Hermanson G. Bioconjugate Techniques, Second Edition

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730 18. Discrete PEG Reagents

5. Purify the biotinylated protein from excess reagent and reaction by-products using dialysis

or gel ﬁ ltration (desalting resin).

3.2. NHS-Chromogenic-PEG

-Biotin

A novel detectable biotinylation reagent containing a hydrophilic PEG spacer is NHS-

chromogenic-PEG

-biotin (also called chromogenic biotin; Thermo Fisher, Solulink; Figure 18.15 ).

Next to the terminal NHS ester of this compound is a bis-aryl hydrazone group created from

the reaction of a 6-hydrazinium nicotinate derivative and a benzaldehyde group to form the

chromogen having an absorbance at 354 nm (   29,000 M

 1

). The NHS ester end can

be used to modify amine-containing molecules and form a stable amide linkage ( Figure 18.16 ).

The spacer arm contains a hydrophilic spacer made from three ethylene oxide units, which

provide water solubility for the compound.

Proteins biotinylated with this reagent will have a characteristic absorbance band at 354 nm,

which can be used to determine accurately the number of biotin groups per molecule. No other

biotinylation compound has such built-in quantiﬁ cation capability. This feature eliminates the

need to consume conjugate by doing a HABA assay to test for the level of biotin incorporation

(Chapter 23, Section 7).

The following protocol is adapted from the manufacturers ’ recommendations.

Protocol

1. Dissolve a protein or other amine-containing molecule to be biotinylated in 0.1 M

sodium phosphate, 0.15 M NaCl, pH 7.2–7.5, at a concentration of 1–10 mg/ml. Note

Figure 18.15 NHS-chromogenic-PEG

-biotin contains an amine-reactive NHS ester that can be used to label

biomolecules through an amide linkage. The chromogenic bis-aryl hydrazone group within the spacer arm of

the reagent allows the degree of biotinylation to be quantiﬁ ed by measuring its absorbance at 354 nm. The

compound also contains a hydrophilic PEG spacer, which provides greater water solubility.

3. Biotinylation Reagents Containing Discrete PEG Linkers 731

Figure 18.16 NHS-chromogenic-PEG

-biotin reacts with amine groups in proteins or other molecules to form

amide bond derivatives.

732 18. Discrete PEG Reagents

that protein solutions that are more dilute than this may require higher levels of bioti-

nylation reagent addition to achieve the same yield of modiﬁ cation.

2. In a fume hood, dissolve NHS-chromogenic-PEG

-biotin in DMF at a concentration of

12.33 mM (2 mg/200 l DMF). With mixing, add a quantity of the reagent to the protein

solution to provide the desired molar excess (i.e., 10- to -20 fold excess).

3. React for 30–60 minutes at room temperature or 2 hours at 4°C.

4. Purify the modiﬁ ed protein from unreacted biotinylation reagent and reaction by-prod-

ucts using dialysis or gel ﬁ ltration. Complete removal of the excess reagent is necessary

to provide accurate measurement of the biotin incorporation level by absorptivity.

5. Measure the absorbance of the biotinylated protein solution at 354 nm. Use the molar

extinction coefﬁ cient for the chromogenic group (   29,000 M

 1

) to determine the

concentration of biotin present. To determine the molar ratio of biotin-to-protein, divide

the molar concentration of biotin by the molar concentration of protein present (which

may be determined by using the Coomassie assay or the BCA assay methods).

3.3. Maleimide–PEG

–Biotin Compounds

Discrete PEG–biotin compounds containing a terminal maleimide group may be used to label sulf-

hydryl-containing proteins and other molecules through thioether bond formation ( Figure 18.17 ).

The targeting of thiol groups in proteins often is used to direct the modiﬁ cation reaction away

from binding sites or active centers in proteins, thus preserving activity. Maleimide reagents in

general are the second most-popular reactive group used for bioconjugation purposes, second only

to NHS esters. Unlike biotinylation compounds containing a hydrophobic hydrocarbon chain

(Chapter 11), the discrete maleimide–PEG-based reagents provide increased hydrophilicity for

modiﬁ ed molecules and maintain solution stability even at high substitution levels.

The maleimide group reacts with thiols in the pH range of 6.5–7.5 to form a stable thioether

linkage with very little cross-reactivity with amines at this pH ( Figure 18.18 ). However, the

maleimide ring is subject to hydrolysis in aqueous solution, and since it is next to an extremely

hydrophilic PEG chain in these reagents, this factor may increase the hydrolysis rate beyond

that typically observed with hydrocarbon-based spacers (Chapter 2, Section 2.2). For this rea-

son, stock solutions of a maleimide–PEG

–biotin compound should be made in highly pure and

dry organic solvent, which then can be added to an aqueous reaction medium to commence the

biotinylation process.

The three maleimide–PEG

–biotin compounds illustrated in this section provide short,

medium, and very long chain spacer options, with the longer chains resulting in the greatest

degree of hydrophilicity of modiﬁ ed molecules. The following protocol is adapted from general

maleimide-based biotinylation methods, as discussed in Chapter 11, Section 2.

Protocol

1. Dissolve a sulfhydryl-containing protein or other thiol-molecule in a thiol-free buffer

within a pH range of 6.5–7.5. The use of 20 mM sodium phosphate, 150 mM NaCl,

pH 7.2, works well for this reaction. The concentration of protein should be in the range

of 1–10 mg/ml. Lower concentrations of protein may result in the need to increase the

molar excess of biotinylation reagent to obtain an acceptable level of modiﬁ cation. If a

thiol is not present on the molecule to be biotinylated, one may be created by disulﬁ de

reduction or through the use of a thiolation reagent (Chapter 1, Section 4.1).

2. Prepare a stock solution of the maleimide–PEG

–biotin compound in DMAC, DMSO, or

DMF (pure and dry solvents only) at a concentration of 10–20 mM.

3. With mixing, add an aliquot of the biotin solution to the protein solution to obtain at

least a 10-fold molar excess over the quantity of protein present. As thiols typically are

present in limiting amounts on proteins, the use of a high-molar reagent ratio is not

required to achieve acceptable yields of biotinylation.

4. React with gentle mixing for 2 hours at room temperature or 4 hours at 4°C.

5. Purify the biotinylated protein by dialysis or gel ﬁ ltration using a desalting resin.

3.4. Hydrazide-PEG

-Biotin

Hydrazide-containing PEG-biotinylation reagents provide reactivity with carbonyl groups

(e.g., aldehydes) to label carbohydrates or glycoproteins via hydrazone bond formation ( Figures

18.19 and 18.20 ). The hydrazide group also may be coupled with carboxylate-containing

3. Biotinylation Reagents Containing Discrete PEG Linkers 733

Figure 18.17 Maleimide–PEG

–biotin compounds of three different discrete PEG sizes are available, including

a PEG

chain that provides a molecular length of over 60 Å.

734 18. Discrete PEG Reagents

Figure 18.18

Maleimide–PEG

–biotin compounds react with thiol-containing molecules to form thioether linkages.

Figure 18.19 Biotin-PEG

-hydrazide is a hydrophilic biotinylation reagent that can be used to modify glycans

or carbohydrates at their reducing end or after periodate oxidation to create aldehydes.

3. Biotinylation Reagents Containing Discrete PEG Linkers 735

Figure 18.20 Biotin-PEG

-hydrazide reacts with aldehyde-containing molecules to form a hydrazone linkage.

molecules using a carbodiimide reaction with EDC (Chapter 3, Section 1) or an active

ester derivative (Chapter 2, Section 1). Like the other discrete PEG reagents, the hydrazide-

PEG

-biotin compound is very hydrophilic and won ’t promote aggregation or precipitation of

labeled proteins. Aldehyde functionalities may be created on glycoproteins or other carbohy-

drates by oxidation using sodium periodate (Chapter 1, Section 2.2, 4.4–4.6) or by modiﬁ -

cation with SFB (Chapter 17, Section 2). The reducing end of sugars or glycans also may be

labeled with these hydrazide reagents to produce a biotin–carbohydrate that is modiﬁ ed at only

a single site.

Hydrazide-PEG

-biotin can be used to label speciﬁ cally glycoproteins on cell surfaces after

mild periodate oxidation of the glycan structures (Wilchek and Bayer, 1987). The hydrophilic

nature of the PEG spacer will prevent the biotinylation reagent from easily penetrating cell

membranes, thus the labeling reaction is restricted to outer membrane glycoproteins. After

biotinylation and cell lysis, the labeled proteins may be detected or isolated using (strept)avidin

reagents (Jang and Hanash, 2003; Ding et al ., 2005; Handlogten et al ., 2005).

736 18. Discrete PEG Reagents

Hydrazide-PEG

-biotin contains a 31.5 Å spacer consisting of four ethylene oxide units,

which effectively imparts water solubility to the reagent. Other hydrazide biotinylation rea-

gents that contain no spacer or a hydrocarbon spacer, such as biotin-hydrazide and biotin-

LC-hydrazide, are water-insoluble and actually will lower the solubility of modiﬁ ed molecules.

Hydrazide-PEG

-biotin can be used to modify molecules without the tendency for aggregation

or precipitation. In addition, the compound is stable in aqueous environments, as it contains

no groups that are easily hydrolysable. A stock solution may be prepared in a water-miscible

organic solvent such as DMAC, DMSO, or DMF to facilitate transfer of a small amount to an

aqueous reaction.

The following protocol describes a method for the periodate oxidation of a glycoprotein

followed by biotinylation of the resultant aldehydes using hydrazide-PEG

-biotin. Chapter 1,

Section 4.6 describes an alternative protocol for the modiﬁ cation of glycans at their reducing

ends with hydrazide compounds.

Protocol

1. Dissolve a glycoprotein to be oxidized in 0.1 M sodium acetate, pH 5.5 (oxidation

buffer), at a concentration of 2–10 mg/ml. PBS at physiological pH may be used for this

reaction, as well. The use of cold buffers for the oxidation step will limit the extent of

carbohydrate oxidation and the potential for protein oxidation.

2. Dissolve sodium meta-periodate in oxidation buffer at a concentration of 20 mM. Protect

from light.

3. Add an equal volume of the glycoprotein solution to the periodate solution with mixing.

4. React for 10–20 minutes with gentle mixing and protected from light.

5. Quench the oxidation reaction by the addition of at least a 4-fold molar excess of

N-acetylmethionine or sodium sulﬁ te over the concentration of periodate in the reaction

mixture (e.g., 40 mM). Pre-dissolve the quencher in buffer at a higher concentration prior

to adding an aliquot of it to the reaction solution. React for 10 minutes. Alternatively,

the oxidation reaction may be stopped by the removal of excess periodate by gel ﬁ ltra-

tion using a desalting column.

6. Prepare a 50 mM solution of hydrazide-PEG

-biotin in DMAC, DMSO, or DMF. Add

a quantity of this solution to the puriﬁ ed, oxidized protein to provide at least a 10-fold

molar excess of biotinylation reagent over the concentration of protein present.

7. React with mixing for 2 hours at room temperature.

8. The hydrazone bond can be reduced to stabilize the linkage by the addition of sodium

cyanoborohydride to a ﬁ nal concentration of 50 mM. React for 30 minutes at room tem-

perature with mixing. All operations with cyanoborohydride should be done in a fume

hood. If the glycoprotein being modiﬁ ed is sensitive to disulﬁ de reduction and potential

denaturation, then this step should be avoided.

9. Purify the biotinylated glycoprotein by gel ﬁ ltration or dialysis.

3.5. Biotin-PEG

-Amine Compounds

Biotin compounds containing a PEG spacer that terminates in a primary amine can be used

for the labeling of carboxylate molecules ( Figure 18.21 ). Activated carboxylates, such as those

containing an NHS ester, spontaneously react with the amines to give amide bond linkages. An

active ester also may be formed in situ by the activation of carboxylates with EDC in the pres-

ence of NHS or sulfo-NHS (Chapter 3, Section 1) ( Figure 18.22 ).

Biotin-PEG

-amine contains a short, two-unit ethylene oxide cross-bridge that provides

a 20.4 Å hydrophilic spacer, which has an amine on its end. Biotin-PEG

-amine is identical

except for one additional ethylene oxide unit. Both compounds are extremely water-soluble

and can be used to label organic molecules or biomolecules containing carboxylates. Bronfman

et al. (2003) used the biotin-PEG

-amine compounds to label nerve growth factor (NGF) pep-

tide on its carboxy lates using EDC-mediated amide bond formation. The biotinylated growth

factor then was used to study receptor internalization in live cells by probing with ﬂ uorescent

streptavidin conjugates.

The following protocol can be used to biotinylate carboxylate-containing molecules in aque-

ous solution using the EDC/sulfo-NHS reaction.

3. Biotinylation Reagents Containing Discrete PEG Linkers 737

Figure 18.21 Biotin-PEG

-amine compounds can be used to modify carboxylate- or aldehyde-containing com-

pounds using a carbodiimide reaction.

738 18. Discrete PEG Reagents

Figure 18.22

Biotin-PEG

-amine can be used to add a biotin label to carboxylate-containing molecules using

the EDC/(sulfo)NHS reaction, which forms a stable amide linkage.

Protocol

1. Dissolve a carboxylate-containing peptide or other molecule in 0.1 M MES, pH 5.0 (reac-

tion buffer). Ideally, this molecule should contain only one carboxylate with no amines

to direct biotinylation to a single site and prevent polymerization of it during the conju-

gation process. However, if a peptide is to be biotinylated that also has amine groups,

then the use of a very high molar excess of the biotin-PEG

-amine reagent during the

reaction will limit the potential for peptide–peptide linking. The concentration of the car-

boxylate molecule should be low if it also has amines presence, but if it only has one or

more carboxylates, then it can be prepared at higher concentration. For example, to use

the biotin-PEG

-amine compounds to biotinylate a protein, the concentration should be

on the order of 1–2 mg/ml so that a large excess of biotinylation agent can be added. For

molecules that are sparingly soluble in aqueous solution, they may be dissolved ﬁ rst in

ethanol and then added to the reaction buffer with mixing to make a ﬁ nal ethanol con-

centration of not more than 50 percent.

2. Dissolve the biotin-PEG

-amine reagent in reaction buffer at a concentration of 25 mM.

3. Add a quantity of the biotin-PEG

-amine solution to the solution containing the car-

boxylate molecule to achieve the desired molar excess. For molecules containing a single

carboxylate to be modiﬁ ed, a 1.5- to 2-fold molar excess may be sufﬁ cient. However, for

proteins or peptides that also contain competing amines, a much larger excess of biotin

compound should be used (e.g., 100-fold excess). For instance, for protein biotinylation,

add 120  l of the biotin-PEG

-amine solution per ml of the solution prepared in Step 1.

4. Immediately before use, dissolve EDC in reaction buffer at a concentration of 25 mM.

Add 12  l of this solution per ml of the combined solution from Step 2. Mix well.

5. React for 2 hours at room temperature or 4 hours at 4°C with gentle mixing.

6. Purify the biotinylated protein or molecule using dialysis or gel ﬁ ltration. For small mol-

ecule biotinylation where these separation methods may not be appropriate, other pro-

cedures may have to be developed, such as reverse-phase chromatography or organic

precipitation techniques.

3.6. Biotin–PEG

–Benzophenone

Biotin–PEG

–benzophenone is a biotinylation reagent with a hydrophilic spacer containing three

ethylene oxide units and a photoreactive group at its end (Quanta BioDesign). The benzophenone

is activated by UV light to an extremely reactive triplet-state ketone, which can insert into C  H,

N H, and other structures, resulting in a covalent bond (Chapter 2, Section 7.2). The reac-

tion is one of the most efﬁ cient photoreactive conjugation mechanisms available (Campbell and

Gioannini, 1979). Thus, this reagent provides a method of adding a biotin group to molecules that

don’t contain typical functionalities useful for bioconjugation. This may include polymeric sur-

faces or organic molecules lacking reactive targets ( Figure 18.23 ).

The presence of the PEG

spacer in this compound provides water solubility to the biotin

arm, whereas the benzophenone group should associate with more hydrophobic regions or sur-

faces, which may be ideal for the biotinylation photoreaction. The reagent can be used by dis-

solving it in an aqueous buffer suitable for use with whatever substance is to be biotinylated.

After mixing this solution with the target molecule or surface, exposure to UV light will initiate

the conjugation reaction. Unlike other photoreactive groups, a benzophenone doesn ’t undergo

decomposition to an inactive form if it doesn ’t couple to target molecules. Instead, it degrades

from the photo-excited state back to its initial state, so it can be once again photolyzed to an

active state. This process increases the likelihood that the benzophenone will couple to a target

molecule during the photoreaction. See Chapter 5, Section 4.3 for an illustration of the benzo-

phenone coupling reaction.

4. Discrete PEG Modiﬁ cation Reagents

Large polymer PEG reagents having molecular weights 2,000 Da have been used for over 20

years as modiﬁ cation agents for biological molecules (Chapter 25). These compounds often are

4. Discrete PEG Modiﬁ cation Reagents 739