for excimer) were selected to investigate the kinetic hydrolysis of substrates and to calculate the E/M ratio. The assays were performed in a reaction mixture of 1 ml (for equilibrium) or 2 ml (for kinetic study). The concentration of substrate was in the range of 2–10 M. Concentrations of fluorescent substrates were determined spectrophotometrically with fully hydrolyzed peptides at 342 nm using 42,000 cm ⫺1 as a molar extinction coefficient (17). To obtain total enzymatic hydrolysates, peptide substrates were incubated in the presence of 3 M trypsin for 5 h. To determine the kinetic parameters (k cat and K m), initial velocities were measured with various substrate concentrations. The change in fluorescence intensity was followed and the slope was determined using SigmaPlot software. The data were fitted to the Michaelis–Menten equation by nonlinear regression analysis.
Labeling of Peptides
RESULTS AND DISCUSSION
Thiol-specific labeling of peptides with the pyrene group was performed as described (16). The labeled peptides were again purified as described above and confirmed by mass spectrometer. Stock solutions of pyrene-labeled substrates were prepared in 100% dimethyl sulfoxide (Me 2SO) and stored at ⫺20°C before use.
The excited-state pyrene molecules have two characteristic emission fluorescence features for monomer and excimer. The monomer (at about 375 nm) and excimer fluorescence (around 460 – 480 nm) is dependent upon the collision rate and the distance between pyrene molecules (17, 18). Utilizing these properties of pyrene, we developed fluorogenic peptide substrates
ASSAY FOR TRYPSIN USING FLUOROGENIC PEPTIDES
249
FIG. 2. Dependence of the E/M ratio on the chain length of substrates. The monomer and excimer fluorescence was determined with 1 M each intact substrate by measuring emission spectra in the range of 360 –500 nm under 342 nm of excitation wavelength, and the E/M ratio (inset) was calculated by selecting the fluorescence intensity at 375 nm (for monomer) and 458 nm (for excimer). Lines a, b, c, and d represent the emission spectra of PS-1, PS-2, PS-3, and PS-4, respectively.
for trypsin. The specific feature of the designed substrates is that they contain two cysteine residues at both the N- and the C-terminus of the peptide for the thiol-reactive modification. The synthesis and labeling of peptides were straightforward. As the final products had limited solubility in water, probably due to the
hydrophobic pyrene moiety, the substrates were dissolved in 100% Me 2SO and used. The structure of the peptide substrate labeled with N-[3-pyrenyl]maleimide at both the N- and the C-terminus is shown in Fig. 1. The emission spectra of synthesized substrates are shown in Fig. 2. The E/M ratio for the 7-mer peptide
FIG. 3. The monomer fluorescence change of total enzymatic hydrolysates (A) and the E/M ratio of intact peptide (B) as a function of substrate concentration. Substrate PS-3 the at indicated concentrations was hydrolyzed by 3 M trypsin for 5 h at 30°C and then the monomer and excimer fluorescence were measured at 375 and 458 nm, respectively, under 342 nm of excitation wavelength. The lines through the data were obtained by linear regression. For other reaction conditions, see Materials and Methods.
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AHN ET AL.
FIG. 4. Measurement of the detection limit of trypsin by substrate PS-3. Trypsin was serially diluted with the reaction buffer (25 mM Tris–HCl, pH 7.6, 100 mM NaCl, 5 mM CaCl 2), 2 M substrate PS-3 was added, and the monomer fluorescence change (at 375 nm under 342 nm of excitation wavelength) was measured after 20 min (■) or 2 h (F).
(PS-1) was the highest and was followed by 8-, 9-, and 10-mer peptides as expected. As a first step toward the characterization of the substrates, we determined the useful substrate concentration range by investigating a possible “inner filter effect” as previously described in FRET analysis (6, 12). This will be attributed to the collisional effects of
the substrates themselves and the liberated pyrene moiety on the intact substrate and/or the products after enzymatic cleavage. Figure 3 shows the monomer fluorescence change of total enzymatic hydrolysates (Fig. 3A) and the E/M ratio of the intact PS-3 peptide (Fig. 3B) as a function of substrate concentration. The monomer fluorescence of hydrolysates increased linearly up to about 25 M with increasing concentration of PS-3 peptide but the E/M ratio was changed from around 15 M. Other peptides also showed features similar to those of PS-3 (results not shown). Therefore, throughout the present work, we kept the substrate concentration below 15 M. As a control experiment, we analyzed the hydrolytic products by reverse-phase HPLC and mass spectrometer, and the cleavage at the Arg-Leu peptide bond was confirmed (results not shown). To establish the enzymatic sensitivity of the present assay system, we determined the detection limit of trypsin by PS-3 substrate. As shown in Fig. 4, the monomer fluorescence increase was linear as a function of enzyme concentration. The detection limit was about 1 nM when the reaction mixture was incubated for 20 min but the limit became lower as the reaction time increased; when incubated for 2 h, a detectable signal change in monomer fluorescence (and also excimer fluorescence) was generated with 0.1 nM trypsin. This high sensitivity is practical for characterizing proteases present at very low concentration (e.g., less than 1 nM level). If this assay system can be extended to other proteases such as hepatitis C virus protease, another type of serine protease, the low detection limit
FIG. 5. Kinetic changes in monomer and excimer fluorescence of pyrene by PS-3 hydrolysis. 0.2 M trypsin was added to the reaction mixture containing 7 M substrate. The emission intensities at 375 and 458 nm under 342 nm of excitation wavelength were chased as described under Materials and Methods.
ASSAY FOR TRYPSIN USING FLUOROGENIC PEPTIDES TABLE 1
Kinetic Parameters for Hydrolysis of Pyrene-Labeled Substrates by Bovine Trypsin Substrate
K m (M)
k cat (s ⫺1)
k cat/K m (M ⫺1 s ⫺1)
PS-1 PS-2 PS-3 PS-4
290 ⫾ 10 100 ⫾ 7 56 ⫾ 2 44 ⫾ 2
3.6 ⫾ 1.4 5.9 ⫾ 2.1 21 ⫾ 3 20 ⫾ 2
1.2 ⫻ 10 4 5.9 ⫻ 10 4 3.8 ⫻ 10 5 4.5 ⫻ 10 5
would be desirable for screening potent inhibitors having K i values in the nanomolar range, as suggested previously (19). Figure 5 shows the progressive fluorescence changes caused by adding trypsin to the PS-3 substrate, which agrees well with our expectation. The cleavage of substrate resulted in dual emission characteristics, an increase in monomer and a decrease in excimer fluorescence proportional to the amount cleaved. But the initial velocity was dependent on the chain length of the substrates used. The kinetic parameters for tryptic cleavage of four substrates are summarized in Table 1. The substrates displayed 5.8- and 6.6-fold variation in k cat and K m, respectively. The substrates PS-3 and PS-4 had significantly elevated activities in the presence of trypsin, emphasizing that the chain length of substrate is important for the substrate– enzyme fit. The k cat/K m parameter for the PS-3 and PS-4 showed more increase than k cat. Two substrates with higher activity also had decreased K m values. This might be ascribed to the increased binding capacity of PS-3 and PS-4 to the protease and/or the enhanced accessibility to the active site of the enzyme compared to other peptides consisting of shorter chain length. The higher K m values of PS-1 and PS-2 seem to be due to the steric hindrance of bulky pyrene moieties. These results indicate that the chain length of substrate is important for the proper substrate– enzyme fit. Considering the synthetic feasibility, the efficient hydrolysis, the fluorescence intensity of monomer/excimer of pyrene, and other factors, we suggest that PS-3 consisting of 9 amino acids is the most practical for measuring the catalytic activity of trypsin. CONCLUSIONS
We developed new fluorogenic peptide substrates for the measurement of proteolytic activity of trypsin utilizing monomer and excimer fluorescence of pyrene incorporated into the substrate. The assay system is
251
sensitive and the preparation of substrate is very simple. As the present substrates contain P⬘ residues, those can be used for S⬘ subsite mapping of proteases. Further investigation will focus on the extension of this assay to other proteases such as NS3 of hepatitis C virus. REFERENCES 1. Collen, D., Lijnen, H. R., De Cokk, F., Durieux, J. P., and Loffet, A. (1980) Biochim. Biophys. Acta 615, 158 –166. 2. Kawabata, S., Miura, T., Morita, T., Kato, H., Fujikawa, K., Iwanaga, S., Takada, K., Kimura, T., and Sakakibara, S. (1988) Eur. J. Biochem. 172, 17–25. 3. Darke, P. L., Leu, C.-T., Davis, L. J., Heimbach, J. C., Diehl, R. E., Hill, W. S., Dixon, R. A., and Sigal, I. S. (1989) J. Biol. Chem. 264, 2307–2312. 4. Le Bonniec, B. F., Myles, T., Johnson, T., Knight, G. C., Tapparelli, C., and Stone, S. R. (1996) Biochemistry 35, 7114 –7122. 5. Grahn, S., Kurth, T., Ullmann, D., and Jakubke, H.-D. (1999) Biochim. Biophys. Acta 1431, 329 –337. 6. Taliani, M., Bianchi, E., Jarjes, F., Fossatelli, M., Urbani, A., Steinku¨ hler, C., De Francesco, R., and Pessi, A. (1996) Anal. Biochem. 240, 60 – 67. 7. Gahn, S., Ullmann, D., and Jakubke, H.-D. (1998) Anal. Biochem. 265, 225–231. 8. Levine, L. M., Michener, M. L., Toth, M. V., and Holwerda, B. C. (1997) Anal. Biochem. 247, 83– 88. 9. Cerretani, M., Renzo, L. D., Serafine, S., Vitelli, A., Gennari, N., Bianchi, E., Pessi, A., Urbani, A., Colloca, S., De Francesco, R., Steinku¨ hler, C., and Altamura, S. (1999) Anal. Biochem. 266, 192–197. 10. Matayoshi, E. D., Wang, G. T., Krafft, G. A., and Erickson, J. (1990) Science 247, 954 –958. 11. Wang, G. T., Chung, C. C., Holzman, T. F., and Krafft, G. A. (1993) Anal. Biochem. 210, 351–359. 12. Holskin, B. P., Bukhtiyarova, M., Dunn, B. M., Baur, P., de Chastonay, J., and Pennington, M. W. (1995) Anal. Biochem. 227, 148 –155. 13. Packard, B. Z., Toptygin, D., Komoriya, A., and Brand, L. (1996) Proc. Natl. Acad. Sci. USA 93, 11640 –11645. 14. Kasha, M. (1991) in Physical and Chemical Mechanisms in Molecular Radiation Biology (Glass, W. A., and Varma, M. N., Eds.), pp. 231–255. Plenum, New York. 15. Fields, G. B., and Noble, R. L. (1990) Int. J. Peptide Protein Res. 35, 161–214. 16. Wu, C. W., and Yarbrough, L. R. (1976) Biochemistry 15, 2863– 2868. 17. Galla, H. J., and Hartmann, W. (1980) Chem. Phys. Lipids 27, 199 –219. 18. Galla, H. J., and Sackmann, E. (1974) Biochim. Biophys. Acta 339, 103–115. 19. Zhang, R., Beyer, B. M., Durkin, J., Ingram, R., Njoroge, F. G., Windsor, W. T., and Malcolm, B. A. (1999) Anal. Biochem. 270, 268 –275.
Development of Peptide Substrates for Trypsin Based on Monomer/Excimer Fluorescence of Pyrene 1 Taeho Ahn,* ,2 Joon-Sik Kim,† Ho-Il Choi,‡ and Chul-Ho Yun§ *Research Institute of Natural Science, †AngioLab, and §Department of Genetic Engineering, Pai-Chai University, Taejon 302-735, Korea; and ‡PepTron, Inc., Daeduk Bio-Community, Taejon 305-390, Korea
Received December 6, 2001; published online June 21, 2002
An assay using fluorogenic peptides based on the monomer/excimer fluorescence features of pyrene was developed to measure the proteolytic activity of trypsin, a serine protease. Two pyrene moieties were incorporated into the respective N- and C-terminus of the peptides as (pyrene)-C-Xaa-C-(pyrene), where Xaa represents amino acid residues of 5-, 6-, 7-, or 8-mer containing the cleavage site of trypsin. The proteolytic cleavage of the substrates led to an increase in monomer fluorescence and a decrease in excimer fluorescence of pyrene. Kinetic parameters (k cat and K m) for the enzymatic hydrolysis of the substrates were successfully determined. The parameters are dependent on the chain length of the substrate and optimal catalytic activity was obtained with substrates that consisted of 9 or 10 amino acid residues. The present assay system is sensitive and the preparation of the substrate is very simple. We suggest that this method may be suitable for high-throughput screening and also applicable to the characterization of other proteases. © 2002 Elsevier Science (USA)
The activity measurement of serine proteases usually utilizes peptide substrates containing a colorimetric or fluorescent leaving group such as 4-nitroaniline or 7-amino-4-methylcoumarin at their C-terminus (1, 2). The enzymatic hydrolysis of these synthetic peptides produces spectral features that enable the quantification of cleavage. These methods are known to be simple in preparation of the substrates and practical for studying the characterization of the protease. How1
This work was supported by the Korea Research Foundation, MOE-KRF Research Professor Grant D00029. 2 To whom correspondence should be addressed at the Research Institute of Natural Science, Pai-Chai University, Taejon 302-735, Korea. Fax: ⫹82-42-520-5385. E-mail: [email protected]. 0003-2697/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
ever, in these substrates the specificity of the amino acid residues at the P⬘ position (counterpart of the S⬘ subsite of enzyme), which is important for the substrate discrimination and catalytic efficiency (3–5), cannot be reflected. Alternatively, other methods utilizing fluorescence resonance energy transfer (FRET) 3, fluorescence polarizations, or radioactive peptides have been suggested (6 –9). In the FRET assay, a pair consisting of energy donor and acceptor, such as 5-(2-aminoethylamino)naphthalene-1-sulfonic acid and 4-(4-dimethylaminophenylazo)benzoic acid, is incorporated into the same peptide. The cleavage of substrate leads to an increase in fluorescence intensity as the acceptor, which quenches the emission energy of donor, becomes separated from the donor. FRET systems are very sensitive methods for the detection of proteolytic activity and have been successfully applied to assays for several proteases (10 –12). However, the syntheses of FRET substrates are usually complex and often result in a decrease in yield depending on the peptide sequences (6). In the present paper, we describe an assay for proteolytic activity of trypsin utilizing monomer and excimer fluorescence of a pyrene moiety incorporated into the N- and C-terminus of the peptide substrate. The proteolytic cleavage of substrates labeled with pyrene produces a dual emission character: an increase in monomer fluorescence and a decrease in excimer emission intensity of pyrene. This assay is sensitive and will be applicable to other proteases. Furthermore, the preparation of substrate is very simple. Very similarly, Packard et al. (13) reported the design of new profluorescent protease substrates utilizing spectral characteristics of xanthene dyes. In that system, the proteo3
Abbreviations used: TAME, p-toluenesulfonyl-L-arginine methyl ester; Me 2SO, dimethyl sulfoxide; FRET, fluorescence resonance energy transfer; E/M ratio, excimer to monomer fluorescence ratio. 247
248
AHN ET AL.
lytic cleavage of substrates which are labeled with dyes on each side of cleavage site produces an increase in the fluorescence intensity on the basis of exciton theory (14). Furthermore, substrate cleavage was also accompanied by spectral changes. But the procedure for peptide derivatization with fluorophores seems to be more complex compared with the present system. MATERIALS AND METHODS
Materials All amino acid derivatives and resins were obtained from Bachem Feinchemikalien (Bubendorf, Switzerland). N-[3-Pyrenyl]maleimide was purchased from Molecular Probes (Eugene, OR). Bovine trypsin (EC 3.4.21.4) was obtained from Calbiochem–Novabiochem AG (La¨ ufelfingen, Switzerland). p-Toluenesulfonyl-Larginine methyl ester (TAME) was from Sigma (St. Louis, MO). The purity of trypsin was checked with SDS–PAGE and used without further purification. All other chemicals were of highest grade commercially available. Design of Peptide Substrates The amino acid sequences used in the present study were as follows: CPARLAC (7-mer, PS-1), CPARLAIC (8-mer, PS-2), CGPARLAIC (9-mer, PS-3), and CGPARLAIGC (10-mer, PS-4). To incorporate a pyrene moiety into the peptide substrates with N-[3-pyrenyl]maleimide, a thiol-reactive probe, all synthesized peptides contained Cys residues at both the N- and the C-terminus. Peptide Synthesis The peptide substrates for trypsin were synthesized on an Applied Biosystems peptide synthesizer (Model 430A) using a standard 9-fluorenylmethyloxycarbonyl protocol (15). Crude peptides were purified by reversephase HPLC using a Phenomenex W-Porex C 18 column, elution being made with an acetonitrile/water gradient (10 –50% of acetonitrile) containing 0.1% trifluoroacetic acid. The sequences of peptides were confirmed by a MilliGen/Biosearch 6600 Prosequencer.
FIG. 1.
Structure of pyrene-labeled substrate (PS-3) for trypsin.
Enzyme Reaction and Fluorescence Measurement The fluorescence was recorded with a Shimadzu RF5301 PC spectrofluorometer equipped with a thermostated cuvette and rapid-mixing apparatus. All experiments were performed at 30°C. The enzyme reaction was started by adding protease to a buffer containing 25 mM Tris–HCl, pH 7.6, 100 mM NaCl, 5 mM CaCl 2, and diluted substrate. The final concentration of Me 2SO in the reaction mixture was less than 1% (v/v). As a control experiment, the catalytic activity of trypsin was measured using TAME in the presence of up to 3% Me 2SO. When excimer (E) and monomer (M) fluorescence of pyrene was measured at equilibrium-state, the excitation wavelength was 342 nm and emission was in the range of 360 –500 nm. Fluorescence intensities at 375 nm (for monomer) and 458 nm (for excimer) were selected to investigate the kinetic hydrolysis of substrates and to calculate the E/M ratio. The assays were performed in a reaction mixture of 1 ml (for equilibrium) or 2 ml (for kinetic study). The concentration of substrate was in the range of 2–10 M. Concentrations of fluorescent substrates were determined spectrophotometrically with fully hydrolyzed peptides at 342 nm using 42,000 cm ⫺1 as a molar extinction coefficient (17). To obtain total enzymatic hydrolysates, peptide substrates were incubated in the presence of 3 M trypsin for 5 h. To determine the kinetic parameters (k cat and K m), initial velocities were measured with various substrate concentrations. The change in fluorescence intensity was followed and the slope was determined using SigmaPlot software. The data were fitted to the Michaelis–Menten equation by nonlinear regression analysis.
Labeling of Peptides
RESULTS AND DISCUSSION
Thiol-specific labeling of peptides with the pyrene group was performed as described (16). The labeled peptides were again purified as described above and confirmed by mass spectrometer. Stock solutions of pyrene-labeled substrates were prepared in 100% dimethyl sulfoxide (Me 2SO) and stored at ⫺20°C before use.
The excited-state pyrene molecules have two characteristic emission fluorescence features for monomer and excimer. The monomer (at about 375 nm) and excimer fluorescence (around 460 – 480 nm) is dependent upon the collision rate and the distance between pyrene molecules (17, 18). Utilizing these properties of pyrene, we developed fluorogenic peptide substrates
ASSAY FOR TRYPSIN USING FLUOROGENIC PEPTIDES
249
FIG. 2. Dependence of the E/M ratio on the chain length of substrates. The monomer and excimer fluorescence was determined with 1 M each intact substrate by measuring emission spectra in the range of 360 –500 nm under 342 nm of excitation wavelength, and the E/M ratio (inset) was calculated by selecting the fluorescence intensity at 375 nm (for monomer) and 458 nm (for excimer). Lines a, b, c, and d represent the emission spectra of PS-1, PS-2, PS-3, and PS-4, respectively.
for trypsin. The specific feature of the designed substrates is that they contain two cysteine residues at both the N- and the C-terminus of the peptide for the thiol-reactive modification. The synthesis and labeling of peptides were straightforward. As the final products had limited solubility in water, probably due to the
hydrophobic pyrene moiety, the substrates were dissolved in 100% Me 2SO and used. The structure of the peptide substrate labeled with N-[3-pyrenyl]maleimide at both the N- and the C-terminus is shown in Fig. 1. The emission spectra of synthesized substrates are shown in Fig. 2. The E/M ratio for the 7-mer peptide
FIG. 3. The monomer fluorescence change of total enzymatic hydrolysates (A) and the E/M ratio of intact peptide (B) as a function of substrate concentration. Substrate PS-3 the at indicated concentrations was hydrolyzed by 3 M trypsin for 5 h at 30°C and then the monomer and excimer fluorescence were measured at 375 and 458 nm, respectively, under 342 nm of excitation wavelength. The lines through the data were obtained by linear regression. For other reaction conditions, see Materials and Methods.
250
AHN ET AL.
FIG. 4. Measurement of the detection limit of trypsin by substrate PS-3. Trypsin was serially diluted with the reaction buffer (25 mM Tris–HCl, pH 7.6, 100 mM NaCl, 5 mM CaCl 2), 2 M substrate PS-3 was added, and the monomer fluorescence change (at 375 nm under 342 nm of excitation wavelength) was measured after 20 min (■) or 2 h (F).
(PS-1) was the highest and was followed by 8-, 9-, and 10-mer peptides as expected. As a first step toward the characterization of the substrates, we determined the useful substrate concentration range by investigating a possible “inner filter effect” as previously described in FRET analysis (6, 12). This will be attributed to the collisional effects of
the substrates themselves and the liberated pyrene moiety on the intact substrate and/or the products after enzymatic cleavage. Figure 3 shows the monomer fluorescence change of total enzymatic hydrolysates (Fig. 3A) and the E/M ratio of the intact PS-3 peptide (Fig. 3B) as a function of substrate concentration. The monomer fluorescence of hydrolysates increased linearly up to about 25 M with increasing concentration of PS-3 peptide but the E/M ratio was changed from around 15 M. Other peptides also showed features similar to those of PS-3 (results not shown). Therefore, throughout the present work, we kept the substrate concentration below 15 M. As a control experiment, we analyzed the hydrolytic products by reverse-phase HPLC and mass spectrometer, and the cleavage at the Arg-Leu peptide bond was confirmed (results not shown). To establish the enzymatic sensitivity of the present assay system, we determined the detection limit of trypsin by PS-3 substrate. As shown in Fig. 4, the monomer fluorescence increase was linear as a function of enzyme concentration. The detection limit was about 1 nM when the reaction mixture was incubated for 20 min but the limit became lower as the reaction time increased; when incubated for 2 h, a detectable signal change in monomer fluorescence (and also excimer fluorescence) was generated with 0.1 nM trypsin. This high sensitivity is practical for characterizing proteases present at very low concentration (e.g., less than 1 nM level). If this assay system can be extended to other proteases such as hepatitis C virus protease, another type of serine protease, the low detection limit
FIG. 5. Kinetic changes in monomer and excimer fluorescence of pyrene by PS-3 hydrolysis. 0.2 M trypsin was added to the reaction mixture containing 7 M substrate. The emission intensities at 375 and 458 nm under 342 nm of excitation wavelength were chased as described under Materials and Methods.
ASSAY FOR TRYPSIN USING FLUOROGENIC PEPTIDES TABLE 1
Kinetic Parameters for Hydrolysis of Pyrene-Labeled Substrates by Bovine Trypsin Substrate
K m (M)
k cat (s ⫺1)
k cat/K m (M ⫺1 s ⫺1)
PS-1 PS-2 PS-3 PS-4
290 ⫾ 10 100 ⫾ 7 56 ⫾ 2 44 ⫾ 2
3.6 ⫾ 1.4 5.9 ⫾ 2.1 21 ⫾ 3 20 ⫾ 2
1.2 ⫻ 10 4 5.9 ⫻ 10 4 3.8 ⫻ 10 5 4.5 ⫻ 10 5
would be desirable for screening potent inhibitors having K i values in the nanomolar range, as suggested previously (19). Figure 5 shows the progressive fluorescence changes caused by adding trypsin to the PS-3 substrate, which agrees well with our expectation. The cleavage of substrate resulted in dual emission characteristics, an increase in monomer and a decrease in excimer fluorescence proportional to the amount cleaved. But the initial velocity was dependent on the chain length of the substrates used. The kinetic parameters for tryptic cleavage of four substrates are summarized in Table 1. The substrates displayed 5.8- and 6.6-fold variation in k cat and K m, respectively. The substrates PS-3 and PS-4 had significantly elevated activities in the presence of trypsin, emphasizing that the chain length of substrate is important for the substrate– enzyme fit. The k cat/K m parameter for the PS-3 and PS-4 showed more increase than k cat. Two substrates with higher activity also had decreased K m values. This might be ascribed to the increased binding capacity of PS-3 and PS-4 to the protease and/or the enhanced accessibility to the active site of the enzyme compared to other peptides consisting of shorter chain length. The higher K m values of PS-1 and PS-2 seem to be due to the steric hindrance of bulky pyrene moieties. These results indicate that the chain length of substrate is important for the proper substrate– enzyme fit. Considering the synthetic feasibility, the efficient hydrolysis, the fluorescence intensity of monomer/excimer of pyrene, and other factors, we suggest that PS-3 consisting of 9 amino acids is the most practical for measuring the catalytic activity of trypsin. CONCLUSIONS
We developed new fluorogenic peptide substrates for the measurement of proteolytic activity of trypsin utilizing monomer and excimer fluorescence of pyrene incorporated into the substrate. The assay system is
251
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