Microplate Enzyme Assay Using Fluorescence

Original Reference: [Include a citable reference even if you got a protocol from another lab or this website]

Protocol Prepared By: Meg Steinweg ([email protected]) & Shawna McMahon ([email protected]) Date Created: July 18, 2007; Last Updated: January 9, 2012

Fluorescence Enzyme Assay Overview Interpreting soil enzyme activity is crucial in understanding enzyme kinetics and the dynamics of belowground ecosystem processes. Extracellular enzymes (EEAs) produced by bacteria and fungi are involved in innumerable biogeochemical processes and are central to facilitating terrestrial ecosystem nutrient cycling. EEA’s have been studied for decades to better understand decomposition and nutrient cycling. More recently, enzyme activity has been studied in relation to ecosystem responses to global change. Enzymes measurements can either be expressed in terms of molarity or activity. Fluorometric enzyme assays use a difference in the fluorescence of substrate from product to measure the enzyme reaction. Fluorescence is when a molecule emits light of one wavelength after absorbing light of a different wavelength. The general concept of the fluorescence enzyme assay is that synthetic substrates bound with a fluorescent dye are added to soil samples (following our protocol). Enzyme activity is measured as the fluorescent dye is released from the substrate by an enzyme-catalyzed reaction; whereas higher fluorescence indicates more substrate degradation compared to lower fluorescence. Two commonly used synthetic fluorescent indicators are 4-methylumbelliferone (MUB) and 7-amino-4-methylcoumarin (MUC). Fluorescence enzyme assays are typically more sensitive than spectrophotometric assays; but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light, so caution is required when handling fluorescent substrates. This assay is applicable to any enzyme for which there is a fluorescently-labeled (MUB or MUC) substrate.

FLUOROMETRIC METHODS ENZYME ASSAYED SUBSTRATE GENERAL FUNCTION _ β-D-cellubiosidase (CB) 4-MUB- β -D-cellobioside Cellulose degradation α-Glucosidase (AG) 4-MUB-α-D-glucopyranoside Sugar degradation β-Glucosidase (BG) 4-MUB-β-D-glucopyranoside Sugar degradation leucine aminopeptidase (LAP) L-leucine-7-amido-4-methylcoumarin hydrochloride Protein degradation N-acetyl-β-Glucosaminidase (NAG) 4-MUB-N-acetyl- β -D-glucosaminide Chitin degradation Phosphatase (PHOS) 4-MUB phosphate Phosphorus mineralization β-Xylosidase (XYL) 4-MUB-B-D- xylopyranoside Hemicellulose degradation

I. Materials and Assay Preparation

A. Equipment

• 15 mL centrifuge tubes • Pipetting reservoirs (one for each standard concentration (6) x two standards = 12 + one for each substrate = 18 for all 6 substrates) • Centrifuge equipped with holders for deep-well plates • Deep-well plates with lids (8 plates for 12 samples—4 each for samples & standards at 4 temps.) • Waring Blender with small stainless steel blending cup • Small glass dishes • Magnetic stir bars and stir plate • 8-channel repeat pipettor (volume distribution at least 200uL-800uL)

B. Reagents & Solutions

* Most substrates are available from Sigma but because product numbers change often, we’ve only provided the recommended amount of substrate to buy • 4-MUB-B-D- xylopyranoside: 100 mg • 4-MUB-β-D-glucopyranoside: 250 mg • 4-MUB-α-D-glucopyranoside: 100 mg • 4-MUB-N-acetyl- β -D-glucosaminide: 100 mg • 4-MUB- β -D-cellobioside: 100 mg • 4-MUB phosphate: 25 g • L-leucine-7-amido-4-methylcoumarin hydrochloride: 25 mg • 7-amino-4-methylcoumarin (MUC): 250 mg • 4-methylumbelliferone (MUB): 250 mg • 50 mM Buffer * Buffer pH should match the soil pH as closely as possible • For acidic and neutral soils: sodium acetate buffer • For basic soils: Tris buffer * http://www.bioinformatics.org/JaMBW/5/4/index.html is a good buffer calculator

C. Fluorescent Substrate Preparation (200 µM; See Notes ii)

Dissolve the fluorescent substrates in deionized water according to the following list: • β-Xylosidase4-MUB-β-D- xylopyranoside: 6.17 mg/100 mL DI H2O • BG: 4-MUB-β-D-glucopyranoside: 6.77 mg/100 mL DI H2O • AG: 4-MUB-α-D-glucopyranoside: 6.77 mg/100 mL DI H2O • NAG: 4-MUB-N-acetyl- β -D-glucosaminide: 7.59 mg/100 mL DI H2O • CB: 4-MUB- β -D-cellobioside: 10 mg/100 mL DI H2O • Phosphatase: 4-MUB phosphate: 5.12 mg/100 mL DI H2O • LAP: L-leucine-7-amido-4-methylcoumarin hydrochloride: 6.5 mg/100 mL DI H2O Stir and use moderate heat to speed dissolution of substrates. Aliquots: Put 11 mL aliquots into 15 mL centrifuge tubes. Storage: Can be stored frozen (-20°C for up to two months). Thaw in warm water. Do not thaw and refreeze

D. Stock Standard Solution Preparation (1 mM)

• Dissolve 17.6 mg of MUB in 100 mL DI H2O (use a volumetric flask). Dissolve 17.5 mg of MUC in 100 mL DI H2O (use a volumetric flask). • Aliquots: Put 5 mL aliquots into 15 mL centrifuge tubes. • Storage: Can be stored frozen (-20°C for up to two months). • Do not thaw and refreeze.

E. Standard Curve

• Dilute standard stock solution (1 mM=1000 µM) to create a standard curve. Start with a 1:10 dilution of the stock solution to create a 100 µM solution. Make all further dilutions from 100 µM. Aim for 30 mL of each standard for a typical assay run with 12 samples.

• 100 µM = 3 mL 1000 µM stock into 27 mL DI H2O (1:10 dilution) • 50 µM = 15 mL 100 µM into 15 mL DI H2O (1:2 dilution) • 25 µM = 15 mL 50 µM into 15 mL DI H2O (1:2 dilution) • 10 µM = 12 mL 25 µM into 18 mL DI H2O (1:10 dilution) • 5 µM = 15 mL 10 µM into 15 mL DI H2O (1:2 dilution) • 2.5 µM = 15 mL 5 µM into 15 mL DI H2O (1:2 dilution)

• Note: To make different amounts of standard, use the following formula: (High Concentration)(x) = (Low Concentration) (Desired Volume)

x = volume of the higher concentration to add to water Add to volume of water = (desired volume – volume of higher concentration)

E.g. to make 80 mL of 25 µM from 50 µM: (50 µM) (x) = (25 µM) (80 mL) x = 40 mL of 50 µM in 40 mL H2O

• Because of background auto fluorescence, a standard curve must be prepared for each sample. See plate layout for clarification

F. Buffer Preparation

• The buffer you select will depend on the pH of the soil. Below are the instructions for two common buffers: sodium acetate (pKa = 4.76) and Tris (pKa = 8.06). • Note that phosphate buffer has a pKa = 7.2 which could work for more neutral/slightly basic soils. However, high phosphate concentrations may interfere with enzyme activity so use with caution.

50 mM Sodium Acetate Buffer (for 4 L volume) 1. Dissolve 16.4 g sodium acetate in 3L DI H2O 2. Bring the volume up to 4L with DI H2O. 3. Adjust the pH to match soil pH using glacial acetic acid or concentrated sodium hydroxide (~10M NaOH). Storage: Buffer should be made fresh, but can be refrigerated (4°C) for up to a week.

50 mM Tris Buffer 1. Dissolve 6.057 g Tris Base (molar mass = 121.14 g/mol) in 900 mL DI H2O. 2. Titrate to pH 8.2 with concentrated HCl. Storage: Buffer should be made fresh, but can be refrigerated (4°C) for a week. G. Logistics

• Standards and substrates can be prepared up to a month in advance, stored frozen. • On the morning of the assay: • Thaw substrates and required standards (MUC only needs to be thawed if you are doing the LAP assay). • Prepare buffer. • Prepare standard curve (see below for dilutions) • This method is best done with two people (One preparing soil slurries and the other preparing plates). • Two people can easily prepare assays for up to 12 soil samples and 8 substrates in morning (at most 2 hrs.) • Expert practitioners can start two sequential sets of assays in a morning. If you start assays in the afternoon, you will likely have to come in late to read the 15°C plates. Be sure to note incubation times for temperature of interest (See Notes):

Temperature Time 4°C TBD ( ~23 hours but may be less) 15°C 6 hours 25°C 3 hours 35°C 1.5 hours

• Preparing samples to be read on the microplate reader takes one person approximately 10 min. per plate. Total analysis time is typically 1.5 hours over the course of a day for a full set of samples. • Preparing samples to be read on the microplate reader takes one person approximately 10 min. per plate. Total analysis time is typically 1.5 hours over the course of a day for a full set of samples.

II. Tips & Tricks

• You may need to alter the plate layout for your experiment. However, we’ve provided the plate layouts we use for doing all the enzymes at once. • Because of differences in background auto fluorescence, you must prepare a standard curve for each sample separately, hence the large standard plates. • When starting, add the standard solutions prior to slurry addition. (Note, this will allow you to incubate the standard plates immediately after the soil slurry is added. This is helpful because they can be read on the microplate before the assay plate. If you don’t do this then there will a time crunch because at 25°C and 4°C, you will need to measure two standard plates and the sample- assay plate at the same time (and it takes ~10 minutes per plate), so you want to spread this out if possible. • Do NOT add the substrates until after all the soil slurries have been added to the wells; otherwise you’ll have uneven incubation time. • The blank graphical representation of the plate may also be useful. Blank graphical representation of the plate:

1 2 3 4 5 6 7 8 9 10 11 12

A

B

C

D

E

F

G

H

Plate Layouts: Enzyme Activity Samples

1 2 3 4 5 6 7 8 9 10 11 12

A S1+ BG S2+ BG S3 + BG S4+ BG S5+ BG S6+ BG S7+ BG S8+ BG S9+ BG S10+ BG S11+ BG S12+ BG

B S1+CB S2+CB S3+CB S4+CB S5+CB S6+CB S7+CB S8+CB S9+CB S10+CB S11+CB S12+CB S1+ S2+ S4+ S5+ S6+ S7+ S8+ S9+ S10+ S11+ S12+ C S3+NAG NAG NAG NAG NAG NAG NAG NAG NAG NAG NAG NAG S1+PHO S2+PHO S3+PHO S4+PHO S5+PHO S6+PHO S7+PHO S8+PHO S9+PHO S10+PHO S11+PHO S12+PHO D S S S S S S S S S S S S S1+ S3+ E S2+XYL S4+XYL S5+XYL S6+XYL S7+XYL S8+XYL S9+XYL S10+XYL S11+XYL S12+XYL XYL XYL F

G

H S1 +LAP S2 +LAP S3 +LAP S4+LAP S5+LAP S6+LAP S7+LAP S8+LAP S9+LAP S10+LAP S11+LAP S12+LAP

• Note: Rows F and G can be used for other MUB based enzyme measurements.

Plate Layouts: MUB and MUC Standard Curve (4C and 25C)

1 2 3 4 5 6 7 8 9 10 11 12 A S1+ std0 S2+ std0 S3+ std0 S4+ std0 S5+ std0 S6+ std0 S7+ std0 S8+ std0 S9+ std0 S10+ std0 S11+ std0 S12+ std0 B S1+std2.5 S2+std2.5 S3+std2.5 S4+std2.5 S5+std2.5 S6+std2.5 S7+std2.5 S8+std2.5 S9+std2.5 S10+std2.5 S11+std2.5 S12+std2.5 C S1+ std5 S2+ std5 S3+ std5 S4+ std5 S5+ std5 S6+ std5 S7+ std5 S8+ std5 S9+ std5 S10+ std5 S11+ std5 S12+ std5 D S1+std10 S2+std10 S3+std10 S4+std10 S5+std10 S6+std10 S7+std10 S8+std10 S9+std10 S10+std10 S11+std10 S12+std10 E S1+ std25 S2+ std25 S3+ std25 S4+ std25 S5+ std25 S6+ std25 S7+ std25 S8+ std25 S9+ std25 S10+ std25 S11+ std25 S12+ std25 F S1+ std50 S2+ std50 S3+ std50 S4+ std50 S5+ std50 S6+ std50 S7+ std50 S8+ std50 S9+ std50 S10+ std50 S11+ std50 S12+ std50 S1+ S2+ 31+ S4+ S5+ S6+ S7+ S8+ S9+ G S10+std100 S11+std100 S12+std100 std100 std100 std100 std100 std100 std100 std100 std100 std100 H

• Note: S1…S12 = samples 1-12; std0 - std100 = standard concentrations III. Procedure

A. Assay Set-Up

1. Prepare standard plates by pipetting 200 µL of appropriate standards into correct wells of MUB or MUC standard plates (oriented in rows). * When you add soil in Step 5, record the time and read these plates after the same amount of elapsed time as the enzyme assay plates at the corresponding temperature. • Prepare two plates for each standard (MUB & MUC) for a total of four plates. One set is incubated at 4°C and the other at 25°C. • The 25°C standard curve is used for all but the 4°C assay (15°C, 25°C & 35°C). 2. Prepare soil slurries by weighing 2.75 grams of field moist soil into blender. • The soil amount can be altered, just be sure to adjust the buffer amount proportionally. • Be sure to measure the gravimetric water content so that dry weight (d.w.) can be calculated—enzyme activity is typically presented on a g-1 soil d.w. basis. 3. Add 91 mL of 50 mM buffer. 4. Blend contents on high for 1 min. 5 Pour contents into a glass bowl, add stir bar and place on stir plate. Mix on low. • Bowl must be wide enough to accommodate the 8-channel pipette. • Stirring gently helps keep soil suspended and minimize variation due to different amounts of soil present in each well. 6. Pipette 800 µL of soil slurry into wells for enzyme activity measurement and standard curves (see plate layouts). Each sample should be in a separate column. 7. Rinse blender with DI H2O or buffer between samples. Repeat Steps 2-7 for each sample. 8. Pipette 200 µL of appropriate 200 µM substrate into correct assays wells (oriented in rows). * Record the time of substrate addition and try to start all plates/substrates as quickly as possible. Note: Steps 1 & 8 are mostly easily performed with only six (6) pipette tips loaded on the repeat pipette (two additions across the plate for a total of 12).

B. Incubation & Analysis

1. Seal the plates with plate mats and mix by hand (invert about 10 times). 2. Place plates in appropriate incubators for required incubation period (See Notes).

Temperature Time 4°C TBD (originally ~23 hours but may be less) 15°C 6 hours 25°C 3 hours 35°C 1.5 hours

3. When incubation is complete, centrifuge plates for 3 min. at 1500 rpm (~350 xg). 4. Transfer 250 µL from each well into corresponding well in a flat-bottomed black 96-well plate. 5. Add 5 µL of 0.5N NaOH to each sample. * Want final pH in wells to be ~10. • Adjust normality as necessary for pH of soil. Note: this step may be omitted without significant loss of sensitivity in high pH soils.

6. Following manufacturer’s instructions for your plate reader, measure fluorescence using the following parameters:

Excitation Wavelength = 365 Emission Wavelength = 450

Additional instructions for the Tecan Infinite M200 microplate reader:

1. Select 10 reads but no multiple reads per well.

2. Open Excel and make sure the correct destination file is open. • The Tecan will put the data in a new worksheet (tab) in whatever spreadsheet is open. Label each tab or put descriptive information in each worksheet as it is generated. Information needed, sample #s, MUB or MUC sample, MUB or MUC standard, gain of the microplate reader. If you are using numbers that don’t mean anything, then do a summary sheet on each workbook that explains the numbers. • i.e. Sample 1 is plot 25 0-5cm, Sample 2 is plot 25 5-15cm, etc…

3. Run the standard plate at optimal gain. The highest measured value should be for the 100 µM standard. Note the optimal gain determined by the machine. If you are doing the 35°C temp then you won’t have the standard information yet, so be careful, you may need to drop the gain more than 5 units. I would suggest dropping it 5 units, run the sample and then drop it again 5 units and run the sample again. That way when you run the 25°C standards 1.5hr later you have another option if the 100 µM standard is still too high for the first 5unit gain drop (another trick of the trade).

4. Set the gain manually to 5 units below the optimal gain from the standard plate for all other plates with the same soil samples. * The gain dramatically changes the measured values. However, it may be necessary to drop the gain even further for some samples if the value exceeds detection limits (indicated by a red OVER in the spreadsheet output). It is CRITICAL that the standard plates are read at the same gain as the samples else the sample values are MEANINGLESS.

IV. Data Analysis & Calculations

A) Standard curve analysis (easy to follow 3 step procedure) 1) Enter standard curve data into spreadsheet (example for MUB)

MUB Standard dilution Standard + Sample (uM) Fluorescence 0 124 2.5 1127 5 2093 10 3967 25 10051 50 19315 100 31709

2) Convert uM standard dilution into umols i.e. convert 100uM standard into umols 200uL in each well = .0002L .0002L * (100umols/L) = .02umols

MUB Standard dilution Standard + Sample (umols) Fluorescence 0 124 0.0005 1127 0.001 2093 0.002 3967 0.005 10051 0.01 19315 0.02 31709

3) Plot standard curves (fluorescence on Y, standard conc. on X) in Excel for the MUB and MUC standards for each soil sample. Calculate the slope, intercept & R2. • We often calculate two standard curves from each standard curve to discriminate resolution for high activity and low activity enzymes, but this isn’t necessary as standard curves are linear. o Low Activity: plot 0-25 µM standards o High Activity: plot 10-100 µM standards . Note: Accept the standard curve if r2 ≥ 0.98.

Slope -- Intercept -- Standard + Sample R2 Fluorescence Low slope 1982918 Low intercept 101.4399 Low R-squared 0.99979 High slope 1414657 High intercept 3854 High R-squared 0.98862

B. Calculating the enzyme activity (easy to follow 4-step procedure; also see notes iii below): The following is a sample data example incubated for 3 hours (at 25C)

Sample Enzyme Fluorescence BG 3491 CB 5373 NAG 4709 PHOS 23849 XYL 7744 AG 5574

1) Use y=mx+b to solve for unknown concentrations (x), where: y = sample fluorescence x = enzyme concentration m = slope from standard curve b = intercept from standard curve sample enzyme concentration Note: x = sample fluorescence – std. curve intercept ÷ std. curve slope

slope Enzyme Sample µmols resolution BG 0.001709 Low CB 0.002658 low NAG 0.002324 low PHOS 0.014134 high XYL 0.003854 low AG 0.00276 low

2) Multiply sample µmols (obtained in step 1) by the volume of buffer used in soil slurry. Example: (sample µmols) * (91) (Note: 91ml is the volume of buffer used for soil slurry in this protocol)

Enzyme Sample µmols BG 0.155554 CB 0.241922 NAG 0.21145 PHOS 1.286209 XYL 0.350732 AG 0.251147

3) Divide sample µmols value (obtained in step 2) by: (incubation time (hrs.) * amount (g) of dry soil used to make slurry * 0.8). Note: 0.8mL is the volume of slurry used in each well. Example: sample µmols step (2) ÷ (incubation hrs.)*(g dry soil used in slurry)*(0.8)

Sample µmols gram dry soil / Enzyme h BG 0.026294 CB 0.040893 NAG 0.035742 PHOS 0.217412 XYL 0.059285 AG 0.042452

4) Multiply the sample µmols value (obtained in step 3) by 1000 to get the correct activity units. Note, this corrects for the actual soil amount in each well. Example: (sample µmols value from step 3) * (1000)  (nmol activity/g dry soil/h)

Sample nmols/gram dry soil / Enzyme h BG 26.29371 CB 40.89288 NAG 35.74206 PHOS 217.412 XYL 59.28535 AG 42.45209

V. Notes

i. The incubation times suggested here may need to be altered for your soils because of differences in activity. If your soils have more activity, you may need to incubate for less time. Thus, we recommend optimizing your incubation times by measuring a ‘test’ sample across each incubation temperature/time (note: for more information see Incubation & Analysis section under Procedure).

ii. We empirically determined that 200 µM substrate concentrations are excessive for our samples. For best practices, substrate concentrations should be optimized by testing a range of concentrations.

iii. It is best to check the background fluorescence on the substrates in buffer solution only. For some substrates the background values are low and do not change significantly with different concentrations. However, it is possible for the substrate - background fluorescence to change considerably; and in these cases, the difference will need to be included in the calculation of activity. a. To test for background substrate fluorescence activity: Add 800uL of buffer to a well. Then add 200µL of substrates. Incubate (for specified length of time based on incubation temperature; note: Incubation & Analysis section). Measure fluorescence after incubation period. If the fluorescence measure is equivalent to the 0µM MUB standard background (i.e. DI water), then overall, background fluorescence is not a concern for that substrate. If the substrate background in much higher than the fluorescence value needs to be subtracted from the sample + substrate fluorescence value prior to calculating enzyme activity. i. Example: background fluorescence – (sample + substrate fluorescence)

VI. Safety

• This is a relatively benign analytical technique. Wear gloves if you are sensitive and consider safety glasses.