1 Introduction

Bacterial surface carbohydrates are the first line molecules when interacting with hosts for colonization and or infection. It is not surprising that human pathogens have developed the ability to exploit these molecular recognition events to evade detection by the host defenses, and to use host receptors to dampen the immune response [1] or to aid in colonization [2]. The study of bacterial lipopolysaccharide and capsule biosynthesis has been explored for many bacterial species by both genetic and biochemical means. The development of biochemical tools has helped clarify biosynthetic pathways and has led to the discovery of glycosyltransferases with potential for synthesis of bioactive glycoconjugates. The species which have received the most attention are the human pathogens, where the structures being produced mimic those found in the host: (1) head groups of common glycolipids (e.g., lacto-N-neotetraose, and gangliosides [3,4,5,6]); (2) glycosaminoglycans (hyaluronic acid and unsulfated chondroitin [7]; (3) Lewis blood group antigens [8] and polysialic acid [9].

An excellent and very detailed review of glycosyltransferase structure and function was published by Lairson et al. which outlines many biochemical features of transferases from various sources [10]. Carbohydrate active enzymes have been classified into families in the sequence database for carbohydrate active enzymes, CAZy [11]. At present this database contains 105 families of glycosyltransferases, and 152 families of glycoside hydrolases obtained from the analysis of over 9200 bacterial genomes (as well as ~800 other organisms). Synthetic assay substrates have been used to characterize many bacterial glycosyltransferases from LPS core, LPS O-Chain, and capsule biosynthetic pathways [12,13,14,15]. Many of the synthetically useful bacterial glycosyltransferase enzymes are found in enzyme families having only bacterial members (e.g., CAZy GT-42 and GT-52) , while a few are found in very large families which have representation from all branches of the tree of life (e.g., CAZy GT-2,GT-4, GT-8).

In this chapter, we outline steps to synthesize several fluorescent acceptor molecules to probe various enzyme activities from bacteria which make human-like glycan linkages. The general principals can be applied to a variety of structures and enzyme families provided a suitable sugar acceptor can be found as a starting material. The assays are sensitive enough to probe crude lysates of bacteria to see if the enzyme activity is present. The assay designs use commercial starting materials and can be adapted to produce a wide variety of assay substrates. We provided examples of simple substrate synthesis, or how sequential enzyme reactions can be used to broaden the utility of the substrates.

A number of pathogens make N-acetyllactosamine, which we have illustrated with the conversion of BODIPY-β-GlcNAc to BODIPY-LacNAc (Fig. 1) using the Helicobacter β-1,4-galactosyltransferase (HP0826). Once the fluorescent boron-dipyrromethene (BODIPY) dye is reacted with the sugar acceptor all of these transformations can be followed by thin layer chromatography . A facile reaction to reduce azides to amines is the Staudinger reaction using triphenylphosphine and water. This gentle reaction (Fig. 2) is easily performed in a biochemistry lab setting. N-Hydroxylsuccinimidyl (NHS) ester reaction uses the newly formed primary amine with an activated BODIPY dye under mild alkaline conditions to produce a stable amide bonded labeled product (Fig. 3). The lactose substrate is a convenient acceptor for most bacterial sialyltransferases; this is illustrated with the conversion of BODIPY-lactose (BDP-Lac) to BODIPY-α2,3-sialyl lactose (BODIPY-GM3) (Fig. 4) using the Campylobacter jejuni α2,3-sialyltransferase (CST-I). The BDP-GM3 substrate can be used for other enzyme reactions, illustrated with the conversion of BODIPY-GM3 to BODIPY-α2,8-α2,3-sialyl lactose (BODIPY-GD3) (Fig. 5) using the Campylobacter jejuni α2,8-sialyltransferase (CST-II).

Fig. 1
figure 1

Chemical structure of BODIPY-LacNAc (BDP-LacNAc). This acceptor can be further used to assay for GT-52 sialyltransferases from Neisseria, as well as GT-42 sialyltransferases from Helicobacter species

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Fig. 2
figure 2

Staudinger ligation reaction. The carbohydrates used were R1 = carbohydrate (i.e., azidoethyl-lactose, azidoethyl-β-d-GlcNAc, azidoethyl-β-d-GalNAc)

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Fig. 3
figure 3

N-Hydroxysuccinimidyl (NHS) ester reaction. R1 = carbohydrate (i.e., aminoethyl-lactose/GlcNAc/GalNAc). The reaction is monitored by TLC

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Fig. 4
figure 4

Chemical structure of BODIPY-α2,3-sialyl lactose (BDP-GM3)

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Fig. 5
figure 5

Chemical structure of BODIPY-α2,8-α2,3-sialyl lactose (BDP-GD3)

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The glycosyltransferase β-1,4-N-acetylgalactosaminlytransferase (CgtA) transfers an N-acetylgalactosamine (GalNAc) molecule to the galactose (Gal) in the αNeu5Ac(2–3)βGal disaccharide to make a GM2 mimic (Fig. 6). Discovered originally by Gilbert et al. [16] to be one of the four enzymes responsible for the biosynthesis of the GT1a ganglioside mimic in the lipooligosaccharide of the gram negative bacterial pathogen Campylobacter jejuni OH4384, an engineered version of the enzyme can be produced in recombinant E. coli. CgtA can be used on small molecules, and glycolipids to build oligosaccharide chains, which mimic the gangliosides GM2 or GD2 [16].

Fig. 6
figure 6

The reaction scheme for CgtA addition of N-acetylgalactosamine to a GM3-like substrate

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Sialic acid terminates oligosaccharide chains on microbial cell surfaces, playing critical roles in host recognition and adherence. The enzymes that transfer the sialic acid moiety from cytidine-5′-monophospho-N-acetyl-neuraminic acid (CMP-NeuAc) to the terminal positions of these key glycoconjugates (Fig. 7) are known as sialyltransferases. The bacterial sialyltransferases are found in four distinct protein families, GT38/42/52/80,100. The GT42 enzymes CstI and CstII have been well studied and have both been examined by X-ray crystallography [17, 18]. Polysialic acid capsules are important virulence factors for a handful of pathogens. This homopolymeric capsular polysaccharide has been studied in bacterial pathogens such as Mannheimia haemolytica serotype A2, Neisseria meningitidis group B/C, and Escherichia coli K1/K92 [19]. This modification is also found in the human host—but so far only on a small group of proteins. The role of this capsule in virulence is multifactorial, including resistance to antimicrobial peptides, evasion from phagocytosis, and escape from an endosome if engulfed [20]. These enzymes normally transfer to a glycolipid acceptor which has a sialic acid/ketodeoxyoctulosonic acid terminus, which means that in vitro we need to have at least two sialic acids on the acceptor before the reaction will proceed [15] (Fig. 8).

Fig. 7
figure 7

The reaction scheme for Cst-II addition of sialic acid to a GM3-like substrate to generate GD3-mimic structures

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Fig. 8
figure 8

The reaction scheme for polysialyltransferase (PST) reactions on GD3-mimic structures

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2 Materials

2.1 BODIPY-Labeled Carbohydrates

  1. 1.

    25 mM sodium borate , pH 8.5.

  2. 2.

    C-18 silica gel columns for concentration and desalting of products. These can be self-packed or purchased from a variety of sources (Sigma-Aldrich).

  3. 3.

    200 mM and 500 mM HEPES, pH 7.5.

  4. 4.

    100 mM MgCl2.

  5. 5.

    100 mM CMP-Neu5Ac: (dissolved in 200 mM HEPES).

  6. 6.

    10 mM UDP-gal.

  7. 7.

    Azido-ethyl carbohydrates: lactose, β-d-N-acetylglucosamine (β-GlcNAc) or α-N-acetylgalactosamine (GalNAc) (Sussex Research Chemicals, Ottawa).

  8. 8.

    Triphenylphosphine.

  9. 9.

    Carbohydrate alkylamines.

  10. 10.

    Tetrahydrofuran (THF).

  11. 11.

    Dimethylformamide (DMF).

2.2 Glycosyltransferases

2.2.1 General Materials for Protein Expression

  1. 1.

    2YT media or components thereof.

  2. 2.

    Baffled Erlenmeyer flask(s).

  3. 3.

    Isopropyl β-d-1-thiogalactopyranoside (IPTG) (Sigma).

2.2.2 General Materials for Enzyme Purification

  1. 1.

    DNase A (Roche).

  2. 2.

    Protease inhibitors (Sigma).

  3. 3.

    Lysozyme (BioShop).

  4. 4.

    Emulsiflex (Avestin).

  5. 5.

    Purification buffer(s), vacuum filter sterilized (see specific materials below).

  6. 6.

    Loose resin or prepacked columns (see specific materials below).

2.2.3 Enzyme-Specific Materials: CgtA

  1. 1.

    50 mM HEPES, 150 NaCl, 10% glycerol, pH 7.2.

2.2.4 Enzyme-Specific Materials: MBP-CstI

  1. 1.

    Buffer A: 20 mM HEPES, 200 mM NaCl, 1 mM EDTA, pH 7.2.

  2. 2.

    Buffer B: 20 mM HEPES, 200 mM NaCl, 1 mM EDTA, 10 mM maltose, pH 7.2.

  3. 3.

    Dialysis Buffer: 20 mM HEPES, 100 mM NaCl, 10% glycerol, pH 7.2.

  4. 4.

    Amylose/Dextrose prepacked column for MBP-Trap purification (GE Healthcare).

2.2.5 Enzyme-Specific Materials: CstII

  1. 1.

    Buffer A: 20 mM Tris, pH 8.3.

  2. 2.

    Buffer B: 20 mM Tris, 1 M NaCl, pH 8.3.

  3. 3.

    Buffer C: 20 mM Tris, 500 mM NaCl, pH 8.3.

  4. 4.

    Q Sepharose prepacked column for anion exchange chromatography (GE Healthcare).

  5. 5.

    S-200 Sephacryl HR gel column for size exclusion chromatography (GE Healthcare).

2.2.6 Enzyme-Specific Materials: MBP-PolyST

  1. 1.

    Buffer A: PBS buffer, pH 7.4.

  2. 2.

    Buffer B: PBS buffer, 2 M NaCl, pH 7.4.

  3. 3.

    Heparin Sepharose prepacked column for affinity chromatography (GE Healthcare).

2.3 Small Molecule Activity Tests and Determination of Specific Activity

All enzyme activity tests and assays require five key components: acceptor substrate, activated donor, metal cofactor, enzyme, and buffer. All reactions are carried out in 50 mM HEPES Buffer, pH 7.2. Certain enzymes require additives, such as detergents, for optimization. Table 1 summarizes reaction components for specific enzymes.

  1. 1.

    Chemical solvents (Caledon, all HPLC grade).

  2. 2.

    Acetonitrile (ACN).

  3. 3.

    Ethyl acetate (EtOAc).

  4. 4.

    Methanol (MeOH).

  5. 5.

    Acetic acid (HOAc).

  6. 6.

    Precoated silica, plastic backed analytical TLC sheets (Millipore Sigma).

Table 1 Enzyme reaction components
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2.4 Analysis by TLC or HPLC

  1. 1.

    Plastic-backed silica TLC plate.

  2. 2.

    TLC running solvent: 4:2:1:0.1 or 3:2:1:0.1 (ethyl acetate–methanol–water–acetic acid).

  3. 3.

    Proteomix WAX-NP3 column (Sepax).

  4. 4.

    10 mM NH4HCO3, 20% methanol.

  5. 5.

    500 mM NH4HCO3, 20% methanol.

  6. 6.

    Dionex HPLC system: DNAPac PA-100 guard column (Dionex).

  7. 7.

    Acetonitrile.

  8. 8.

    2 M ammonium acetate pH 7.0.

3 Methods

3.1 BODIPY-Labeled Carbohydrates

3.1.1 Purification Techniques for BODIPY-Labeled carbohydrates Using C-18 Silica Columns

  1. 1.

    Wash 2 × 1 mL C-18 columns by running 20 mL of methanol (MeOH) and 20 mL of water through the columns, successively.

  2. 2.

    Slowly load reaction mixture onto the C-18 column. BODIPY-glycoside should be retained on the column. Wash this column with 10 mL of water.

  3. 3.

    Elute BODIPY-glycoside with approximately 4 mL of MeOH.

3.1.2 Purification Using Preparative Glass-Backed TLC Plates

  1. 1.

    Reduce the total volume of the BODIPY-labeled mixture to approximately 80 μL using a vacuum concentrator.

  2. 2.

    The concentrated BODIPY-labeled mixture is then layered onto a preparative glass-backed TLC plate (20 cm × 20 cm × 1 mm thick) and allowed to completely dry (see Note 1 ).

  3. 3.

    Develop TLC plate using the corresponding solvent mix (Table 2).

  4. 4.

    Allow the developed TLC plate to completely dry in the dark at room temperature (RT) for 10–24 h.

  5. 5.

    Once TLC plate is completely dry, scrape off the BODIPY-labeled band of interest (the mobility of products is slightly different based on it being a monosaccharide or disaccharide) carefully with a Scoopula and transfer into a 50 mL conical tube (Table 3).

  6. 6.

    Wash the silica with 6 × 14 mL of warm water (40 °C) or until it is completely clear. Centrifuge at 1751 × g at RT for 5 min and gently decant the supernatant without including silica. Keep the supernatant as it contains your sample.

  7. 7.

    Purify supernatant by following the method described in the Subheading 3.1.1.

  8. 8.

    Determine the absorbance at 504 nm (ABS504nm). Use MeOH to dilute sample and as a blank. Using ABS504nm, calculate the final concentration of the sample (Extinction coefficient = 80,000 cm−1 M−1).

  9. 9.

    Completely dry down sample using a vacuum concentrator; store the final product at −20 °C.

Table 2 TLC solvent mix ratios used for each BODIPY-labeled substrate
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Table 3 Rf values of BODIPY-labeled carbohydrates in different solvents
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3.1.3 Reduction of the Azide to an Amine and Labeling the Amine with BODIPY-NHS

  1. 1.

    In a microcentrifuge tube, add 10 mg of azido-ethyl carbohydrate (lactose, β-GlcNAc or GalNAc) and dissolve in 500 μL of tetrahydrofuran (THF) and sonicate for 15 min to help dissolve mixture.

  2. 2.

    Add 10 mg of triphenylphosphine into the mixture and sonicate for 1 h. Add 50 μL of water to the mixture to completely dissolve everything.

  3. 3.

    Leave reaction mixture in the dark constantly rotating for 24 h at RT.

  4. 4.

    Manual reverse phase purification of reaction mixture: Wash 2 × 1 mL C-18 silica columns by running 20 mL of methanol (MeOH) and 20 mL of water through the columns, successively. Slowly load reaction mixture onto the C-18 column and then elute with water. The flow-through contains the primary amine product. Collect four 500 μL water fractions in microcentrifuge tubes. The primary amine should be in the flow-through, and some of the early water wash fractions.

  5. 5.

    Using a vacuum concentrator, the fractions are taken to dryness (see Note 2 ).

  6. 6.

    Pool the dried down fractions of the carbohydrate alkylamine by resuspension in 500 μL of sodium borate (25 mM, pH 8.5) and 220 μL of dimethylformamide (DMF).

  7. 7.

    In a separate microcentrifuge tube dissolve 5 mg of BODIPY-NHS ester in 300 μL of DMF.

  8. 8.

    Mix components from items 6 and 7. In a single microcentrifuge tube and incubate at RT while constantly rotating the mixture for 1–2 h.

  9. 9.

    Monitor the progress of the reaction using thin layer chromatography on plastic-backed TLC plates (silica 60) and develop using the solvent mix (Table 2, Fig. 9). The product will have a lower mobility than the free BODIPY compound. Refer to Table 3 for approximate RF values of BODIPY-β-Lactose, BODIPY-α-GalNAc and BODIPY-β-GlcNAc.

  10. 10.

    Purify BODIPY-labeled carbohydrates following the method described in Subheading 3.1.2, steps7–9.

Fig. 9
figure 9

TLC analysis of various reactions with BDP-glycosides. (a) BDP-Lac synthesis from the aminoethyl-lactose made by a Staudinger reaction. Lane 1: starting material, BODIPY-NHS; Lanes 2–3, NHS reaction. During the reaction progress, aminoethyl-lactose is labeled with BODIPY. Lanes 2–3 time points: 55 min and 100 min (b) BDP-GM3 preparative synthesis from BDP-Lac. During the reaction progress, a sialic acid moiety is transferred onto the BDP-Lac resulting in the gradual accumulation of BDP-GM3. Lanes 1–4 time points: 0 min, 15 min, 35 min, and 80 min. (c) BDP-GD3 preparative synthesis from BDP-GM3: During the reaction progress, a sialic acid moiety is transferred onto the BDP-GM3 resulting in the gradual accumulation of BDP-GD3 (target substrate) and BDP-GT3. Lanes 1–3 time points: 5 min, 15 min, and 50 min

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3.1.4 BODIPY-α2, 3-Sialyl Lactose (BODIPY-GM3)

  1. 1.

    For a 1 mL reaction, mix the following final concentrations in a microcentrifuge tube: 2 mM BODIPY-lactose; 50 mM HEPES, pH 7.5; 10 mM MgCl2; 3 mM CMP-Neu5Ac; 0.067 mg/mL CSTI enzyme; and water to make up the final 1 mL volume (see Note 3 ).

  2. 2.

    Incubate reaction mixture at 37 °C.

  3. 3.

    Monitor the progress of the reaction using plastic-backed TLC plates and develop using the solvent mix (Table 3). Usually after 2 h, reaction is ~98% complete (see Note 4 ).

  4. 4.

    Apply reaction mixture to Sep-Pak columns by following the method described in Subheading 3.1.1. BDP-GM3 will elute with MeOH.

  5. 5.

    Purify BDP-labeled carbohydrates following the method described in Subheading 3.1.2.

3.1.5 BODIPY-α2,8-α2,3-Sialyl Lactose (BDP-GD3)

  1. 1.

    For a 1 mL reaction, mix ingredients to achieve the following final concentrations in a microcentrifuge tube: 2 mM BDP-GM3, 50 mM HEPES, pH 7.5; 10 mM MgCl2; 2 mM CMP-Neu5Ac; 0.1 mg/mL CST-II enzyme; and water to make up the final 1 mL volume (see Note 5 ).

  2. 2.

    Follow steps 2–5. From Subheading 3.1.4 (BODIPY-GM3).

3.1.6 BODIPY-N-Acetyllactosamine (BDP-LacNAc)

  1. 1.

    For a 1 mL reaction, mix ingredients to achieve the following final concentrations in a microcentrifuge tube: 0.5 mM BDP-β-GlcNAc, 2 mM UDP-Gal, 50 mM HEPES, pH 7.5; 2 mM MgCl2; 0.75 mg/mL β-1,4-galactosyltransferase (HP0826) crude lysate [21]; and water to make up the final 1 mL volume (see Note 6 ).

  2. 2.

    Incubate reaction mixture at 30 °C.

  3. 3.

    Monitor the progress of the reaction using plastic-backed TLC plates and develop using the solvent mix; EtOAc–MeOH–H2O–HOAc (7:2:1:0.1). Usually after 1.5 h, reaction is ~100% complete. Refer to Table 3 for approximate Rf values.

  4. 4.

    Purify reaction mixture using C-18 silica columns following the method described in Subheading 3.1.1.

  5. 5.

    Determine the final concentration and store the purified sample following the method described in steps 7–9. Of Subheading 3.1.2.

3.2 Glycosyltransferases

3.2.1 Production of Recombinant Glycosyltransferases

All enzymes listed are produced recombinantly in E. coli cells. Plasmid constructs and preferred cell type/strain for production can be found in published literature [17, 19, 22].

  1. 1.

    Using a liquid starter culture, inoculate 250 mL sterile 2YT media in a 1-L baffled Erlenmeyer flask to an OD600 of 0.01.

  2. 2.

    Grow cells at 37 °C until the culture reaches an OD600 between 0.3 and 0.6 or is entering exponential phase of growth.

  3. 3.

    Induce the culture with IPTG to a final concentration of 0.5 mM.

  4. 4.

    Return flask to incubator and reduce the temperature accordingly: CgtA (25 °C), MBP-CstI (25 °C),CstII (30 °C), MBP-PST (20 °C).

  5. 5.

    Continue culture growth for 16–20 h.

  6. 6.

    Harvest cells by centrifugation at 5000 × g for 20 min at 4 °C,decant the supernatant and store the cell pellet at −20 °C until needed.

3.2.2 Purification of Recombinant Glycosyltransferases

Purification of the glycosyltransferases is completed efficiently to preserve the specific activity of the target enzyme. It is vital to do the lysis of the cells and subsequent purification on the same day, and to keep the samples on ice or cold during these processes. Purification buffers and resins are different for each enzyme; however, the general method of purification is similar. In the case of CgtA, this enzyme is used as a clarified lysate and has been found to be inactivated during purification.

  1. 1.

    Resuspend the cells in 15 mL/g of cells with appropriate buffer (see Subheadings 3.1.3–3.1.5). Commercial additives such as DNase, RNase A, protease inhibitors, and/or lysozyme can be added according to the supplier’s directions. We do recommend protease inhibitors for bacterial extracts.

  2. 2.

    Lyse cells with an Emulsiflex at 15000 psi.

  3. 3.

    Centrifuge lysate at 17000 × g for 30 min at 4 °C.

  4. 4.

    Decant the supernatant and ultracentrifuge at 215619 × g for 1 h at 4 °C.

  5. 5.

    Filter supernatant through 0.2 μm syringe filter.

3.2.3 CgtA Purification

  1. 1.

    Aliquot clarified lysate and store at −80 °C, use as needed.

3.2.4 MBP-CstI Purification

  1. 1.

    Purify the enzyme on a 5-mL MBP-trap with a linear gradient of 0–100% Buffer B over 5 column volumes (CV).

  2. 2.

    Dialyze the peak fractions at 4 °C and concentrate as needed.

  3. 3.

    Aliquot and store the purified enzyme at −80 °C, use as needed.

3.2.5 CstII Purification

  1. 1.

    Purify using 5 mL Q Sepharose column (anion-exchange) with a linear gradient of 0–50% Buffer B over 5 CV.

  2. 2.

    Peak fractions (fractions showing activity) are combined and concentrated to <5 mL, then purified using size-exclusion chromatography on a 125 mL Sephacryl S-200 HR gel filtration column with a Buffer C isocratic gradient.

  3. 3.

    Peak fractions are collected and concentrated as needed.

  4. 4.

    Aliquot and store the purified enzyme −80 °C, use as needed.

3.2.6 MBP-PST Purification

  1. 1.

    Purify the enzyme on a 5 mL heparin Sepharose column with a linear gradient of 0–70% Buffer B over 5 CV.

  2. 2.

    Peak fractions (fractions showing activity) are combined and concentrated to a max of 2 mg/mL (if needed).

  3. 3.

    For long term storage add DTT and glycerol to a final concentration of 1 mM DTT and 10% glycerol.

  4. 4.

    Aliquot and store the purified enzyme −80 °C, use as needed.

3.3 Small Molecule Activity Tests and Determination of Specific Activity

Activity tests can be completed as needed throughout the lysis/purification protocols. It may be useful to first test the activity of crude lysates before purification, to ensure an active enzyme was produced. After purification activity tests can be completed on individual fractions, subsequently these “peak fractions” (fractions with high activity) can be combined to achieve a maximum specific activity of the enzyme prep. For specific activity determination, the concentration of the enzyme should be known.

3.3.1 Enzyme Activity Tests

All enzyme reactions have a similar setup (Table 4), using the components listed in Table 1. Read the enzyme specific notes (Table 5) to ensure the reaction is tailored to the target enzyme. Complete the following steps to confirm enzyme activity. A negative control tube should always be prepared with all components except the activated donor.

  1. 1.

    Add all components, except the enzyme, listed in Table 4 to a small Eppendorf tube.

  2. 2.

    If suggested, add additive components from Table 5.

  3. 3.

    Mix components well, spin tube at low speed to collect liquid.

  4. 4.

    Add suggested amount of enzyme (Table 4) to mixture and stir gently with pipette tip.

  5. 5.

    Place tube in incubator at 30 °C for 30 min (see Note 7 ).

  6. 6.

    Quench the reaction with a 1:1 volume ratio of 80% ACN. Spin tube at low speed to collect liquid.

  7. 7.

    Spot all reactions (including negative control) on TLC sheet and resolve in solvent containing 4:2:1:0.1 ratio of EtOAc–MeOH–H2O–HOAc.

  8. 8.

    Image plate with UV or Blue light transilluminator.

Table 4 General set-up for test reactions
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Table 5 Suggested additive components
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3.3.2 Determination of Specific Activity

Specific activity is more accurately calculated with pure enzyme after the concentration of the enzyme is determined. It can be calculated crudely using the TLC plates and Image software such as Image Lab (Bio-Rad), which measures conversion rate by densitometry of the reactant and product “spots” (more qualitative measure) but is best calculated by HPLC on an appropriate column, which measures conversion rate by integration of the reactant and product peaks on a chromatogram (more quantitative measure). For more information see section TLC and HPLC Analysis. Reactions that have a conversion rate in the linear range for the enzyme, usually between 30 and 60%, give a more precise value for specific activity. As such two experiments should be conducted to determine the optimal variables for the calculation. First, the correct dilution or concentration of the enzyme should be defined (this is a value within the linear range of conversion). Second, a time course should be completed to achieve a linear range of conversion (0–100%) over a period of 0–180 min. Analysis of TLC plates or HPLC chromatograms will yield conversion rates (ideally <35%) which can substitute into the equation below to determine the specific activity of the enzyme. The average specific activities of CSTI, CSTII and MBP-PST are listed in Table 6 (see Note 8 ).

Table 6 Average specific activity values for each enzyme discussed
Full size table

3.3.3 Equation to Determine Enzyme activity and Specific Activity of an Enzyme

$$ \mathrm{Enzyme}\kern0.5em \mathrm{activity}\kern0.5em \left(\mathrm{units}/\mathrm{mL}\right)=\frac{\left(\%\kern0.5em \mathrm{conversion}\right)\left(\mathrm{mM}\kern0.5em \mathrm{substrate}\right)\left(\upmu \mathrm{L}\kern0.5em \mathrm{reaction}\kern0.5em \mathrm{volume}\right)}{\left(\min \kern0.5em \mathrm{of}\kern0.5em \mathrm{reaction}\left)\right(\upmu \mathrm{L}\kern0.5em \mathrm{enzyme}\right)}\times 1000. $$
$$ \mathrm{Specific}\kern0.5em \mathrm{activity}\kern0.5em \left(\mathrm{units}/\mathrm{mg}\right)=\frac{\mathrm{Enzyme}\kern0.5em \mathrm{activity}\kern0.5em \left(\mathrm{units}\kern0.5em {\mathrm{mL}}^{-1}\right)}{\mathrm{Enzyme}\kern0.5em \mathrm{concentration}\kern0.5em \left(\mathrm{mg}\kern0.5em {\mathrm{mL}}^{-1}\right)} $$

3.4 Experiments

3.4.1 Experiment 1, Enzyme Dilution Series, Fixed Time Point

  1. 1.

    Follow steps 1 –8 from above (Enzyme Activity Tests), in 6 tubes using the 30 min fixed time point analysis. Reactions in each tube will contain a different dilution of enzyme (the negative control will be absent of enzyme), outlined in Table 7 below.

  2. 2.

    Conversion % of each tube can be determined by TLC or HPLC.

  3. 3.

    Plot the results, dilutions expressed as reciprocals on the x axis and conversion % on the y axis.

  4. 4.

    Choose a dilution within the linear range of the curve and determine the new concentration (mg mL−1). If dilutions do not fall within a linear range, increase or decrease dilutions as needed.

Table 7 Enzyme dilution series for Experiment 1
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3.4.2 Experiment 2: Linear Range of Conversion, Time Variable

  1. 1.

    Follow steps 1–8 from above (Enzyme Activity Tests), in 7 tubes. Each tube will be quenched at a different time point (disregard the fixed time point in step 5). The negative control will be the time zero 0 time point. It is helpful to also prepare a donor-less negative control. See time points outlined in Table 8.

  2. 2.

    Substrate conversion % of each tube can be determined by TLC or HPLC.

  3. 3.

    Plot the results, time in minutes on the x axis and conversion % on the y axis (see Notes 9 and 10 ).

  4. 4.

    Choose a time point within the linear range of conversion and resulting in <35% conversion, substitute the parameters from this reaction into the equation to determine enzyme activity and specific activity using the equation above (see Note 8 ).

Table 8 Time point series for Experiment 2
Full size table

3.5 Analysis by TLC or HPLC

3.5.1 Analysis of Small Molecule tests Using TLC

  1. 1.

    Spot 0.5 μL of the stopped reaction onto the TLC plate.

  2. 2.

    Wait for spots to dry completely; a blow-dryer can be used to quickly dry spots.

  3. 3.

    Develop by placing the plate within a chamber with solvent covering the bottom. The solvent for separating BDP-Lac, BDP-GM3, BDP-GM2, BDP-GD3 and BDP-GT3 is 4:2:1:0.1 (ethyl acetate–methanol–water–acetic acid). The solvent for separating BDP-GD3 and BDP-Sia oligomers is 3:2:1:0.1 (ethyl acetate–methanol–water–acetic acid).

    Spots on TLC plates are visualized on a Bio-Rad EZDOC gel scanner, using either the Blue light or the UV transilluminator tray (see Figs. 10 and 11).

Fig. 10
figure 10

Thin layer chromatography of a polysialyltransferase (PST) in vitro enzyme assay. A) Synthesis of BDP-GM3 with CstI; B) Synthesis of BDP-GD3 with CstII

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Fig. 11
figure 11

Thin layer chromatography of a polysialyltransferase in vitro enzyme assay using BODIPY-GD3 as the substrate. An increase in chain length can be seen during the process of polysialylation over time

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3.5.2 Analysis of BDP-Glycoside Assays Using HPLC with Fluorescence Detection

  1. 1.

    Use a Proteomix WAX-NP3 at 42 °C on an HPLC.

  2. 2.

    Monitor at excitation 504 nm and emission 514 nm (for BDP-labeled compounds).

  3. 3.

    Use the following buffers: A, 10 mM NH4HCO3 + 20% methanol; B, 500 mM NH4HCO3, 20% methanol.

  4. 4.

    Dilute samples with Buffer A until peaks are within range of the detector (5–15 pmol).

  5. 5.

    Products are separated by a gradient from 0–100% B at 0.3 mL/min over 20 min.

3.6 Analysis of Polysialylated Compounds Using HPLC with Fluorescence Detection

  1. 1.

    Dilute the reactions 1:100 with 12.5% acetonitrile.

  2. 2.

    Apply 10 μL of stopped reaction sample, corresponding to 25 pmol of acceptor, to a DNAPac PA-100 guard column (Strong anion exchanger) for separation.

  3. 3.

    Chromatography is conducted at 0.5 mL min−1 with a column temperature of 40 °C, with the mobile phases consisting of acetonitrile (M1) and 2 M ammonium acetate pH 7.0 (M2). Products are separated using an elution gradient from 0 to 50% M2 over 15 min (Fig. 12).

Fig. 12
figure 12

HPLC strong anion exchanger chromatography of a polysialyltransferase in vitro enzyme assay. BDP-GD3 was modified with one or more additional sialic acids. An increase in chain length can be seen during the process of polysialylation, and the individual peaks are the addition of single monomers to the growing polymer

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4 Notes

  1. 1.

    Make sure sample is completely dried on TLC before continuing, or else you will not get a clear separation.

  2. 2.

    Dried down fractions of the carbohydrate alkyl amine should be stored in −20 °C freezer if not in use.

  3. 3.

    BODIPY-β-lactose must be completely dried down before use in this reaction. A speed vacuum concentrator can be used to dry down the substrate.

  4. 4.

    Do not incubate this reaction at 37 °C for very long periods of time as the CST-I enzyme will convert GM3 back to lactose through the reverse reaction.

  5. 5.

    BODIPY-GM3 must be completely dried down before use in the reaction.

  6. 6.

    BDP-β-GlcNAc must be completely dried down before use in the reaction.

  7. 7.

    To visualize conversion rate over a period of time, 1–2 μL samples can be removed from tubes and quenched (e.g., 5, 10, 20, 40, 60 min).

  8. 8.

    Specific activity is usually not determined for the CgtA enzyme since it is used as a crude clarified lysate and the exact concentration of the enzyme in unknown.

  9. 9.

    This plot should have a linear range. If it is not linear please see Note 10 ; troubleshoot and then repeat the experiment.

  10. 10.

    Factors that affect linear range of conversion are (1) too low/too high or fluctuating substrate concentration; (2) too low/too high or fluctuating incubator temperature; (3) inconsistent reaction volume; (4) inconsistency between tubes (pipetting, timing, order of addition).