Hermanson G. Bioconjugate Techniques, Second Edition

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530 11. Biotinylation Reagents

5-(Biotinamido)pentylamine is able to participate in the acyltransferase reaction, becoming cov-

alently attached to protein substrates at their glutamine residues ( Figure 11.13 ). Lee et al. (1988)

used this biotinylation reagent to quantify factor XIII in plasma. Transglutaminase activ-

ity resulted in the modiﬁ cation of an N , N-dimethylcasein substrate which was subsequently

detected by an avidin–biotin assay procedure. The assay may be done in microplates using

wells coated with the substrate protein and quantifying the enzyme activity with streptavidin-

alkaline phosphatase (Slaughter et al., 1992). Jeon et al. (1989) subsequently applied the assay

to the measurement of transglutaminase activity in cells. Components biotinylated in cellular

systems also can be isolated by use of afﬁ nity chromatography on immobilized avidin (Lee

et al ., 1992).

4. Photoreactive Biotinylation Agents

Biotin derivatives containing a photoreactive group provide nonselective biotinylation potential

at certain reactive hydrogen sites or nucleophilic groups. They can be used to incorporate an

avidin binding, biotin group into molecules that do not possess amines, sulfhydryls, or other

easily modiﬁ able functional groups. Many of these photoreactive derivatives utilize the phe-

nyl azide type of photosensitive group, which can be activated by exposure to UV light to an

intermediate nitrene or the nucleophile-reactive dehydroazepine (Chapter 2, Section 7.1 and

Chapter 5, Section 3). However, additional photoreactive groups that also are useful include a

psoralen ring system and a benzophenone group. A psoralen-based biotinylation agent is pre-

sented in this section, while a benzophenone-containing one with a water-soluble PEG spacer is

discussed in Chapter 18, Section 3.6.

Figure 11.13 5-(Biotinamido)pentylamine can be used to label glutamine residues in proteins by enzymatic

action of transglutaminase.

4.1. Photobiotin

Perhaps the most common photoreactive biotin derivative is N-(4-azido-2-nitrophenyl)-amino-

propyl-N -(N-D-biotinyl-3-aminopropyl)-N -methyl-1,3-propanediamine, simply called photoac-

tivatable biotin or photobiotin (Forster et al., 1985) (Thermo Fisher). The compound contains

a 9-atom diamine spacer group on the biotin valeric acid side chain at one end, while the other

end of the spacer terminates in an aryl azide reactive group. The presence of a nitro group on the

phenyl azide ring allows for photoactivation at higher UV wavelengths approaching the visible

region of the spectrum, thus avoiding potential breakdown of biological molecules through UV

exposure. Photolyzing with light at a wavelength of 350 nm causes rapid activation with nitrene

formation. The nitrene can couple to replaceable hydrogen sites in target molecules, add to dou-

ble bonds within Van der Waals distance, or undergo ring expansion to the dehydroazepine.

If ring expansion occurs, the principal target group for coupling is a nucleophile, such as a primary

amine ( Figure 11.14 ).

Photobiotin has been used to biotinylate numerous macromolecules, including proteins

and nucleic acids. The biotinylation of alkaline phosphatase was done with complete reten-

tion of activity (Forster et al., 1985). Tubulin was labeled with photobiotin and detected on

dot blots down to a level of 10 pg of sample using an avidin–enzyme conjugate (Lacey and

Grant, 1987). DNA and RNA were labeled for use in hybridization assays (Forster et al .,

1985; Keller et al., 1989). For instance, photobiotin-modiﬁ ed probes have been used to detect

ﬂ avivirum RNA in infected cells (Khan and Wright, 1987), to detect single-copy genes and

low-abundance mRNA (McInnes et al., 1987), for the diagnosis of barley yellow dwarf virus

(Habili et al., 1987), to assay luteinizing hormone  mRNA in individual gonadotropes (Childs

et al., 1987), and to perform DNA mapping using a cross-hybridization technique (Chetrit

et al ., 1989).

Photobiotin can be dissolved in water or buffer at a concentration of 1 mg/ml and stored in

the dark at 20°C until needed. As long as no exposure to light is permitted, the compound is

stable for at least 1 year under these conditions.

The protocol for modifying DNA probes with photobiotin can be found in Chapter 27,

Section 2.3. It is based on the method of Forster et al. (1985). The following method is a sug-

gested protocol for the modiﬁ cation of proteins using a photoreactive biotin derivative. Some

optimization may be necessary to obtain the best incorporation levels.

4. Photoreactive Biotinylation Agents 531

532 11. Biotinylation Reagents

Figure 11.14 Photobiotin can be made to couple spontaneously with nucleophiles by exposure to UV light. The

phenyl azide ring undergoes ring expansion to a highly reactive dehydroazepine intermediate, which can react

with amines.

Protocol for Labeling Proteins with Photobiotin

1. Dissolve the protein to be biotinylated at a concentration of at least 1 mg/ml in water or

dilute buffer at neutral pH.

2. In subdued light, dissolve photobiotin (Thermo Fisher) in water at a concentration of

1 mg/ml.

3. Add a quantity of photobiotin solution to the protein solution to give at least a 5-fold

molar excess of biotinylation reagent.

4. Place in an ice bath and irradiate from above (about 10 cm away) for 15 minutes using a

sunlamp (such as Philips Ultrapnil MLU 300 W, General Electric sunlamp RSM 275 W,

or National Self-Ballasted BHRF 240–250 V 250 W W-P lamp).

5. Remove excess photobiotin by dialysis or gel ﬁ ltration using a desalting column.

4.2. Psoralen-PEO

-Biotin

Psoralen-PEO

-biotin is a photoreactive biotinylation reagent containing a psoralen group at

one end and a triethylene glycol (PEG-based) spacer in the middle (Thermo Fisher). This com-

pound is water-soluble due to the presence of the hydrophilic PEG arm. It is able to photo-

insert into double-stranded DNA and to a lesser extent into double-stranded regions of RNA.

The reaction occurs upon exposure to UV light in the range of 320–400 nm, which forms an

excited triplet state intermediate that can insert in certain double bond structures, especially at

the 5,6-double bond of thymine bases.

The psoralen ring system can intercalate within double-stranded DNA or RNA and induce

the formation of adducts with adjacent thymine bases ( Figure 11.15 ). The furan-side and

pyrone-side of the tricyclic rings in psoralen both can form cycloaddition products with the

5,6-double bond of thymine residues, which results in crosslinks between the DNA strands

with a PEG-biotin label sticking out.

Psoralen-PEO

-Biotin has been used to label double-stranded DNA for detection using

(strept)avidin reagents (Henriksen et al., 1991; Wygrecka et al., 2007). The psoralen photore-

active group provides better insertion yields than typical phenyl azide-based systems, such as

the standard photobiotin probe discussed previously in this section.

Protocol

1. Dissolve the DNA sample to be modiﬁ ed at a concentration of 20–100 g/ml in 10 mM

Tris, 1 mM EDTA, pH 7.4. Note: The sample may be heated to denature and solubilize

genomic DNA and then cooled to form dsDNA for modiﬁ cation.

2. Dissolve the Psoralen-PEO

-biotin reagent in DMF at a concentration of 20 mM (use a

fume hood). Protect from light.

3. Add a quantity of the Psoralen-PEO

-biotin solution to the DNA solution to result in a

ﬁ nal concentration of 200  M. Mix well.

4. Expose the solution to long wavelength UV light at about 365 nm (Philips TL 20W/09

UV light works well) for 10–30 minutes. The solution may be cooled on ice to prevent

heating during the irradiation process.

4. Photoreactive Biotinylation Agents 533

534 11. Biotinylation Reagents

5. Precipitate the sample to remove unreacted biotinylation reagent by adding 0.1 M potas-

sium acetate and ethanol (1:2 ratio). Centrifuge and wash the biotinylated DNA pellet with

ethanol, then dry it under nitrogen. The puriﬁ ed sample may be dissolved in water or buffer.

5. Active Hydrogen-Reactive: p -Aminobenzoyl Biocytin, Diazotized

p-Aminobenzoyl biocytin contains a 4-aminobenzoic acid amide derivative off the  -amino

group of biocytin ’s (Section 3, this chapter) lysine residue (Thermo Fisher). The aromatic amine

can be treated with sodium nitrite in dilute HCl to form a highly reactive diazonium group

(Figure 11.16 ), which is able to couple with active hydrogen-containing compounds. A diazo-

nium reacts rapidly with histidine or tyrosine residues within proteins, forming covalent diazo

Figure 11.15 The photoreactive compound psoralen-PEO

-biotin can intercalate into double stranded DNA or

RNA segments and covalently link to thymine bases via a photoreaction process.

bonds (Wilchek et al., 1986) ( Figure 11.17 ). It also can react with guanidine residues within

DNA at position 8 of the base (Rothenberg and Wilchek, 1988) ( Figure 11.18 ). Biotinylation

via diazo linkages is reversible by treatment with a 10-fold molar excess of Na

(sodium

dithionite) in 50 mM Tris, pH 8.5 (Gorecki et al., 1971) (Chapter 2, Section 6.1 and Chapter 4,

Section 9).

The procedure for creating the diazonium derivative of p-aminobenzoyl biocytin and cou-

pling to a protein or a nucleic acid is as follows.

Figure 11.16 The aminophenyl group of this biotin derivative can be transformed into a diazonium reactive

group by treatment with sodium nitrite in dilute HCl.

5. Active Hydrogen-Reactive: p-Aminobenzoyl Biocytin, Diazotized 535

536 11. Biotinylation Reagents

Protocol

Formation of the diazonium derivative:

1. Dissolve 2 mg of p-aminobenzoyl biocytin (Thermo Fisher) in 40 l of 1 N HCl (concen-

tration of 50 mg/ml). Cool the solution on ice.

2. Dissolve 7.7 mg of sodium nitrite in 1 ml of ice-cold water. Prepare fresh.

3. Mix 40 l of the p-aminobenzoyl biocytin solution with 40 l of the sodium nitrite

solution.

4. React for 5 minutes on ice to create the diazonium derivative.

5. Stop the reaction by the addition of 35 l of 1 N NaOH. Use immediately for biotinylation.

Biotinylation of proteins on blots using the diazonium derivative of p-aminobenzoyl biocytin.

1. Dilute the diazonium derivative of p-aminobenzoyl biocytin with 0.2 M sodium borate,

pH 8.4, to a concentration of 10  g/ml.

Figure 11.17 The diazonium group of p-diazobenzoylbiocytin can react with tyrosine or histidine residues in

proteins to form diazo bonds.

2. Transfer proteins onto a nitrocellulose membrane using any appropriate procedure,

including dot blotting the protein solution onto the surface.

3. Incubate the membrane with the biotin derivative at a ratio of 1 ml/cc

of membrane.

4. React for 1 hour at room temperature.

5. Wash the membrane thoroughly with 0.1 M Tris, 0.15 M NaCl, pH 7.5.

6. Block nonspeciﬁ c sites on the membrane with an appropriate blocking component (such

as BSA) and detect the biotinylated proteins using an avidin or streptavidin conjugate.

6. Glycan Biotinylation Reagents

Biotinylated oligosaccharides are convenient probes of carbohydrate interactions, because

the biotin label can be captured or detected using an avidin or streptavidin derivative. For

instance, immobilized streptavidin can be used to purify glycoconjugates that have been labeled

Figure 11.18 The diazonium group of p-diazobenzoylbiocytin can couple to the C-8 position of guanidine

bases in nucleic acids, forming diazo bonds.

6. Glycan Biotinylation Reagents 537

538 11. Biotinylation Reagents

with a biotin group, potentially isolating glycoproteins or carbohydrate binding proteins.

Enzyme- or ﬂ uorescently-labeled avidin or streptavidin can be used to probe for biotin-labeled

carbohydrates in cells or tissue samples. In addition, a biotinylated glycan can be displayed

on avidin or streptavidin to make an immunogen for developing speciﬁ c antibodies to the

carbohydrate.

Complex glycans on glycoproteins or other carbohydrate bearing molecules can be modiﬁ ed

with a biotinylation reagent using a number of reaction strategies. Oxidation with sodium meta

periodate can be used to create aldehyde residues from diols on sugars, and this technique has

been used to speciﬁ cally modify sialic acids on glycans by reductive amination with a biocytin-

hydrazide compound (see Section 3.2, this chapter, Bayer et al., 1988). Other procedures make

use of released glycans from glycoproteins or other glycoconjugates, which contain reducing

ends upon cleavage. The reducing ends then can be reacted with amine- or hydrazide-contain-

ing biotinylation compounds to couple with the open aldehyde group at the reducing end,

thus forming a hydrazone linkage. The hydrazone bond may be reduced to stabilize the bond

(recommended when reacting with amine-containing biotin compounds) or left as the unre-

duced hydrazone, which typically is done when coupling with hydrazide-biotin compounds.

Alternatively, the reducing end of a carbohydrate can be reacted with an amine to form a glyc-

osylamine derivative without opening the acetal ring, thus better preserving the native structure

of a glycan, which is important in some studies involving protein interactions.

A recent addition to the methods of glycan biotinylation makes use of the Staudinger ligation

reaction with a phosphine-biotin derivative (see also Chapter 17, Section 5). Carbohydrates

containing azide derivatives have been modiﬁ ed with this biotin compound to probe for glyco-

conjugates in vivo. This reaction is particularly useful for doing cell-based assays, because the

ligation reaction is completely orthogonal to any biological reactions or interactions.

The following sections describe ﬂ uorescent biotinylation reagents that can be used to study

carbohydrate function and interactions.

6.1. Biotinylated Aminopyridine

BAP (biotinylated aminopyridine or 2-amino-(6-amidobiotinyl)pyridine) is a derivative of D -

biotin made by reacting the NHS ester of this vitamin with 2,6-diaminopyridine (DAP) in large

molar excess, typically done using a carbodiimide EDC/NHS reaction ( Figure 11.19 ). The

resultant compound has ﬂ uorescent properties due to the present of the aminopyridine ring,

and its remaining free amine group may be used to modify reducing saccharides and glycans

by reductive amination ( Figure 11.20 ). BAP can be used to label oligosaccharides under mild

conditions and without the carbohydrate structural degradation that results using periodate

oxidation of carbohydrates.

Rothenberg et al. (1993) demonstrated the utility of BAP for highly sensitive ﬂ uorescence

detection and separation of oligosaccharides by reverse phase HPLC, with limits of detection

down to about the 50-femtomole level (low picomole levels if using a cuvette reader with a

1 cm path length). Toomre and Varki (1994) subsequently published an improvement on the

synthesis and use of the BAP reagent. In addition, the biotin group of BAP-labeled glycans can

be used to create neoglycoproteins by interaction with tetrameric avidin or streptavidin mol-

ecules. The resultant glyco-complexes have been shown to be potent immunogens for evoking

an IgG immune response in mice toward the glycan components (Srikrishna et al. , 2001).

BAP-modiﬁ ed glycans also can be used to probe for receptors or binding proteins, which

then can be detected by use of streptavidin conjugates or isolated by afﬁ nity chromatography

on immobilized streptavidin or immobilized monomeric avidin (Thermo Fisher). The use of

immobilized monomeric avidin is convenient, because the biotinylated glycans can be released

by elution with acid pH or by using a solution containing biotin. BAP-carbohydrate adducts

have been shown to be high afﬁ nity binders of both streptavidin and avidin, despite the rela-

tively short spacer afforded by the diaminopyridine-biotin linker (Toomre and Varki, 1994).

The biotin group of BAP makes the compound somewhat hydrophobic, but attached to gly-

cans the conjugates should display good water solubility due to the abundance of hydroxyl

groups in addition to potentially having other charged groups on the sugars. BAP solubility in

water was reported to be about 1 mg/ml, but with the addition of less than 1 percent DMSO,

this solubility can be increased more than 10-fold (Toomre and Varki, 1994).

Fluorescence of the diaminopyridine group allows detection of conjugates down to the pico-

mole range, with excitation and emission maxima at 345 and 400 nm, respectively. For detec-

tion of BAP and its conjugates, the optimal buffer environment is less than pH 5, because its

ﬂ uorescent properties are pH dependent. A preferred buffer is sodium acetate at pH 4.

After BAP conjugation to saccharides or glycans, separation of the conjugates and unre-

acted BAP can be ﬂ uorescently followed using size exclusion chromatography (SEC) on a TSK-

G3000PW column, which successfully resolves most of the lower molecular weight conjugate

species, including single-sugar adducts through 3-sugar carbohydrates. BAP-glycan conjugates

containing more than 3 sugars elute early in the separation and do not resolve into discrete

peaks, as smaller adducts do. Unreacted BAP elutes last in the SEC separation.

Figure 11.19 The synthesis of BAP can be done by reacting an excess of diaminopyridine with biotin in the

presence of EDC and NHS.

6. Glycan Biotinylation Reagents 539