Fluorescence Anisotropy Assay for the Traceless Kinetic Analysis of

Apr 24, 2008 - A novel fluorescence polarization assay based on the natural fluorophore epicocconone has been developed. This assay allows the rapid a...
0 downloads 0 Views 108KB Size
Download PDF
Anal. Chem. 2008, 80, 4170–4174

Fluorescence Anisotropy Assay for the Traceless Kinetic Analysis of Protein Digestion Felix Cleemann and Peter Karuso* Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, 2109, Australia A novel fluorescence polarization assay based on the natural fluorophore epicocconone has been developed. This assay allows the rapid and accurate determination of enzyme kinetic parameters as well as inhibition constants through the measurement of fluorescence anisotropy on the actual substrate of the protease. It takes advantage of epicocconone’s ability to reversibly react with proteins to form an internal charge-transfer complex that is highly fluorescent. The protein–substrate is labeled in situ without the need for prior incubation and/or derivatization steps, which saves time and effort compared to methods employing specifically labeled protein–substrates. The assay can be carried out in 96- or 384-well plates, making it suitable for high-throughput applications in drug development and biotechnology. Proteolytic enzymes are catalysts for the hydrolysis of amide bonds in peptides and proteins, but their role in cells is far more complex. Members of this class of enzymes are involved in numerous important physiological processes such as protein turnover, digestion (and digestability), fertilization, cell signaling, cell differentiation and growth, wound healing, immunological defense, cancer, and apoptosis.1 Many proteases are validated drug targets, and more will be identified in coming years. Consequently, the study of proteolysis phenomena plays a central role in biochemical, pharmaceutical, and agricultural research and in the food industry. The most commonly used technique to study proteolytic phenomena relies on the cleavage of short synthetic peptide analogues. Cleavage of the scissile bond results in the release of a colorimetric or fluorescent tag, such as 7-amino-4-methylcoumarin, that allows the continuous measurement of protease activity.2,3 Internally quenched peptide substrates have proven to be of utility for the determination of primary sequence specificity.4 A long-range energy transfer between a donor fluorophore on one end of the peptide and a suitable acceptor on the other end is diminished upon cleavage of the peptide. However, the use of synthetic fluorogenic protease substrates fails to address issues of recognition sequence accessibility and protease-protein inter* To whom correspondence should be addressed. Phone: +612-9850-8290. Fax: +612-9850-8313. E-mail: [email protected]. (1) Turk, B. Nat. Rev. Drug Discovery 2006, 5, 785–799. (2) Zimmerman, M.; Ashe, B.; Yurewicz, E. C.; Patel, G. Anal. Biochem. 1977, 78, 47–51. (3) Zimmerman, M.; Yurewicz, E.; Patel, G. Anal. Biochem. 1976, 70, 258– 262. (4) Maly, D. J.; Huang, L.; Ellman, J. A. ChemBioChem 2002, 3, 16–37.

4170

Analytical Chemistry, Vol. 80, No. 11, June 1, 2008

actions because the substrate bears little resemblance to the actual target(s) of the protease. The use of fluorescein isothiocyanate (FITC)-labeled casein as substrate in protease assays is advantageous, as it more closely resembles the natural substrate of the protease.5 Although the method is very sensitive, it requires a high degree of labeling that might alter the reactivity of the substrate substantially. Moreover, it can only be applied to protein–substrates that contain multiple cysteine residues. Alternatively, proteolysis of native protein–substrates can be monitored by time-consuming analysis of quenched reactions using high-performance liquid chromatography,6 capillary electrophoresis,7 polyacrylamide gel electrophoresis,8 or related approaches. Protease assays based on fluorescence anisotropy (FA) present an exception to this limitation as the hydrolysis of proteins can be monitored continuously, in a high-throughput format. FA is sensitive to changes in the rotational motion of fluorescently labeled molecules.9 The steady-state anisotropy r of fluorescently labeled molecules in solution can be calculated by the Perrin equation (eq 1), where τ is the fluorescent lifetime, θ is the rotational correlation time, and r0 is the fundamental anisotropy in the absence of rotational diffusion.

r )

r0 1+τ / θ

(1)

The rotational correlation time θ is directly related to the volume V of the rotating unit by

θ)

ηV RT

(2)

where η is the viscosity of the solution, T is the temperature in Kelvin, and R is the gas constant. Proteolytic breakdown of a large fluorescently labeled protein into smaller peptide fragments is accompanied by depolarization of the solution as the molecular volume V of the hydrolysis products decreases. (5) Twining, S. S. Anal. Biochem. 1984, 143, 30–34. (6) Profumo, A.; Turci, M.; Damonte, G.; Ferri, F.; Magatti, G.; Cardinali, C.; Cuniberti, C.; Rocco, M. Biochemistry 2003, 42, 12335–12348. (7) Olsen, K.; Otte, J.; Skibsted, L. H. J. Agric. Food Chem. 2000, 48, 3086– 3089. (8) Mohanty, A. K.; Simmons, C. R.; Wiener, M. C. Protein Expression Purif. 2003, 27, 109–114. (9) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: New York, 2006. 10.1021/ac7025783 CCC: $40.75  2008 American Chemical Society Published on Web 04/24/2008

Currently used FA assays require protein substrates that are specifically labeled with fluorescein,10–12 tetramethylrhodamine,13 BODIPY dyes,14,15 5-(4,6-dichlorotriazinyl)aminofluorescein,16 or bisarsenical fluorophores that selectively bind to recombinant proteins.17 The additional derivatization step is often costly and time-consuming, rendering these methods unsuitable for routine applications. Because the fluorescent label remains bound to the hydrolysis products it can potentially interfere with the proteolytic enzyme and downstream analyses by HPLC or mass spectrometry. The natural product epicocconone is known to react reversibly with proteins to form an internal charge-transfer (ICT) complex that is highly fluorescent in the relatively hydrophobic environment around proteins but is in rapid equilibrium with unconjugated, nonfluorescent dye.18–20 This mechanism has been exploited for accurate and sensitive protein quantification as well as for real-time monitoring of tryptic digestion as part of proteomic analysis.21,22 Upon enzymatic digestion of protein samples treated with epicocconone, a concentration-dependent decrease in fluorescence is observed. However, monitoring of fluorescence leads to complex behavior as there is a time-dependent increase in fluorescence as the epicocconone reacts with the protein–substrate as well as a decrease in fluorescence as the protein is hydrolyzed. A third process is the slow photoisomerization (or degradation) of the ICT complex, all of which conspire to make kinetic analysis difficult. This work describes the development of a novel real-time protease assay based on FA that allows simple first-order enzyme kinetic analysis of nonderivatized natural substrates by in situ reversible labeling with epicocconone. To explore the scope of the method we have analyzed different protein–substrates, enzymes, and pH’s. Finally, the utility of this method to determine enzyme kinetic parameters (KM, Vmax) as well as inhibition constants (Ki, IC50) in a high-throughput format was investigated. EXPERIMENTAL SECTION Bovine serum albumin (A3059), R-casein (C-6780), apo-transferrin (T2036), carbonic anhydrase (C7025), elastin (E6527), N-tosyl-L-lysine chloromethyl ketone hydrochloride (90182), bicine (B3876), BES (B9879), dithiothreitol (D0632), iodoacetamide (I6125), SDS (L4509), and EDTA (E5134) were purchased from Sigma-Aldrich (St. Louis, MO). Epicocconone (0.5 mg/mL in DMSO) and FluoroProfile were obtained from FLUOROtechnics Pty. Ltd. Sydney, Australia. Epicocconone was used for all proteolysis assays and was freshly (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22)

Bolger, R.; Checovich, W. BioTechniques 1994, 17, 585–589. Maeda, H. J. Biochem. 1980, 88, 1185–1191. Sem, D. S.; McNeeley, P. A. FEBS Lett. 1999, 443, 17–19. Kim, J. H.; Shin, H. J.; Cho, H.; Kwak, S. M.; Cho, H.; Kim, T. S.; Kang, J. Y.; Yang, E. G. Anal. Chim. Acta 2006, 577, 171–177. Jolley, M. E. J. Biomol. Screening 1996, 1, 33–38. Schade, S. Z.; Jolley, M. E.; Sarauer, B. J.; Simonson, L. G. Anal. Biochem. 1996, 243, 1–7. Levine, L. M.; Michener, M. L.; Toth, M.; Holwerda, B. C. Anal. Biochem. 1997, 247, 83–88. Blommel, P. G.; Fox, B. G. Anal. Biochem. 2005, 336, 75–86. Bell, P. J. L.; Karuso, P. J. Am. Chem. Soc. 2003, 125, 9304–9305. Coghlan, D. R.; Mackintosh, J. A.; Karuso, P. Org. Lett. 2005, 7, 2401– 2404. Panda, D.; Datta, A. J. Chem. Sci. 2007, 119, 99–104. Karuso, P.; Crawford, A. S.; Veal, D. A.; Graham, G. B. I.; Choi, H.-Y. J. Proteome Res. 2008, 7, 361–366. Mackintosh, J. A.; Veal, D. A.; Karuso, P. Proteomics 2005, 5, 4673–4677.

prepared as a working solution by diluting the stock 100-fold in the corresponding assay buffer. FluoroProfile was used for all protein quantifications. Dithiothreitol, prepared at a concentration of 200 mM in bicine buffer (100 mM, pH 8.5), was used to reduce protein samples and for neutralizing iodoacetamide. Iodoacetamide, prepared at a concentration of 1 M in bicine buffer (100 mM, pH 8.5), was used for alkylating protein samples. R-Chymotrypsin (Sigma-Aldrich, C4129) was prepared at a concentration of 1.5 mg/mL in bicine (100 mM, pH 8.5). Papain (Sigma-Aldrich, P4762) was prepared at a concentration of 10 mg/mL in BES buffer (100 mM, pH 7.0) containing 1 mM dithiothreitol and 2 mM EDTA. Pepsin (Sigma-Aldrich, P6887) was prepared at a concentration of 1.2 mg/mL in phosphate buffer (100 mM, pH 2.2). Preparation of Protein Samples for Digestion. Protein samples were dissolved in 100 mM bicine (pH 8.5) at concentrations of 7–10 mg/mL. To 100 µL of each protein sample 1 µL of 10% SDS and 5 µL of 200 mM dithiothreitol were added. The samples were incubated at 70 °C for 10 min. Iodoacetamide (1 M; 4 µL) was added, and the solutions were incubated at room temperature for 45-60 min. Residual iodoacetamide was neutralized with dithiothreitol (200 mM; 20 µL) and incubation at room temperature for 45-60 min. The reduced and alkylated protein samples were diluted 10-fold in the corresponding assay buffer. Bicine buffer (100 mM, pH 8.5) was used for papain and R-chymotrypsin digestions, acetate buffer (100 mM, pH 5.5) was used for R-chymotrypsin digestion, and phosphate buffer (100 mM, pH 2.2) was used for pepsin digestion. Real-Time Monitoring of Fluorescence Anisotropy during Protein Digestion. For each digestion experiment 4 wells of a black 96-well microtiter plate (BD Biosciences) were used. These four wells contained 100 µL of protein sample in duplicate and 100 µL of digestion buffer control (i.e., no protein) in duplicate. A freshly prepared solution of epicocconone (100 µL) was added to each well, and the plate was preincubated in the fluorescence plate reader (Spectramax M5, Molecular Devices) at room temperature for 50 min. Protease (5 µL) was added to one protein sample and one control well. Assay buffer (5 µL) was added to each of the remaining two wells, and the fluorescence reading was begun immediately. In the fluorescence polarization mode the fluorescence intensity parallel to the excitation plane (IP) and perpendicular to the excitation plane (IS) were recorded separately (excitation 540 nm, emission 630 nm for red fluorescence and excitation 430 nm, emission 540 nm for green fluorescence). All experiments were carried out at room temperature (25 °C). See the Supporting Information (section 1) for a graphical representation of the process. Data Analysis. The fluorescence progress curves, IP and IS separately, were manipulated by subtracting controls. The fluorescence values obtained from the “buffer only” control were subtracted from the sample containing protein only. Similarly, the fluorescence reading obtained from the well containing protease and buffer was subtracted from the fluorescence readings obtained from the wells containing protein and protease. Fluorescence anisotropy (r) was calculated by using the equation 3. r ) (IP - IS)/(IP - 2IS ) Analytical Chemistry, Vol. 80, No. 11, June 1, 2008

(3) 4171

Figure 1. Time-dependent change of fluorescence intensity (open squares) and fluorescence anisotropy (open triangles) during incubation of BSA with epicocconone (25 °C; pH 8.5, 100 mM bicine buffer).

Duplicates were averaged, and the progress curves were fitted to a single-exponential decay (Prism, version 4.0b, GraphPad Software, San Diego CA) to obtain the rate constants. In cases where the positive control (all reagents except protease) showed a marked decrease in FA, a more accurate analysis was achieved by fitting this data to a single-exponential decay. A two-phase exponential was then fitted to the digestion curves, while fixing the rate constant for the first exponential to that of the positive control. This effectively subtracted any change of anisotropy unassociated with proteolysis (see the Supporting Information). Enzyme Kinetics. The concentration of reduced and alkylated bovine serum albumin (BSA) was determined using the FluoroProfile protein quantification kit.22 The protein solution was diluted out to the required concentration range in bicine buffer (100 mM, pH 8.5), mixed with epicocconone in a ratio of 1:1 (v/v), and 200 µL of the solution was transferred to each well of a 96-well plate. Finally, 5 µL of papain (50-fold dilution in assay buffer) was added. Each concentration was repeated in triplicate, and the fluorescence readings were started immediately (excitation 540 nm, emission 630 nm). Enzyme Inhibition. A stock solution of N-tosyl-L-lysine chloromethyl ketone (TLCK) in DMSO (50.9 mM) was diluted out to the required concentration range in bicine buffer (100 mM, pH 8.5). Five different concentrations (2-fold dilutions) were used. Aliquots (10 µL) were withdrawn and preincubated with 90 µL of papain (10-fold dilution in assay buffer) for 30 min at room temperature. In each well of a 96-well plate 100 µL of reduced and alkylated BSA (4.8 µM) and 100 µL of epicocconone were mixed. Finally, 5 µL of the preincubated enzyme solution was added (in triplicate for each concentration of inhibitor) and the fluorescence reading was immediately started (excitation 540 nm, emission 630 nm). RESULTS AND DISCUSSION In Situ Protein Labeling with Epicocconone. In initial experiments, reduced and alkylated BSA was incubated with epicocconone at pH 8.5 in bicine buffer. The time-dependent change in fluorescence intensity and fluorescence polarization was recorded (Figure 1). During a period of 50 min fluorescence gradually increases upon formation of the red-fluorescent ICT complex. This was expected as the formation of the ICT complex is a chemical process with its own rate constant.19 After the maximum intensity is reached (17–70 min), the fluorescence slowly decreases with time due to photobleaching 4172

Analytical Chemistry, Vol. 80, No. 11, June 1, 2008

Figure 2. First-order exponential decay of fluorescence polarization after adding the protease R-chymotrypsin to the epicocconone-labeled BSA sample (25 °C; pH 8.5, 100 mM bicine buffer). Open triangles are control readings with no added protease, and open squares are with chymotrypsin.

and/or decomposition of the fluorescent adduct (data not shown). In contrast, r reaches a constant value shortly after addition of epicocconone to the protein, reflecting the fact that anisotropy is normalized to the total fluorescence intensity. However, a large excess of free epicocconone in the solution can cause unstable anisotropy due to some background fluorescence carried over from the green into the red from the unconjugated dye. The highest anisotropy was obtained at a ratio of 10:1 BSA/epicocconone where the concentration of unbound epicocconone is negligible. Control experiments showed a simple exponential relationship between the concentration of epicocconone and anisotropy (see the Supporting Information). The influence of epicocconone on the catalytic activity of chymotrypsin was studied by determining the rate of hydrolysis for the fluorescent substrate glutaryl-L-phenylalanine 7-amido-4-methylcoumarin in the presence of varying amounts of epicocconone. Under the standard assay conditions ([epicocconone] ) 6.1 µM) chymotrypsin is reduced in its activity by 30%. Although high concentrations of epicocconone inhibit the enzyme, the assay can be carried out without loss in accuracy at dye concentrations ([epicocconone] ) 1.5 µM) that do not affect enzymatic activity (see the Supporting Information Figure S3). Monitoring of Protein Digestion by Fluorescence Anisotropy. Addition of the protease R-chymotrypsin to the preincubated protein sample resulted in a time-dependent decrease in fluorescence polarization that could be fitted to a first-order exponential (Figure 2). As BSA is cleaved, small labeled peptides are produced which rotate more quickly and thus depolarize the light more readily as it passes through the solution. In the absence of protease the epicocconone-labeled BSA gave a stable anisotropy, indicating no decrease in average molecular size of the epicocconone adduct. The value of r has a theoretical maximum of 0.4, but in practice this theoretical limit is rarely obtained and values of ∼0.3 can be expected for the best fluorophores.9 The values obtained in this assay (generally >0.3) are close to the theoretical maximum, indicating that epicocconone is an optimal choice of fluorophore for anisotropy measurements. This can be attributed to the long Stokes’ shift which minimizes interference from intrinsically fluorescent samples, the essentially nonfluorescent unconjugated dye (in the red), and the short fluorescent lifetime (∼1 ns), typical of small organic fluorophores. The enzymatic rate constants determined from fluorescence decay (k ) 4.0 ± 1.2 × 10-2 µM/min) and fluorescence polarization

Figure 3. Time-dependent change of fluorescence intensity after simultaneous addition of R-chymotrypsin and epicocconone to BSA (25 °C; pH 8.5, 100 mM bicine buffer). Open triangles are control readings with no added protease and show a time-dependent increase in total fluorescent signal. Open squares are with chymotrypsin added and show more complex behavior.

(k ) 3.6 ± 0.2 × 10-2 µM/min) are in good agreement. This result is of particular interest because independent validation by SDS-PAGE proved the correlation between the rate of hydrolysis and fluorescence in real-time monitoring of tryptic digestion.21 Under the investigated conditions (Supporting Information Figure S4) chymotrypsin can be detected to a limit of 1.1 × 10-3 units (one unit hydrolyzes 1 µmol of BSA in bicine buffer, 100 mM, pH 8.5 at 30 °C). Control experiments with no protease showed that preincubation of BSA and epicocconone is not necessary to monitor digestion by FA, as the first-order rate constant is independent of the length of the incubation time. It is also of note that the standard error for the rate constant is much smaller when measured by anisotropy because multiple processes are operating when measuring total fluorescence. The more complex behavior of fluorescence intensity under these experimental settings is the consequence of an overlap of three different processes (Figure 3). Fluorescence increase, caused by formation of the ITC complex, is accompanied by fluorescence decay due to proteolysis and photoisomerization/decomposition. To demonstrate the applicability of this FA assay toward different protein–substrates we repeated the digestion experiment with apo-transferrin (MW 77 kDa), soluble bovine elastin (MW 3–60 kDa), and R-casein (MW 24.5 kDa). In all cases digestion could conveniently be monitored by fluorescence polarization. Even in case of relatively small proteins such as R-casein a high fluorescence polarization (r ) 0.29) was initially obtained (see the Supporting Information). The possibility of monitoring digestion of complex protein mixtures was investigated by subjecting samples of bovine blood plasma and yeast cell proteome to hydrolysis by R-chymotrypsin. The decrease in fluorescence polarization followed an exponential decay and could also be fitted to a simple first-order exponential (Figures 4 and 5). These data indicate that proteolysis of complex mixtures can be followed by a change in anisotropy. Effect of pH on the Assay. Many dyes applied in fluorescence (polarization) assays show a high sensitivity toward changes in pH, rendering them incompatible with the optimum pH of the enzyme of interest. To study the effect of different buffers and pH we monitored BSA digestion by (a) R-chymotrypsin in pH 5.5 acetate buffer and (b) pepsin in pH 2.2 phosphate buffer. Changing the assay buffer from pH 8.5 to pH 5.5 resulted in a reduced overall anisotropy (r ) 0.22 compared to r ) 0.33 at pH

Figure 4. First-order exponential decay of fluorescence polarization after adding the protease R-chymotrypsin to an epicocconone-labeled sample of bovine plasma proteome (30 °C; pH 8.5, 100 mM bicine buffer). Open triangles are control readings with no added protease, and open squares are with chymotrypsin.

Figure 5. First-order exponential decay of fluorescence polarization after adding the protease R-chymotrypsin to an epicocconone-labeled sample of yeast (Saccharomyces cerevisiae) cell proteome (30 °C; pH 8.5, 100 mM bicine buffer). Open triangles are control readings with no added protease, and open squares are with chymotrypsin.

8.5 using BSA as substrate) and a more rapid decrease of anisotropy in the absence of protease. This can be explained by the fact that the red-fluorescent ITC complex is in equilibrium with the free epicocconone and that this equilibrium lies further to the left at acidic pH. Nevertheless, the rate constant of hydrolysis could be accurately determined by taking the background signal into consideration in the subsequent data analysis. At pH 2.2 the intensity of the red fluorescence is too low for precise measurements of fluorescence polarization. Fortunately, at acidic pH the inherent weak green fluorescence of epicocconone is markedly increased in the hydrophobic environment around proteins. Upon excitation at 430 nm and detection at 540 nm a stable anisotropy value could be obtained. As expected, the addition of pepsin was accompanied by a concentration-dependent decrease of fluorescence polarization (Supporting Information). Steady-State Enzyme Kinetic Studies. Hydrolysis of BSA by the cysteine protease papain was chosen as a model system to study the assay’s applicability in steady-state kinetic measurements of enzymatic reactions. In order to determine the enzyme kinetic parameters KM and Vmax the concentration of BSA was varied between 24.0 µM and 750 nM. Initial velocities were determined from exponential fit of the progress curves and fitted to the Michaelis–Menten equation (Figure 6). The kinetic parameters KM ) 1.7 ± 0.2 µM and Vmax ) 5.5 ± 0.2 × 10-4 µM/s are in good agreement with the values obtained from the simultaneous measurement of fluorescence intensity (KM ) 1.4 ± 0.3 µM and Vmax ) 9.0 ± 0.5 × 10-4 µM/s). Again the Analytical Chemistry, Vol. 80, No. 11, June 1, 2008

4173

log[I] against vi/v0. A control experiment using (Z)-Arg-aminomethylcoumarin (7.5 µM, pH 8.5, 100 mM bicine buffer) as substrate gave an IC50 of 370 nM. The fact that the IC50 values do not match closely can be explained by the competitive nature of the inhibition. TLCK competes with the substrates for the binding site of the enzyme. A difference in substrate affinity will result in different IC50 values as the FA method used a real protein (BSA) as substrate, not a pseudosubstrate ((Z)-Arg-aminomethylcoumarin).

Figure 6. Papain-catalyzed hydrolysis of BSA (25 °C; pH 8.5, 100 mM bicine buffer). Plot of the initial velocities against substrate concentration and fit of the data to the Michaelis–Menten equation.

Figure 7. Reaction progress curves, measured by fluorescence anisotropy, after incubation of papain with increasing amounts of the irreversible inhibitor TLCK (25 °C; pH 8.5, 100 mM bicine buffer).

standard errors are lower when using FA than when using fluorescence. Enzyme Inhibition Studies. Inhibition of papain by the irreversible inhibitor TLCK was chosen to investigate the possibility of studying inhibition processes by means of FA.23 The concentration of BSA was kept constant (2.4 µM), and the enzyme was preincubated with increasing amounts of inhibitor before adding the substrate. The experiment was conveniently carried out on a 96-well plate with all samples in triplicate, showing that our method is suitable for high-throughput applications in, for example, drug discovery of specific protease inhibitors. As expected, the initial reaction rates determined by FΑ dropped with increasing amounts of inhibitor (Figure 7). An IC50 value of 50.1 ± 0.1 nM was determined using nonlinear regression by fitting the data to a sigmoidal dose–response curve plotting (23) Wolthers, B. FEBS Lett. 1969, 2, 143–145.

4174

Analytical Chemistry, Vol. 80, No. 11, June 1, 2008

CONCLUSIONS In summary, we have developed a fluorescence polarization assay based on the natural fluorophore epicocconone. This assay allows the rapid and accurate determination of enzyme kinetic parameters as well as inhibition constants through the measurement of FA on the actual substrate of the protease. The protein substrate is labeled in situ but reversibly without the need for prior incubation and/or derivatization steps which saves time and effort compared to methods employing specifically labeled protein substrates. Moreover, epicocconone does not interfere with subsequent mass spectrometric analysis.21 Unlike the use of synthetic peptide analogues, the screening of proteases with unknown substrate specificity becomes possible. The option to scan for red fluorescence at basic pH and for green fluorescence at acidic pH offers the possibility to study a wide range of proteolytic enzymes at their optimum pH on any proteinaceous substrate or even complex mixtures (e.g., foods or entire proteomes). All experiments can be carried out in 96- or 384-well plate format, thus rendering the assay suitable for high-throughput applications in biochemical and pharmaceutical research and the food industry to measure digestibility. ACKNOWLEDGMENT The authors thank the Alexander von Humboldt Foundation for a Feodor Lynen-Fellowship for F.C. SUPPORTING INFORMATION AVAILABLE Effects of varying experimental conditions and nonlinear leastsquares fitting methods. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review December 20, 2007. Accepted March 11, 2008. AC7025783