A Continuous Fluorometric Assay for Trypsin Based on ... - CSJ Journals

doi:10.1246/cl.130713 Published on the web September 18, 2013

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A Continuous Fluorometric Assay for Trypsin Based on Melittin and the Noncovalent-binding-induced Pyrene Excimer Na Xu,1,2 Yue Li,1,3 Hong-Wei Li,1 and Yuqing Wu*1 State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012, P. R. China 2 School of Materials Science and Engineering, Jilin Institute of Chemical Industry, Jilin 132022, P. R. China 3 School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, P. R. China 1

(Received August 7, 2013; CL-130713; E-mail: [email protected]) A continuous fluorometric assay system based on melittin and a pyrene derivative 1 for trypsin and its inhibitor screening is successfully developed by taking advantage of the noncovalent-binding-induced pyrene excimer. The 1melittin assembly and its disassembly after further addition of trypsin are confirmed by the fluorescence changes at 475 nm. Such a system can be applied to other proteases of melittin, and essentially, the concept based on the noncovalent-binding-induced continuous fluorometric assay can be extended to other biochemical analyses. Trypsin, a digestive enzyme, is formed initially as the trypsinogen and can self-cleave to yield a more active form as needed.1,2 It is responsible for the degradation of proteins into peptides and amino acids that are readily absorbed through the intestinal mucosa. Trypsin is a class of protease that plays a key role in controlling the pancreatic exocrine function, and high levels of expression of trypsin correlate directly to some types of pancreatic diseases.35 Therefore, the inhibition of trypsin is considered relevant to medicinal chemistry for the diagnosis and treatment of diseases such as pancreatitis, pulmonary emphysema, rheumatoid arthritis, and asthma.6,7 Therefore, new convenient assays for trypsin and its inhibitor screening are highly desired for developing efficient applications in proteomics. Several methods for trypsin assay have been reported recently;812 however, most are time-consuming or require specialized instruments. Fluorometric methods were reported for trypsin assays based on doubly labeled substrate peptides,1315 and by using a water-soluble conjugated polymer, label-free fluorescence assays for trypsin were also established.16,17 Nevertheless, convenient label-free fluorometric assays for trypsin and inhibitor screening still remain limited.1822 A continuous assay with the ensemble of a tetraphenylethene compound and Arg6 for trypsin and inhibitor screening was successfully developed by Xue et al.18 In the present study, we will report a new label-free continuous fluorometric assay for trypsin and its inhibitor screening by taking advantage of the “turn-off” fluorescence behavior of the noncovalent-bindinginduced pyrene excimer. In our previous study, we reported a unique protein-labeling system with a fast fluorometric switch from the pyrene monomer to the excimer.23 It involved noncovalent binding of the ideally spaced positively charged residues in melittin with an easily available fluorescence probe, N-[4-(1-pyrenyl)butanoyl]-L-tryptophan (1), in exploiting the principle of pyrene excimer formation. Probe 1 is easily synthesized by using a wellestablished procedure.23,24 When excited at 354 nm, the solution

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Figure 1. Illustration of the formation of the pyrene excimer induced by the noncovalent binding between probe 1 and melittin and the consequent disassembly of it in the presence of trypsin. of 1 is dark blue, and it changes to a lighter blue color after the addition of melittin, which is ascribed to the transformation from the pyrene monomer to the excimer. However, the following addition of proteases such as trypsin will be reverse with time being ascribed to the hydrolysis of melittin (Figure 1). Such a phenomenon can be used to quantitatively assay trace amounts of trypsin and further to screen its inhibitors with high throughput. This fluorometric method can also be used to assay other target proteases of melittin, such as cathepsins and matrix metalloproteinases (MMPs). Probe 1 is synthesized by using the previously established method,23,24 and the detailed experimental procedures are listed in ESI.25 The previous results confirmed the direct interaction between 1 and melittin, where the changes in the maximum emission (max) both for Trp19 in melittin and pyrene in 1 were indicated.23 Based on the quenching of Trp19 in melittin, the binding constant (Kb) between 1 and melittin was evaluated to be 4.6 © 1017 with a stoichiometry of 3.6, indicating that there were about four binding sites for 1 at the surface of a melittin. In addition, the interaction of 1 with melittin was also confirmed by directly measuring the fluorescence emission of 1 after it was bound to melittin, and the fluorescence intensity ratio I475/I376 served as a measurement of the pyrene excimer formation.23 Herein, we will demonstrate that the assembly of 1 and melittin, 1melittin, can be employed to further establish a continuous fluorometric assay for a series of proteases such as trypsin and cathepsins. It illustrates that the hydrolysis of melittin by protease will destabilize the aggregated pyrene in 1melittin, and consequently, the fluorescence of the pyrene excimer at 475 nm will be diminished or will even disappear finally. As shown in Figure 2, the initial PBS buffer solution (pH 8.5) of 1 (5.0 ¯M) and melittin (5.0 ¯M) shows strong fluorescence emission at 475 nm, which is attributed to the pyrene excimer.24 However, the fluorescence intensity starts to decrease after incorporating trypsin into the solution, and more fluorescence reduction is observed by prolonging the incubation time, as

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Figure 2. Time-dependent fluorescence spectra of 1melittin (5.0 ¯M/5.0 ¯M) at 37 °C in the absence and presence of trypsin (50.0 ¯g mL¹1) with time.

Figure 3. (left) The time-course plots of the fluorescence intensity at 475 nm in the presences of three different concentrations of trypsin; (right) the trypsin concentrationdependent intensity decrease rate of I475 at the first 20 s after trypsin addition. shown in Figure 2. The fluorescence reduction observed for the pyrene excimer upon the addition of trypsin can also be distinguished by the naked eye under an excitation of 365 nm. In the inset of Figure 2, photos of the solution containing 1melittin in the absence and presence of trypsin are compared. In addition, it should be noted that as control, the fluorescence variation of solo 1 upon the addition of trypsin can be neglected under identical conditions (data not shown). The hydrolysis of melittin should depend strongly on the concentration of trypsin in the system. Therefore, three concentrations of trypsin ®10.0, 25.0, and 50.0 ¯g mL¹1® are employed to hydrolyze melittin in the 1melittin system separately. The ongoing processes are also monitored by measuring the fluorescence intensity changes of the pyrene excimer at 475 nm (I475) with time. Figure 3 shows the time-course plot of I475 in the presence of three different concentrations of trypsin. The corresponding decrease rates of I475 at the first 20 s after trypsin addition are evaluated (Figure 3, right). It is obvious that the fluorescence intensity of I475 decreases more rapidly in the presence of higher concentrations of trypsin. The fluorescence reduction for the pyrene excimer at 475 nm in incubation with trypsin should be ascribed to the disassembly of the pyrene aggregation.23,24 In addition, the quenching of the plot without trypsin, which cannot be eliminated from the system with endeavor, may attribute to the combination of collisional or dynamic quenching, static quenching, etc., as small molecules bound to the protein close to the fluorophore can decrease its quantum yield gradually. In addition, the “turn-off” response of the pyrene excimer emission at 475 nm induced by trypsin may also depend on the concentrations of either melittin or 1 in the 1melittin system. Four solutions of 1melittin constructed from different concentrations of melittin are prepared separately, and in each solution, Chem. Lett. 2013, 42, 15281530

Figure 4. (left) The fluorescence intensity of I475 with hydrolysis time measured at 37 °C for solutions containing 5.0 ¯M probe 1, 50.0 ¯g mL¹1 trypsin, and different concentrations of melittin; (right) the melittin concentration-dependent intensity decrease rate of I475 at the first 20 s after trypsin addition.

Figure 5. (left) The fluorescence intensity of I475 with hydrolysis time measured at 37 °C for solutions containing 5.0 ¯M melittin, 50.0 ¯g mL¹1 trypsin, and different concentrations of probe 1; (right) the probe concentration-dependent intensity decrease rate of I475 at the first 20 s after trypsin addition. the concentrations of probe 1 and trypsin are fixed at 5.0 ¯M and 50.0 ¯g mL¹1, respectively. The fluorescence intensity of I475 with hydrolysis time at 37 °C is also measured and presented for each solution (Figure 4, left), illustrating a lingering reduction of I475 according to the higher concentration of the involved melittin. The decrease rates of I475 in the first 20 s after trypsin addition indicate that they are getting slower as the increase in melittin concentration (Figure 4, right). Actually in this case, the changes in the initial reaction rate are not the true changes in the activity of trypsin, because the substrate (i.e., melittin) is enhanced, but the fluorescence probe 1 is not changed, and hence, the hydrolysis rate cannot be evaluated accurately according to the fluorescence change of I475. In addition, the effects of the concentrations of probe 1 on the diminishing of I475 are also studied by using fixed concentrations of melittin and trypsin at 5.0 ¯M and 50.0 ¯g mL¹1, respectively. The fluorescence intensity change of I475 with hydrolysis time at 37 °C is measured for each solution (Figure 5, left), which illustrates that the extinction rate of I475 increases according to higher concentrations of the involved probe 1. The corresponding extinction rates of I475 for different concentrations of probe 1 are also evaluated from the observation at the first 20 s after trypsin addition (Figure 5, right), which increases with an increase in the concentration of probe 1. Of note is that the residual emission of the pyrene excimer intensity after 100 s at high concentrations of 1 should be attributed to the excessive pyrene excimer, which cannot be disassembled even after all the trypsin has been used up or all the melittin has been digested by trypsin.



1530 implications for diagnostic methods on pancreatic diseases and anticarcinogenic drug discovery.

Figure 6. (left) The time-course plots of I475 for 1melittin (5.0 ¯M/5.0 ¯M) in the presence of trypsin (50 ¯g mL¹1) and four different concentrations of KSTI (0.0, 0.5, 1.2, and 2.0 ¯g mL¹1); (right) the plot of the inhibition efficiency of KSTI toward trypsin versus the concentration of KSTI at 0.0, 0.5, 1.2, and 2.0 ¯g mL¹1. The hydrolysis of melittin by trypsin should be retarded when the corresponding inhibitor of trypsin is added to the solution. Accordingly, a small fluorescence reduction of I475 for the solution containing 1melittin will be expected. Therefore, the assembly of 1melittin and trypsin can be further used to screen the inhibitors of trypsin. Kunitz soybean trypsin inhibitor (KSTI, T2327) from Glycine max is selected to demonstrate the usefulness of the assembly to screen trypsin inhibitors.26 Similarly, the fluorescence intensity at 475 nm for the complex of 1melittin (5.0 ¯M/5.0 ¯M) and trypsin (50.0 ¯g mL¹1) in the presence of different amounts of KSTI (0.0, 0.5, 1.2, and 2.0 ¯g mL¹1) is recorded with time at 37 °C. Figure 6 shows the time-course plots of I475 for the assembly in the presence of four different concentrations of KSTI. As expected, in the absence of KSTI, I475 decreases remarkably with time, but in the presence of KSTI, the degree of fluorescence reduction for the ensemble is reduced gradually. Based on the plot of the inhibition efficiency versus the concentration of KSTI, the corresponding IC50 value of KSTI from soybean toward trypsin is estimated to be 1.41 ¯g mL¹1 (Figure 6, right). The IC50 value is a little different from those determined with other assay methods.26 However, it is still reasonable because the reported IC50 values are affected by several parameters such as the concentrations of trypsin and the substrate. All these results obviously indicate that the assembly of 1melittin can be utilized not only for trypsin activity assay but also for further inhibitor screening. In summary, we have successfully developed a new application of the fluorescence probe of N-[4-(1-pyrenyl)butanoyl]-L-tryptophan (1), a continuous assay method with the assembly of 1melittin for trypsin and its inhibitor screening by taking advantage of the fluorescence switch between the pyrene monomer and the excimer. The formation of the pyrene excimer is driven by the specific interactions between 1 and melittin, which can be disassembled after further addition of trypsin. Compared to the reported assay methods for trypsin, this new fluorometric assay has the following advantages besides being label-free and having continuous features: (1) the assay can be carried out in pure aqueous or buffer solutions, (2) the response time is acceptable, and (3) probe 1 is easily prepared and melittin is commercially available. Thus, this new assay method should be cost-effective and practically applicable. Therefore, this fluorometric assay is useful for the activity assay of protease and high-throughput screening of its inhibitors that may have

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The present research was financially supported by the projects of the Natural Science Foundation of China (Nos. 20934002, 91027027, and 21373101), Jilin Province Natural Science Foundation (No. 20070926-01), and the State Key Laboratory of Supramolecular Structure and Materials, Jilin University. References and Notes 1 D. Schomburg, I. Schomburg, A. Chang, Springer Handbook of Enzymes, 2nd ed., Springer-Verlag, New York, 2002, pp. 1124. 2 M. Hirota, M. Ohmuraya, H. Baba, J. Gastroenterol. 2006, 41, 832. 3 H. Rinderknecht, Dig. Dis. Sci. 1986, 31, 314. 4 P. G. Lankisch, S. Burchard-Reckert, D. Lehnick, Gut 1999, 44, 542. 5 M. F. Byrne, R. M. Mitchell, H. Stiffler, P. S. Jowell, M. S. Branch, T. N. Pappas, D. Tyler, J. Baillie, Can. J. Gastroenterol. 2002, 16, 849. 6 D. Leung, G. Abbenante, D. P. Fairlie, J. Med. Chem. 2000, 43, 305. 7 J. V. Olsen, S.-E. Ong, M. Mann, Mol. Cell. Proteomics 2004, 3, 608. 8 J. M. Artigas, M. E. Garcia, M. R. Faure, A. M. Gimeno, Postgrad. Med. J. 1981, 57, 219. 9 S. Regnér, J. Manjer, S. Appelros, C. Hjalmarsson, J. Sadic, A. Borgström, Pancreatology 2008, 8, 600. 10 R. E. Ionescu, S. Cosnier, R. S. Marks, Anal. Chem. 2006, 78, 6327. 11 V. M. Koritsas, H. J. Atkinson, Anal. Biochem. 1995, 227, 22. 12 M. B. Walker, A. C. Retzinger, G. S. Retzinger, Anal. Biochem. 2006, 351, 114. 13 W. H. Farmer, Z. Yuan, Anal. Biochem. 1991, 197, 347. 14 M. F. Kircher, R. Weissleder, L. Josephson, Bioconjugate Chem. 2004, 15, 242. 15 K. E. S. Dean, G. Klein, O. Renaudet, J.-L. Reymond, Bioorg. Med. Chem. Lett. 2003, 13, 1653. 16 L. An, Y. Tang, F. Feng, F. He, S. Wang, J. Mater. Chem. 2007, 17, 4147. 17 J. H. Wosnick, C. M. Mello, T. M. Swager, J. Am. Chem. Soc. 2005, 127, 3400. 18 W. Xue, G. Zhang, D. Zhang, D. Zhu, Org. Lett. 2010, 12, 2274. 19 X. Gu, G. Yang, G. Zhang, D. Zhang, D. Zhu, ACS Appl. Mater. Interfaces 2011, 3, 1175. 20 W. Xue, G. Zhang, D. Zhang, Analyst 2011, 136, 3136. 21 M. Wang, G. Zhang, D. Zhang, D. Zhu, B. Z. Tang, J. Mater. Chem. 2010, 20, 1858. 22 K. Xu, F. Liu, J. Ma, B. Tang, Analyst 2011, 136, 1199. 23 Y. Li, H.-W. Li, L.-J. Ma, Y.-Q. Dang, Y. Wu, Chem. Commun. 2010, 46, 3768. 24 L.-J. Ma, Y.-F. Liu, Y. Wu, Chem. Commun. 2006, 2702. 25 Supporting Information is available electronically on the CSJ-Journal Web site, http://www.csj.jp/journals/chem-lett/ index.html. 26 R. Roychaudhuri, G. Sarath, M. Zeece, J. Markwell, Biochim. Biophys. Acta, Proteins Proteomics 2004, 1699, 207.

