Enzyme assay and activity fingerprinting of hydrolases with the red ...

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Jul 17, 2008 - 2Protéus SA, 70 Allée Graham Bell, Nımes 30000, France. Correspondence should be addressed to J.-L.R. ([email protected]) ...

PROTOCOL

Enzyme assay and activity fingerprinting of hydrolases with the red-chromogenic adrenaline test Viviana S Fluxa´1, Denis Wahler2 & Jean-Louis Reymond1 1Department

of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, Berne CH-3012, Switzerland. 2Prote´us SA, 70 Alle´e Graham Bell, Nıˆmes 30000, France. Correspondence should be addressed to J.-L.R. ([email protected]).

© 2008 Nature Publishing Group http://www.nature.com/natureprotocols

Published online 17 July 2008; doi:10.1038/nprot.2008.106

The adrenaline test for enzymes is a colorimetric enzyme assay based on the quantification of periodate-sensitive reaction products such as 1,2-diols and 1,2-aminoalcohols by back-titration of the oxidant with adrenaline to produce adrenochrome as an easily detectable red product. The test uses commercial reagents and is suitable for screening the activity of various hydrolases. It is demonstrated here for testing epoxide hydrolases, lipases and esterases, and for activity fingerprinting of these enzymes across substrate series. The complete assay requires 2–3 h.

INTRODUCTION Assays for enzyme activity play a key role in the search for novel enzymes, which are in great demand as components of consumer products (e.g., laundry detergents), industrial processes (e.g., fine chemical synthesis), diagnostics and analytical reagents (e.g., reporter enzymes in immunoassays). In the daily use of enzymes, these assays are required to test enzyme activities in various samples and to investigate the substrate specificity of enzymes1,2. The substrate specificity measured across different substrates forms an activity fingerprint characteristic of the enzyme3. This fingerprint provides an additional tool for identification besides activity. Closely related enzymes may display the same activity but clearly distinguishable activity fingerprints. Activity fingerprinting methods have been reported in various formats including microarrays4 (in particular, for proteases5–7 and lipases8, solid-supported arrays9, etc.) or using substrate cocktails analyzed by HPLC10–12 or by mass spectrometry13–15. However, simple (MTP)-based assays are the most straightforward to implement because they require only minimal instrumentation. Activity fingerprinting of single enzymes as described here is similar to the multi-enzyme profiling methods used in microbiology for strain identification, such as the apizym system16,17. Ideally, enzyme activity assays should produce a simple signal such as a color reaction, as a robust, inexpensive and simple system with commercially available reagents. In addition, the assay should be readily adapted to various substrates. The adrenaline test for enzymes described herein provides such a robust system for the case of hydrolytic enzymes and can be used for both highthroughput screening and activity fingerprinting18. In this test, the fast reaction of adrenaline with sodium periodate to form the red dye adrenochrome can be used to quantitate sodium periodate independent of the presence of proteins or co-solvents at any pH between 2 and 10 in aqueous buffers (Fig. 1). The reaction allows us to measure various periodate-sensitive functional groups such as 1,2-diols and 1,2-aminoalcohols by back titration of sodium periodate (periodate-sensitive groups are oxidized by periodate, which is converted to iodate). These functional groups are produced by the enzyme reactions from nonoxidizable precursors such as the esters or amides of 1,2-diols and amino-alcohols. In general, sodium periodate is a rather selective oxidizing agent; it will react with 1,2-diols, 1,2-aminoalcohols, 2-hydroxyketones, catechols, 1270 | VOL.3 NO.8 | 2008 | NATURE PROTOCOLS

2-aminophenols, thiols and thioethers, but other functional groups are generally unreactive. Thus, the red color indicates the absence of enzyme reaction (control). Conversion of substrate to product by the enzyme results in consumption of periodate and the absence of adrenaline oxidation in the third step and no color, which can be judged qualitatively or measured quantitatively by spectrophotometry. In the various implementations of the adrenaline test, a variety of substrates can be used and an activity fingerprint can be generated using MTPs. Interfacial lipolytic activities under biphasic conditions using various vegetable oils as substrates, which release glycerol upon hydrolysis, can also be detected. It should be mentioned that lipases and esterases have also been profiled in MTPs using pH indicators; however, the pH system must be very carefully controlled in this case19. The adrenaline assay presented here has the advantage of allowing versatile reaction conditions in

O O

O

O

Lipase or esterase

O

OH HO

OH

O (Glycerol)

(Tributyrin) Epoxide hydrolase

O

OH OH

(Phenylethylene glycol)

(Styrene oxide)

2. P consumes NaIO4

1. Enzyme

P

Pox.

(NaIO4-sensitive product)

(Oxidized product)

S (NaIO4-resistant substrate)

NaIO3 + H2O NaIO4

3. The remaining NaIO4 oxidizes adrenaline

MeHN HO

HO O

OH OH

(–)-Adrenaline

N Me

O

Adrenochrome

 max = 490 nm

Figure 1 | Principle of the adrenaline test for enzymes.

© 2008 Nature Publishing Group http://www.nature.com/natureprotocols

PROTOCOL terms of pH, temperature and compatibility, with an interesting range of useful enzymatic reactions in the field of hydrolases (lipases, epoxide hydrolases (EHs), proteases, etc.), some of which are not easy to measure otherwise (EHs). The method employs commercially available and inexpensive reagents and substrates and is suitable for both qualitative (visual) and quantitative measurements of activity for screening and fingerprinting. This protocol describes the procedure for testing and fingerprinting the enzymatic activity of EHs for a set of epoxides and of lipases and esterases using vegetable oils, tributyrin and ethylene glycol bisoctanoate. The general setup for water-soluble substrates uses 10 mM of substrate in the presence of 1 mM NaIO4, such that the first 10% of conversion will be detected. The remaining NaIO4 is then back-titrated with 1.5 mM adrenaline. Under biphasic conditions (e.g., oils), the setup is similar, but the enzyme substrate is used in larger excess. The use of 1 mM of NaIO4 results in the formation of 1 mM or less adrenochrome, which provides an easily detectable gradient in the red color. Adrenaline is used in excess for back titration to ensure rapid and quantitative detection of the remaining periodate. Although 10 mM substrate gave the best results in our hands, it is possible to use higher or lower substrate concentrations at will. The assay is compatible with a variety of substrates within one class, for example, various esters of 1,2-diols or various epoxides, and can be easily applied to measure activity fingerprints of enzymes by setting up parallel assays under identical conditions but with different substrates. This is demonstrated here at the example of activity fingerprinting of an EH using 24 different commercially available epoxides. For the fingerprinting experiment, the protocol also describes how to convert the fingerprint to a grayscale graphical pattern for visual inspection. The general method can be readily transferred to other enzymes by adapting the substrate and reaction conditions. For example, series of polyol acetates derived from carbohydrates and other commercially available polyols can be used for activity fingerprinting of esterases20.

MATERIALS REAGENTS . Enzymes to test (partially purified enzyme preparation or crude enzyme extract can be used) . Adrenaline ((–)-epinephrine), 97% (51-43-4, Fluka-Biochemika, cat. no. 02250) . NaIO4, 99.8% (7790-28-5, Sigma-Aldrich, cat. no. 311448) . KH2PO4, 499.0% (7778-77-0, Sigma, cat. no. P5379) . K2HPO4, 499.0% (16788-57-1, Sigma, cat. no. P5504) . NaCl, 499.5% (7647-14-5, Fluka, cat. no. 71381) . KCl, 499.5% (7447-40-7, Sigma-Aldrich, cat. no. 84426) . HCl puriss, 437% (7647-01-0, Riedel-de Hae¨n, cat. no. 30720) . NaOH, 98% (1310-73-2, Sigma-Aldrich, cat. no. S5881) . Na2HPO4, 499.5% (10028-24-7, Riedel-de Hae¨n—Fluka, cat. no. 30412) . Acetonitrile, 99.8% (75-05-8, Aldrich, cat. no. 271004) . 1-Butene oxide purum (S1) (106-88-7, Fluka, cat. no. 19930) . Butyloxirane purum (S2) (1436-34-6, Fluka, cat. no. 20390) . 2-Hexyloxirane, 96% (S3) (2984-50-1, Aldrich, cat. no. 260258) . 2-Octyloxirane, 95% (S4) (2404-44-6, Aldrich, cat. no. 260339) . 2-Phenyloxirane, 97% (S5) (96-09-3, Fluka, cat. no. 77950) . 2-(3-Butenyl)oxirane, 97% (S6) (10353-53-4, Aldrich, cat. no. 260347) . 2-(5-Hexyl)oxirane, 97% (S7) (19600-63-6, Acros, cat. no. 335140050) . 2-(7-Octenyl)oxirane, 96% (S8) (85721-25-1, Aldrich, cat. no. 410829) . 2-(Isopropoxymethyl)oxirane purum (S9) (4016-14-2, Fluka, cat. no. 50039) . 2-(Butoxymethyl)oxirane, 96% (S10) (2426-08-6, Fluka, cat. no. 19937) . 2-(Isobutoxymethyl)oxirane, 97% (S11) (3814-55-9, Aldrich, cat. no. 473650)

Experimental design The adrenaline test is an endpoint measurement that quantifies the amount of reaction product formed after a selected reaction time. The oxidation steps involved in the measurements are fast and quantitative in the concentration ranges described here. The reproducibility of the measurement is determined by the pipetting accuracy of the experimenter regarding all reagents involved, in particular, the amount of enzyme, substrate and sodium periodate. The typical data fluctuation in MTP experiments with liquid transfer operations using pipettes is ±10%. The accuracy will improve with the precision of the pipetting hardware and by statistical averaging over repeated experiments (duplicates or more). It is also important to note that data accuracy also primarily depends on the careful preparation of all stock solutions (weights and volumes). The assay is described as a qualitative test of activity for the purpose of high-throughput screening or activity fingerprinting, where relative reactivities must be determined. It is in principle possible to convert the obtained color signal to product concentration by carrying out appropriate calibration of the experiment with measured amount of product under identical conditions. In this case, the accuracy will also depend on the purity of the reference product and the precision of the reference solution used for calibration. In all assays, it is important to include a nonactive enzyme preparation as a reference to test whether the adrenochrome signal is stable in the absence of enzyme (these are the ‘blanks’ in the experiments below). The absence of color, that is, the absence of adrenochrome formation, without enzyme may indicate adverse reactions of assay components in the assay (e.g., glycerol, glucose, Tris, which undergo oxidation with NaIO4). This point should be taken into consideration when testing crude cell culture extract, as periodate-sensitive components may be present. Furthermore, the substrate itself may be unstable in the assay medium, either due to its intrinsic chemical reactivity or due to the presence of unwanted enzyme impurities in the assay.

. 2-(Tert-butoxymethyl)oxirane, 97% (S12) (7665-72-7, Fluka, cat. no. 19941) . Octyl/decyl glycidyl ether, technical grade (S13) (68609-96-1, Aldrich, cat. no. 412821)

. 2-((Allyloxy)methyl)oxirane, 97% (S14) (106-92-3, Fluka, cat. no. 5959) . Benzyl glycidyl ether, 99% (S15) (2930-05-4, Fluka, cat. no. 469785) . 2-(Phenoxymethyl)oxirane, 85% (S16) (122-60-1, Fluka, cat. no. 78556) . 7-Oxabicyclo[4.1.0]heptane, 98% (S17) (286-20-4, Fluka, cat. no. 29260) . 6-Oxabicyclo[3.1.0]hexane, 98% (S18) (285-67-6, Fluka, cat. no. 29826) . 1,3-Butadiene diepoxide, 99% (S19) (1464-53-5, Fluka, cat. no. 9366) . 1,4-Butanediyl diglycidyl ether, 95% (S20) (2425-79-8, Fluka, cat. no. 19005) . Neopentyl glycol diglycidyl ether, technical grade (S21) (17557-23-2, Aldrich, cat. no. 338036)

. 1,4-Cyclohexanedimethanol diglycidyl ether, technical grade (S22) (14228-73-0, Aldrich, cat. no. 338028)

. Trans-2,3-dimethyloxirane, 97% (S23) (21490-63-1, Fluka, cat. no. 41031) . Cis-2,3-epoxybutane, 97% (S24) (1758-33-4, Aldrich, cat. no. 294047) . Olive oil (O1), common commercial, suitable for consumption . Sunflower oil (O2), common commercial, suitable for consumption . Grapeseed oil (O3), common commercial, suitable for consumption . Tributyrin, 97% (T) (60-01-5, Fluka, cat. no. 91012) . Ethylene glycol dioctanoate, 98% (G) (627-86-1, Sigma, cat. no. D8907) EQUIPMENT

. Microplate spectrophotometer equipped with a temperature regulator (SpectraMax 250 Microplate Spectrophotometer; Molecular Devices) (see EQUIPMENT SETUP) . Corning 96-well, half-area polystyrene MTPs (Corning, cat. no. CLS3695)

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PROTOCOL

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. Thermal adhesive sealing films for microplates (Sigma, AlumaSeal 96 foil, cat. no. F4550) . Disposable pipette tips (Eppendorf; 10 ml, cat. no. 022492004; 200 ml, cat. no. 022492039; 1,000 ml, cat. no. 022492055) . Single-channel pipette adjustable volume (Eppendorf; 0.5–20 ml, cat. no. 022471902; 10–100 ml, cat. no. 022472003; 20–200 ml, cat. no. 022472054; 100–1,000 ml, cat. no. 022472101) . Multichannel pipette (Eppendorf, 30–300 ml, cat. no. 022452045) . Shaker at 1,200 r.p.m., such as Eppendorf Thermomixer compact (Eppendorf, cat. no. 5350 000.013) . Vortex mixer REAGENT SETUP Epoxides S1–S24 100 mM stock solutions (S1–S24) Dissolve 0.5 mmol of the corresponding substrate in 5 ml of acetonitrile. Epoxide solutions can be stored for up to 8 weeks at 20 1C. Tributyrin 100 mM stock solution (T) Dissolve 293 ml of tributyrin (r ¼ 1.032 g ml–1) in acetonitrile to a final volume of 10 ml. Solution can be stored for up to 8 weeks at 20 1C. Ethylene glycol dioctanoate 100 mM stock solution (G) Dissolve 314.5 mg ethylene glycol dioctanoate in 10 ml of acetonitrile. Solution can be stored for up to 8 weeks at 20 1C. L-Adrenaline salt (1 M HCl) 10 mM stock aqueous solution In a 10-ml volumetric flask, add 18 mg of L-adrenaline ((–)-epinephrine) in 7 ml of water. Using a glass pipette, add 98 ml of HCl 37% to the volumetric flask. Dilute with water up to 10 ml. ! CAUTION HCl is corrosive. For storage, seal the flask with a stopper and paraffin film and store at 4 1C for up to 8 weeks. PBS buffer, pH 7.4 (buffer A) Dissolve 8.0 g of NaCl and 0.2 g of KCl in 300 ml of water. Add 1.44 g of Na2HPO4 and 0.24 g of KH2PO4, then dilute with water

to make a final volume of 1,000 ml. PBS buffer can be used, but any buffer that is not sensitive to sodium periodate is suitable. Do not use Tris, Bis-Tris or triethanol amine. Glycerol, which is sometimes present in enzyme preparations as stabilizer, must also be avoided, although trace amounts will have little influence. The reaction of adrenaline with sodium periodate is independent of proteins or co-solvents at any pH between 2 and 10 (ref. 20). Potassium phosphate buffer, 20 mM, pH 7.2 (buffer B) Make a solution of 40 mM KH2PO4 in water (Solution A) and a 40 mM K2HPO4 in water (Solution B). Mix 28 ml of solution A with 72 ml of solution B, then dilute with water to 200 ml. Measure the pH with a pH meter and adjust if necessary with 1 M HCl or 1 M NaOH. Store for up to 3 months at room temperature (up to 25 1C). Enzyme stock solution 1 mg ml–1 (E1) Dilute 2 mg of the enzyme to 1 mg ml–1 in 2 ml of buffer20. Enzyme stock solution 100 lg ml–1 (E2) Using a micropipette, add 500 ml of E1 in 4,500 ml of buffer. Enzyme stock solution 10 lg ml–1 (E3) Using a micropipette, add 500 ml of E2 in 4,500 ml of buffer. 10 mM NaIO4 in water Dissolve 21.4 mg of NaIO4 salt in 10ml of distillated water. Prepare fresh. Olive oil 1 (O1) Dispense 100 ml of vegetable oil into a microtest tube. Used pure, no further treatment. Sunflower oil 2 (O2) Dispense 100 ml of vegetable oil into a microtest tube. Used pure, no further treatment. Grapeseed oil 3 (O3) Dispense 100 ml of vegetable oil into a microtest tube. Used pure, no further treatment. EQUIPMENT SETUP Spectrophotometer setup Set the spectrophotometer to an absorption wavelength of 490 nm and 3 readings per well.

PROCEDURE Optical density measurement of red dye adenochrome for enzyme activity fingerprinting 1| This step can be performed by using option A (homogeneous assay, e.g., EH activity fingerprinting) or option B (combined homogeneous and heterogeneous assays, e.g., lipases and esterases on vegetable oils, tributyrin and ethylene glycol bisoctanoate). (A) Hydrolitic activity fingerprinting of EHs toward 24 epoxides TIMING 3 h 7 min (i) Preprofiling Experiment: finding the right enzymatic concentration. Choose the temperature for your procedure. This depends on the hydrolytic activity from the enzyme. Room temperature is a good staring point. If you have previously observed poor activity at this temperature with other reagents, we suggest trying 37 1C. (ii) Prepare buffer A, epoxides S1–S24 100 mM solutions (S1–S24), L-adrenaline salt (1 M HCl) 10 mM stock aqueous solution, enzyme stock solution 1 mg ml–1 (E1), enzyme stock solution 100 mg ml–1 (E2), enzyme stock solution 10 mg ml–1 (E3), NaIO4 10 mM in water. (iii) In a 15-ml Falcon tube, mix 6,300 ml of buffer A and 900 ml of a freshly prepared aqueous 10 mM NaIO4 solution using a micropipette. (iv) Using a multichannel pipette, add 80 ml of the solution prepared in Step A(iii) to 88 wells of a 96-well MTP as shown in Figure 2 (all assays are carried out in duplicate: red ¼ S1, S5, S9, S13, S15, S17, S18, S19, S21, S23, S24, enzyme at 100 mg ml–1; yellow ¼ S1, S5, S9, S13, S15, S17, S18, S19, S21, S23, S24, enzyme at 10 mg ml–1; blue ¼ S1, S5, S9, S13, S15, S17, S18, S19, S21, S23, S24, enzyme at 1 mg ml–1; white ¼ control, buffer, NaIO4, epoxide, no enzyme). (v) Using a micropipette, add 10 ml of E1 solution to two wells of the microplate A for each of the 11 substrates (S1, S5, S9, S13, S15, S17, S18, S19, S21, S23, S24, red wells; Fig. 2) to be tested. 1 2 3 4 5 6 7 8 9 10 11 12 (vi) Using a micropipette, add 10 ml of E2 solution to two S5 S9 S13 S15 S17 S18 S19 S21 S23 S24 A S1 wells of the microplate A (yellow wells; Fig. 2) for S5 S9 S13 S15 S17 S18 S19 S21 S23 S24 B S1 each of the 11 substrates to be tested. S5 S9 S13 S15 S17 S18 S19 S21 S23 S24 C S1 S5 S9 S13 S15 S17 S18 S19 S21 S23 S24 (vii) Using a micropipette, add 10 ml of E3 solution to two D S1 S5 S9 S13 S15 S17 S18 S19 S21 S23 S24 E S1 wells of the microplate A (blue wells; Fig. 2) for each S5 S9 S13 S15 S17 S18 S19 S21 S23 S24 F S1 of the 11 substrates to be tested. S5 S9 S13 S15 S17 S18 S19 S21 S23 S24 G S1 (viii) Using a micropipette, add 10 ml of S1 solution to S5 S9 S13 S15 S17 S18 S19 S21 S23 S24 H S1 eight wells of the microplate A (S1 wells; Fig. 2). m CRITICAL STEP When adding substrate solutions, Figure 2 | Diagram of 96-well microplate A for placement of enzyme and using the same micropipette and tip, pipette up and epoxides solutions at different concentrations. Red ¼ enzyme at 100 mg ml–1; down 2–3 times to ensure proper mixing between all yellow ¼ enzyme at 10 mg ml–1; blue ¼ enzyme at 1 mg ml–1. White ¼ reagents. control, buffer, NaIO4, epoxide, no enzyme.



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PROTOCOL 1 2 3 4 5 6 7 8 9 10 11 12 (ix) Using a micropipette, add 10 ml S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 A of S5 solution to eight wells of S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 B the microplate A (S5 wells; S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 C Fig. 2). S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 D (x) Using a micropipette, add 10 ml S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 E of S9 solution to eight wells of S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 F the microplate A (S9 wells; S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 G Fig. 2). S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 H (xi) Using a micropipette, add 10 ml of S13 solution to eight wells Figure 3 | Diagram of 96-well MTP (microplate C) for placement of buffer, NaIO4, epoxides (S1–S24), enzyme and L-adrenaline. Orange: buffer, NaIO4, epoxide solution, enzyme. White ¼ control, buffer, NaIO4, of the microplate A (S13 wells; epoxide solution. Fig. 2). (xii) Using a micropipette, add 10 ml of S15 solution to eight wells of the microplate A (S15 wells; Fig. 2). (xiii) Using a micropipette, add 10 ml of S17 solution to eight wells of 96-well MTP (S17 wells; Fig. 2). (xiv) Using a micropipette, add 10 ml of S18 solution to eight wells of the microplate A (S18 wells; Fig. 2). (xv) Using a micropipette, add 10 ml of S19 solution to eight wells of the microplate A (S19 wells; Fig. 2). (xvi) Using a micropipette, add 10 ml of S21 solution to eight wells of the microplate A (S21 wells; Fig. 2). (xvii) Using a micropipette, add 10 ml of S23 solution to eight wells of the microplate A (S23 wells; Fig. 2). (xviii) Using a micropipette, add 10 ml of S24 solution to eight wells of the microplate A (S24 wells; Fig. 2). (xix) Place a thermal adhesive sealing film that is non-permeable to gas on the microplate A and incubate for 30 min at the selected temperature (Step A(i)). (xx) Meanwhile, prepare a second 96-well MTP (microplate B). By using a multichannel pipette, add 15 ml of L-adrenaline 10 mM stock solution to 88 (11  8) wells of the microplate B, following the placement from Figure 2. (xxi) Remove the adhesive film from the incubated microplate A and using a multichannel pipette, transfer 85 ml of each well to the corresponding well in microplate B, which contains the L-adrenaline solution. (xxii) Incubate microplate B for a further 5 min; set the spectrophotometer for optical density (OD) measurement (l ¼ 490 nm). m CRITICAL STEP Adrenochrome is unstable and polymerizes to brown and finally insoluble black products upon prolonged standing in solution. To quantify periodate concentration by absorbance evaluation, measurements must be done in the first 15 min. (xxiii) Place the MTP in the spectrophotometer and measure the OD. (xxiv) Save the OD measurements. At this stage, you can decide if you have found the right enzymatic concentration to proceed with the hydrolytic activity fingerprinting (see ANTICIPATED RESULTS for an example). ? TROUBLESHOOTING (xxv) Enzyme profiling under optimal conditions. In a 15-ml Falcon tube, mix 7 ml of buffer A and 1 ml of aqueous 10 mM NaIO4 solution using a micropipette. Using a multichannel pipette, add 80 ml of the mixture to all wells of the microplate C as shown in Figure 3 (all assays done in duplicate: orange ¼ assay; white ¼ control, buffer, NaIO4, epoxide, no enzyme). (xxvi) Using a micropipette, add 10 ml of enzyme solution (the optimal concentration was found in Steps A(i–xxiii) to two wells of the microplate C for each substrate to be tested (orange wells; Fig. 3). (xxvii) Using a micropipette, add 10 ml of each epoxide solution (S1–S24) to four wells of the microplate C, as described in the Figure 3. m CRITICAL STEP When adding substrate solutions, using the same micropipette and tip, pipette up and down 2–3 times to ensure proper mixing between all reagents. (xxviii) Place a thermal adhesive sealing film that is non-permeable to gas on microplate C and incubate for 30 min at the selected temperature. (xxix) Prepare a new 96-well MTP (microplate D). Using a multichannel pipette, add 15 ml of L-adrenaline 10 mM stock solution to all wells of the microplate D. (xxx) Remove the adhesive film from the incubated microplate C and using a multichannel pipette, transfer 85 ml of each well to the corresponding well in microplate D, which contains the L-adrenaline solution. (xxxi) Incubate for a further 5 min and set the spectrophotometer for OD measurement (l ¼ 490 nm). (xxxii) Place the microplate C in the spectrophotometer and measure the OD. See ANTICIPATED RESULTS for an example of a plate. (xxxiii) Save the OD measurements. Go to Step B(xxii) to create a graphical fingerprinting pattern.

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PROTOCOL 1 2 3 4 5 6 7 8 9 10 11 12 (B) Lipases and/or esterases activity A on vegetable oils, tributyrin and ethylene glycol bisoctanoate TIMING 2 h B (i) Prepare tributyrin 100 mM C stock solution (T), ethylene D glycol dioctanoate 100 mM E stock solution (G), L-adrenaline F salt (1 M HCl) 10 mM stock aqueous solution, enzyme stock G T T T T T T T T solution 1 mg ml–1 in buffer B H G G G G G G G G (E1), enzyme stock solution 100 mg ml–1 in buffer B (E2), Figure 4 | Diagram for the placement of buffer NaIO4, tributyrin (T), ethylene glycol bisoctanoate (G) enzyme stock solution 10 mg and enzymes in the 96-well microplate E. Red ¼ enzyme at 100 mg ml–1; yellow ¼ enzyme at 10 mg ml–1; ml–1 in buffer B (E3) and NaIO4 blue ¼ enzyme at 1 mg ml–1; white ¼ control, buffer, NaIO4, epoxide, no enzyme. 10 mM in water and vegetable oils (O1–O3). (ii) Prepare potassium phosphate buffer, 20 mM, pH 7.2 (buffer B). ? TROUBLESHOOTING (iii) In a 15-ml Falcon tube, mix 7,940 ml of buffer B and 1,170 ml of a freshly prepared aqueous 10 mM NaIO4 solution using a micropipette. Mix properly. (iv) Using a micropipette, add 310 ml of the mixture prepared in Step B(iii) to 24 microtest tubes of 1.5 ml capacity. (v) Using a micropipette, add 80 ml of the mixture prepared in Step B(iii) to 16 wells of a 96-well MTP (microplate E) (Fig. 4). (vi) Using a micropipette, add 40 ml of E1 solution to six microtest tubes (red tubes; Fig. 5) for each oil to be tested at 100 mg ml–1 of enzyme. (vii) Using a micropipette, add 10 ml of E1 solution to four wells of the microplate E (red wells; Fig. 4) for each tributyrin and ethylene glycol bisoctanoate to be tested at 100 mg ml–1 of enzyme. (viii) Using a micropipette, add 40 ml of E2 solution to six microtest tubes (yellow tubes; Fig. 5) for each oil to be tested at 10 mg ml–1 of enzyme. (ix) Using a micropipette, add 10 ml of E2 solution to four wells of the microplate E (yellow wells; Fig. 4) for tributyrin and ethylene glycol bisoctanoate to be tested at 10 mg ml–1 of enzyme. (x) Using a micropipette, add 40 ml of E3 solution to six microtest tubes (blue tubes; Fig. 5) for each oil to be tested. (xi) Using a micropipette, add 10 ml of E3 solution to four wells of the microplate E (blue wells; Fig. 4) for tributyrin and ethylene glycol bisoctanoate to be tested at 1 mg ml–1 of enzyme. (xii) Using a micropipette, distribute 50 ml of each vegetable oil in the corresponding microtest tubes as shown in Figure 5 (O1–O3). Close all microtest tubes. (xiii) Using a micropipette, add 10 ml of tributyrin 100 mM (T) to eight wells of microplate E (T wells; Fig. 4). (xiv) Using a micropipette, add 10 ml of ethylene glycol bisoctanoate 100 mM (G) to eight wells of microplate E (G wells; Fig. 4). (xv) Set the shaker at 1,200 r.p.m. and incubate the microtest tubes for 30 min (T and G do not require incubation under agitation). Meanwhile, set the spectrophotometer (l ¼ 490 nm). (xvi) Prepare a second 96-well MTP (microplate F) and using a micropipette, add 15 ml of a 10 mM aqueous solution of L-adrenaline to 40 wells of the plate as indicated in Figure 6. (xvii) Using a micropipette, transfer 85 ml from each microtest tube to each of the corresponding wells of the microplate F containing 15 ml of the adrenaline solution (Fig. 6).

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1 O1

O1 O2 O3

O3

O3 O1

O2

O2

O2

O1

O1

O1

O1

O3 O2

O1 O2

O2

O3

O3 O2

O3

O3

Figure 5 | Diagram for the placement of buffer NaIO4, vegetable oils and enzymes in the microtest tubes. Red ¼ O1–O3, enzyme at 100 mg ml–1; yellow ¼ O1–O3, enzyme at 10 mg ml–1; blue ¼ O1–O3, enzyme at 1 mg ml–1; white ¼ control, buffer, NaIO4, epoxide, no enzyme. All assays are done in duplicate. 1274 | VOL.3 NO.8 | 2008 | NATURE PROTOCOLS

2

3

4

5

6

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Figure 6 | Diagram of 96-well MTP F. Placement of L-adrenaline 10 mM stock solution. A quantity of 15 ml in each well is marked in gray. Upper block ¼ tests with vegetable oil. Lower block ¼ tests with tributyrin and ethylene glycol bisoctanoate.

© 2008 Nature Publishing Group http://www.nature.com/natureprotocols

PROTOCOL (xviii) Using a micropipette, transfer 85 ml of each T and G well on microplate E to each of the corresponding wells of the microplate F, which contains 15 ml of the adrenaline solution (Fig. 6). m CRITICAL STEP The average time needed to fill 24 wells with the last component should not go past 2 min. A quick mixing with the micropipette at the moment of addition is sufficient to ensure a homogeneous solution. (xix) Incubate for 5 min and set the spectrophotometer for OD measurement (l ¼ 490nm). m CRITICAL STEP Adrenochrome is unstable and polymerizes to brown and finally insoluble black products upon prolonged standing in solution. To quantify periodate concentration by absorbency evaluation; measurements have then to be done in the first 15 min. (xx) Place the 96-well MTP F in the spectrophotometer and measure the OD. (xxi) Save the OD measurements. Proceed to Step B(xxii) to create a graphical fingerprint pattern. (xxii) Once the OD is measured, diminish values from the Figure 7 | Fingerprint pattern for the measurement of A. niger EH, assaying in –1 corresponding noise value (blanks) by subtracting the adrenaline test (50 mg ml , 26 1C). average of the blank from the sample OD. (xxiii) Reduce data between 0 and 255 integer values by setting the maximal measured value to 255. Calculate as follows: (255  ‘value to reduce’)/‘maximum value’ ¼ reduced value. Round the result to the nearest integer value. ‘Maximum value’ is the maximum value read from the optical densities obtained through all individual measurements done for the fingerprint, and ‘value to reduce’ is each of the other values that you need to transform to a 0–255 integer scale. (xxiv) Invert (mirror image) the values from 255–0 to 0–255. This way, as 255 codes for white and 0 for black, the maximum activity result will be represented as black, and the absence of activity will be seen as the absence of color. (xxv) Save this file in csv format (coma-separated values). (xxvi) Open the file in a text editor and write the following input: P2 ‘#. of columns’ space ‘no. of rows’ 255 Your values

(refers to the maximum value)

(xxvii) Save the file as pgm (portable graymap) by simply replacing the .csv ending with .pgm. pgm files are visible in graphic programs such as Adobe Photoshop. Once opened, you may change the format to jpg or bmp.



TIMING Optical density measurement of red dye Adenochrome for enzyme activity fingerprinting: (A) Hydrolitic activity fingerprinting of epoxides hydrolases toward 24 epoxides Preprofiling experiment: finding the right enzymatic concentration (Steps A(i–xxiv)): Step 1A(i–ii), 30 min; Steps 1A (iii–xviii), 35 min; Steps 1A(xix), 32 min; Steps 1A(xx–xxiv), 29 min Enzyme profiling assay under optimal conditions (Steps A(xxv– xxxiii)): Steps 1A(xxv–xxvii), 8 min; Step 1A(xxviii), 32 min; Steps 1A(xxix–xxxiii), 21 min (B) Lipases and/or esterases activity on vegetable oils, tributyrin and ethylene glycol bisoctanoate Step 1B(i): 30 min; Steps 1B(i–xiv): 27 min; Step 1B(xv): 32 min; Steps 1B(xvi–xx): 21 min Create a graphical fingerprint pattern (Steps (xxii–xxvii)): Figure 8 | Activity fingerprint obtained with the adrenaline test for A. niger Steps 1B(xxii–xxvii), 10 min EH (using 24 epoxide substrates). NATURE PROTOCOLS | VOL.3 NO.8 | 2008 | 1275

PROTOCOL ? TROUBLESHOOTING Troubleshooting advice can be found in Table 1. TABLE 1 | Troubleshooting table.

© 2008 Nature Publishing Group http://www.nature.com/natureprotocols

Step 1A(xxiv)

1B(ii)

Problem Choose the most suitable concentration for your fingerprint profile assay

Possible reasons

Possible solutions An optimal enzymatic concentration is exemplified in Figure 7, where maxima and minima can be rapidly identified

Decoloration is observed in control wells Buffer or media components consume the without enzyme (colorless solution in periodate blanks)

Increase the amount of NaIO4 to consume the oxidizable component. This will work if the component is constant

No activity is observed in the presence of enzyme (red solution)

Low enzyme concentration, low temperature, poor enzymatic activity

Increase the temperature by 5 1C or increase the enzyme concentration by a factor of 10

No activity is observed in the presence of enzyme (red solution)

The substrate(s) chosen is (are) not suitable Try a different substrate compatible with for your enzyme the test

No activity is observed in the presence of enzyme (red solution)

The enzyme is deactivated by the periodate Use a modified setup by adding the (e.g., cysteine proteases) periodate after incubation with the enzyme

Enzyme activity seems too high (colorless solutions, no modulation in activities while fingerprinting)

The enzyme solution contains a periodatesensitive component (glycerol, glucose, etc.)

Confirm the effect by testing if the decoloration occurs without substrate and without any incubation. The problem may be solved by dialysis

Enzyme activity seems too high (colorless solution, no modulation in activities while fingerprinting)

Enzyme activity saturation, high enzyme concentration, high temperature, strong enzymatic activity

Lower the enzyme concentration by a factor of 10 or lower the assay temperature by 5 1C

Solution remains red after increasing temperature and enzymatic concentration

Enzyme shows no activity

Change the pH range of your reaction. The adrenaline test works between pH values of 2 and 10

Enzyme is not active at pH 7.2

Use the optimal pH and buffer for the enzyme. Take care of avoiding oxidizable buffers

Using phosphate buffer, vary the pH between 5.7 and 8. See reagents section for other buffers

ANTICIPATED RESULTS Visual appearance of the assay The visual appearance of the MTP is shown using the example of profiling hydrolytic activities of Aspergillus niger EH under the optimal conditions found (50 mg ml–1 enzyme, 26 1C) (optimized conditions will vary depending on the nature of the enzyme). In Figure 7, each well contains a different epoxide substrate (10 mM) as described in PROCEDURE. Wells that are colored in red indicate an absence of reaction, for example, the control at lower right. Colorless wells indicate that epoxide hydrolysis has taken place. This photo provides a good overview of the color ranges that will be obtained with the assay. In general, obtaining good photos of the plates can be quite difficult, but inspection by eye is very easy. The color is then quantified using an MTP reader. Create a fingerprint from assay As an example, the following steps describe how we obtained the fingerprint pattern for A. niger EH from the OD data. Inversion of data and reduction to a 0 (white) to 255 (black) integer scale (Step B(xxiii)): 215 90 63 38

23 248 183 251

165 251 101 255

48 92 83 94

1276 | VOL.3 NO.8 | 2008 | NATURE PROTOCOLS

68 50 55 59

206 23 74 94

PROTOCOL Change the input and save as pgm file (Step B(xxvii)): P2 6 4 255 220, 46, 174, 68, 87, 212 106, 250, 252, 109, 71, 46 81, 190, 117, 100, 73, 92 60, 252, 255, 109, 79, 109 A. niger EH

© 2008 Nature Publishing Group http://www.nature.com/natureprotocols

Open pgm file in Photoshop or a similar graphic program (Step B(xxvii)) (Fig. 8).

Min. 100

10

Max. 1

µg ml–1

100

10

1

µg ml–1

01 01 Grayscale fingerprints to display activity data The purpose of converting the primary data to grayscale activity 02 02 fingerprints is to facilitate visual inspection of the data. The 03 03 fingerprint display of the data shown in Figure 8 above for activity fingerprinting of EH is shown in Figure 9 (upper). In T T short, the data show that this particular EH reacts with a variety G G of primary epoxides of type R-CHOCH2, except for position D2, E2, D4 and E4, which contain secondary epoxide (cyclohexene oxide, CVL TBE cyclopentene oxide, cis- and trans-2-butene oxide). The data obtained for activity fingerprinting of Chromobacterium viscosum Figure 9 | Activity fingerprints obtained with the adrenaline test for A. niger lipoprotein lipase (CVL) and Thermoanaerobium brockii esterase EH (upper) using 24 epoxide substrates, and for TBE and CVL using three vegetable oils, tributyrin and ethylene glycol bis-octanoate (lower). (TBE) are shown in Figure 9 (lower). The data show that the lipase CVL does not convert tributyrine at all but show good reactivity on the other substrates. By contrast, the esterase TBE shows comparable activity on all substrates. Nevertheless, in both cases, significant activities are only observed for the higher enzyme concentrations used.

Published online at http://www.natureprotocols.com Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions 1. Reymond, J.L. in Enzyme Assays: High-throughput Screening, Genetic Selection and Fingerprinting (Ed. Reymond, J.L.) (Wiley-VCH, Weiheim, 2005). 2. Reymond, J.L. & Babiak, P. Screening systems. Adv. Biochem. Eng. Biotechnol. 105, 31–58 (2007). 3. Reymond, J.L. & Wahler, D. Substrate arrays as enzyme fingerprinting tools. Chembiochem 3, 701–708 (2002). 4. Uttamchandani, M., Huang, X., Chen, G.Y.J. & Yao, S.Q. Nanodroplet profiling of enzymatic activities in a microarray. Bioorg. Med. Chem. Lett. 15, 2135–2139 (2005). 5. Salisbury, C.M., Maly, D.J. & Ellman, J.A. Peptide microarrays for the determination of protease substrate specificity. J. Am. Chem. Soc. 124, 14868–14870 (2002). 6. Diamond, S.L. Methods for mapping protease specificity. Curr. Opin. Chem. Biol. 11, 46–51 (2007). 7. Gosalia, D.N. & Diamond, S.L. Printing chemical libraries on microarrays for fluid phase nanoliter reactions. Proc. Natl. Acad. Sci. USA 100, 8721–8726 (2003). 8. Grognux, J. & Reymond, J.L. A red-fluorescent substrate microarray for lipase fingerprinting. Mol. Biosyst. 2, 492–498 (2006). 9. Babiak, P. & Reymond, J.L. A high-throughput, low-volume enzyme assay on solid support. Anal. Chem. 77, 373–377 (2005). 10. Goddard, J.P. & Reymond, J.L. Enzyme activity fingerprinting with substrate cocktails. J. Am. Chem. Soc. 126, 11116–11117 (2004).

11. Yang, Y.Z. & Reymond, J.L. Protease profiling using a fluorescent domino peptide cocktail. Mol. Biosyst. 1, 57–63 (2005). 12. Park, S. & Shin, I. Profiling of glycosidase activities using coumarin-conjugated glycoside cocktails. Org. Lett. 9, 619–622 (2007). 13. Basile, F., Ferrer, I., Furlong, E.T. & Voorhees, K.J. Simultaneous multiple substrate tag detection with ESI-ion trap MS for in vivo bacterial enzyme activity profiling. Anal. Chem. 74, 4290–4293 (2002). 14. Yang, M., Brazier, M., Edwards, R. & Davis, B.G. High-throughput massspectrometry monitoring for multisubstrate enzymes: determining the kinetic parameters and catalytic activities of glycosyltransferases. Chembiochem 6, 346–357 (2005). 15. Yu, Y., Ko, K.S., Zea, C.J. & Pohl, N.L. Discovery of the chemical function of glycosidases: design, synthesis, and evaluation of mass-differentiated carbohydrate libraries. Org. Lett. 6, 2031–2033 (2004). 16. Gruner, E., Vongraevenitz, A. & Altwegg, M. The Api Zym system—a tabulated review from 1977 to date. J. Microbiol. Methods 16, 101–118 (1992). 17. Sicard, R. et al. Multienzyme profiling of thermophilic microorganisms with a substrate cocktail assay. Adv. Synth. Catal. 347, 987–996 (2005). 18. Wahler, D. & Reymond, J.L. The adrenaline test for enzymes. Angew. Chem. Int. Ed. Engl. 41, 1229–1232 (2002). 19. Liu, A.M.F. et al. Mapping the substrate selectivity of new hydrolases using colorimetric screening: lipases from Bacillus thermocatenulatus and Ophiostoma piliferum, esterases from Pseudomonas fluorescens and Streptomyces diastatochromogenes. Tetrahedron Asymmetry 12, 545–556 (2001). 20. Wahler, D., Boujard, O., Fabrice, L. & Reymond, J.-L. Adrenaline profiling of lipases and esterases with 1,2-diol and carbohydrate acetates. Tetrahedron 60, 703–710 (2004).

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