Hermanson G. Bioconjugate Techniques, Second Edition

Подождите немного. Документ загружается.

540 11. Biotinylation Reagents

Alternatively, separations can be done by anion-exchange chromatography using a column

packed with the HPLC support TSK-DEAE-2SW, which effectively resolves negatively charged

carbohydrates, such as those containing sialic acid residues. BAP–glycan conjugates will elute

according to their degree of negative charge character. Sulfate-containing sugars in general will

interact more strongly with the matrix and have longer retention times than those containing

only carboxylates.

BAP may be prepared by the reaction of NHS-biotin with DAP. The biotinylation compound

is commercially available (Thermo Fisher) or it may be formed in situ by reaction of D -biotin

with EDC and NHS. An optimized protocol for the preparation of the reactive intermediate

ester and the ﬁ nal BAP compound can be determined from Rothenberg et al. (1993) with mod-

iﬁ cations by Toomre and Varki (1994). Basically, a solution of 0.3 M DAP is prepared in 40 ml

of 50 mM MES, pH 6.5, and 10 ml of 0.1 M D-biotin in DMSO is added. The reaction is initi-

ated by the addition of EDC and NHS to a ﬁ nal concentration of 150 and 50 mM, respectively.

The reaction was allowed to continue overnight at room temperature with mixing before puri-

ﬁ cation of BAP on a C

sample prep cartridge. The reaction mixture was applied to the car-

tridge and reaction by-products and DAP were removed by washing with water and 10 percent

acetonitrile. BAP was ﬁ nally eluted in high purity by washing with 50 percent acetonitrile.

The conjugation of BAP to oligosaccharides can be done by the following protocol based on

the method of Toomre and Varki (1994).

Figure 11.20 BAP can be used to label the reducing end of released glycans by reductive amination in the pres-

ence of a reducing agent.

Protocol

1. Dissolve an oligosaccharide or glycan having a reducing end to be modiﬁ ed in 2:1 pyri-

dine/glacial acetic acid (vol/vol) with a total reaction volume of 10–100 l. If the car-

bohydrate initially is insoluble in the reaction solution, a prior dissolution in a minimal

amount of DMSO or water can be done and then an aliquot transferred to the reaction

medium.

2. Add to the solution a 50-fold molar excess of BAP over the estimated amount of carbo-

hydrate present in the reaction mixture.

3. Heat at 80°C in a sealed Reactivial (Thermo Fisher) for 1 hour.

4. Add to the reaction mixture an equal volume of the reducing agent borane-dimethylamine

(BDA) complex, which was previously prepared in the reaction buffer at a concentration

of 125 mg/ml.

5. React for another hour at 80°C.

6. Purify the BAP–glycan conjugate from unreacted glycans by use of a C

sample prep

cartridge, as described above for the synthesis of BAP. Separation of excess BAP from the

conjugates may be done by SEC or anion exchange chromatography, depending on the

size of carbohydrates being modiﬁ ed and their intrinsic charge. Follow the separations

by visualization of BAP ﬂ uorescence with a hand-held UV lamp or through the use of a

ﬂ uorescence detector.

6.2. Biotinyl- L -3-(2-naphthyl)-alanine hydrazide

BNAH is a biotin-hydrazide derivative containing a UV absorbing and ﬂ uorescent naphthalene

group (biotinyl- L-3-(2-naphthyl)-alanine hydrazide) (Leteux et al., 1998). Unlike BAP described

previously, BNAH has a hydrazide group for coupling to the reducing end of carbohydrates,

instead of an amine. While both groups can be successfully conjugated to an aldehyde of a

released glycan, the biotin–amine compounds require a reducing agent to stabilize the resultant

hydrazone bond.

BNAH may be coupled to reducing sugars without reduction, since the linkage formed between

a hydrazide and an aldehyde is much more stable than that with an amine.

6. Glycan Biotinylation Reagents 541

542 11. Biotinylation Reagents

In some cases, the ability to modify glycans at the reducing end without reduction preserves

the carbohydrate ’s native structure sufﬁ ciently to allow interactions with proteins that would

otherwise not interact if the bond were reduced. Therefore, depending on the ultimate use of

the biotinylated carbohydrate, using a hydrazide mediated conjugation process can have advan-

tages over the use of amine-biotin compounds.

In the case of BNAH, however, it was determined that the resultant linkage with the reduc-

ing end of an oligosaccharide or glycan was not a hydrazone bond, but a glycosylhydrazide

derivative, which preserves the pyranose ring structure of the sugar ( Figure 11.21 ). This ﬁ nding

is the main reason a BNAH modiﬁ ed glycan effectively displays a near-native conformation

at the reducing end. In addition, the biotin label in this conﬁ guration is reversible and can be

released by incubation under acidic conditions, thus allowing recovery of the carbohydrate.

Another advantage of the BNAH derivative is that the conjugation reaction with reducing

sugars can be done in aqueous conditions and in an environment that permits carbohydrate

and biotinylation reagent solubility. Modiﬁ ed carbohydrates may be stored for at least 1 year

at  20°C in a solution of water/methanol (9:1 v/v) without degradation.

The following protocol is based on the method of Leteux et al. (1998).

Figure 11.21 BNAH contains a hydrazide group that can be used to label the reducing end of released glycans

through the formation of a hydrazone bond.

Protocol

1. Dissolve 100 nmol of the carbohydrate to be modiﬁ ed and 500 nmol of BNAH in 25  l

of methanol (20 mM BNAH solution).

2. Evaporate the solution to dryness and re-dissolve in 25 l of either an acidic solution of

methanol/water/acetic acid (74:8:8, v/v) or in a neutral solution consisting of methanol/

water (9:1, v/v), depending on the relative solubility of the carbohydrate.

3. React for at least 5 hours at 60°C if using the acidic reaction solution or for a total of

16 hours at 60°C if using the neutral solution.

4. Puriﬁ cation of the conjugates may be done by reverse phase HPLC separation. Dry

the reaction solution under a nitrogen stream and reconstitute in a minimum volume

of acetonitrile/water (1:1, v/v). Apply the sample to a 5 m C

-silica HPLC column

(250  4.6 mm, Nucleosil). Elute with a gradient of water to acetonitrile at a ﬂ ow rate of

1 ml/minute over a time course of 30 minutes. Free BNAH and BNAH-glycan derivatives

can be monitored by absorbance at 275 nm. The conjugate peak also will be positive for

carbohydrate by reaction with orcinol, which can be detected by spray after spotting a

small eluted sample on a TLC plate.

6.3. Biotin-PEG-Phosphine

Saxon and Bertozzi (2000) reported on the synthesis and use of a novel biotinylation com-

pound containing a phosphine group for coupling to azide-containing molecules. The rea-

gent has a biotin handle at one end, a tetraethyleneglycol (PEG) spacer imparting increased

water solubility in the middle, and a 3-(diphenylphosphino)-4-(methoxycarbonyl)benzamide

group on the other end. Biotin-PEG-phosphine reacts with azide derivatives of amino acids,

sugars, and cross-linkers to form an intermediate aza-ylide, which spontaneously rearranges

in aqueous solution to create a stable amide bond ( Figure 11.22 ). This reaction is a modiﬁ ed

Staudinger ligation that can be used to target azide containing glycans or proteins in vivo .

Cells grown in the presence of azide analogs of certain amino acids or sugars will incor-

porate these derivatives into proteins or carbohydrates through enzymatic synthesis using

6. Glycan Biotinylation Reagents 543

544 11. Biotinylation Reagents

the native cell machinery. Azides thus displayed on biomolecules are unreactive with other

substances typically found within the cell, but the azide derivatives may be targeted for con-

jugation using the modiﬁ ed Staudinger reaction (see Chapter 17, Section 5 for additional infor-

mation on this reaction and its use in biological labeling).

Biotin-PEG phosphine can be used to label glycans or proteins that have been modiﬁ ed to

contain azide groups. The beautiful speciﬁ city of this reaction and its lack of toxic side reac-

tions or additives make it suitable for use in living organisms, including cells and animals. The

reaction is completely orthogonal to functional groups and reactions found in living systems, so

the biotinylation process proceeds with no cross-reactions with other biomolecules. In addition,

no cell toxicity has been observed due to the phosphine or the phosphine oxide by-product of

the coupling reaction. The phosphine also doesn ’t appear to be capable of reducing disulﬁ des

within proteins.

Prescher et al. (2004) have shown that mice fed with the peracetylated azido-mannose sugar

derivative Ac

ManNAz efﬁ ciently convert it through deacetylation by cytosolic esterases into

Figure 11.22 Azido-sialic acid-containing glycans can be labeled in vivo with biotin-PEG-phosphine using the

Staudinger ligation reaction, which forms an amide bond.

an azido sialic acid (SiaNAz), which then gets incorporated enzymatically into cell-surface gly-

cans. Biotinylation of these aberrant carbohydrates provides a method of detecting or isolating

glycoproteins or other glycoconjugates. For instance, ﬂ uorescently labeled streptavidin can be

used to image the cell-surface structures containing the biotinylated azido-sugar derivatives.

Alternatively, immobilized streptavidin or immobilized monomeric avidin can be used to purify

biotinylated azido-glycoconjugates after cell lysis and provide a method for studying glycopro-

tein function and interactions.

The following protocol is based on the methods of Saxon and Bertozzi (2000) and Prescher

et al. (2004).

Protocol

1. Treat and grow cells in the presence of 20 M Ac

ManNAz for at least 3 days. The

azide-mannose derivative may be solubilized in 70 percent DMSO as a more con-

centrated stock solution and then an aliquot added to the media containing the cells.

Alternatively, mice may be treated with the azide-sugar derivative in aqueous DMSO at a

level of 100–300 mg/kg, using an injection of 200 l administered intraperitoneally daily

for 7 days.

2. When working with cells, ﬁ rst wash them several times with PBS, pH 7.4, to remove any

remaining Ac

ManNAz from the media. Alternatively, if working with animals, isolate

the tissue type desired and prepare the cells to be labeled in PBS, pH 7.4.

3. To label cell-surface azide-glycans, the cells are reacted for 1 hour using a ﬁ nal concen-

tration of 1 mM biotin-PEG-phosphine reagent dissolved in PBS, pH 7.4.

4. Wash the cells several times with PBS, pH 7.4, to remove excess biotinylation compound.

5. The labeled glycans may be analyzed by cell sorting after staining with ﬂ uorescently

modiﬁ ed streptavidin. Alternatively, the cells may be lysed and the labeled glycans iso-

lated using immobilized streptavidin.

6. Glycan Biotinylation Reagents 545

546

Modiﬁ cation of proteins and other molecules with a radioactive element provides a means

of detection that can be extremely sensitive for assay, localization, and imaging applications.

Among the most common radiolabels for biological studies are

H, and the iso-

topes of iodine,

125

I and

131

I. The unstable isotopes of carbon, phosphorus, sulfur, and hydrogen

are all  emitters, releasing particulate radiation consisting of either positrons or electrons.

To measure labeled molecules containing  emitters often necessitates tedious sample manipulation

including tissue homogenization and mixing with scintillation cocktails for subsequent counting.

The radioactive isotopes of iodine, by contrast, are both  emitters, providing a much eas-

ier route to measurement than -particle-emitting radioisotopes. High-energy electromagnetic

radiation can be detected directly without the need for intermediate scintillation cocktails.

Iodine-131 was the ﬁ rst unstable iodine isotope to be used for labeling protein molecules (Li,

1945; Pressman and Keighley, 1948). The

131

I isotope decays by both 



(electron) and  emission.

The speciﬁ c activity of this element can be as high as 6,550 Ci/mmol, providing extraordinary

sensitivity for detecting labeled molecules.

Iodine-125 decays by electron capture followed by  emission. However, the maximum

energy of

125

I electromagnetic energy emission can be as little as one-tenth to one-third that of

131

I (Wilbur, 1992; Powsner, 1994). The greater energy intensity of

131

I emission actually can be

a disadvantage, since  rays emanating from it are more penetrating, requiring increased precau-

tions and greater protective equipment. In addition, the relatively short half-life of

131

I (8.1 days)

as compared to

125

I (60 days) necessitates that labeled compounds be prepared more often, since

activity losses will be severe upon storage. Because

125

I is not a particle emitter, its use in vivo for

imaging applications limits radiation damage to surrounding proteins, cells, and tissues.

These factors make

125

I the iodine label of choice for radiolabeling biological molecules.

Its commercial availability from a number of suppliers at relatively low cost further adds to

its popularity. Even though it has lower speciﬁ c activity than

131

I, iodine-125 still provides

much greater sensitivity than

S, or

H in labeling biomolecules. In fact, the use of

a radioactive iodine label can create probes that have 150-fold more sensitivity than tritiated

molecules and as much as 35,000 times the detectability of

C-labeled molecules (Bolton and

Hunter, 1986).

Radioiodination is the process of chemically modifying a molecule to contain one or more

atoms of radioactive iodine. Early studies on protein modiﬁ cation determined that iodine in

Iodination Reagents

aqueous solution formed a reactive ion, H



( Figure 12.1 ), that is capable of modifying

tyrosine side chains, the imidazole groups of histidine, and either modifying sulfhydryl groups

or catalyzing their oxidation to disulﬁ des ( Figure 12.2 ). Most methods now utilize a chemical

agent to create the reactive iodine species, thus driving the reaction at much greater rates.

There are two main methods of radioiodination that are commonly employed to modify pro-

teins and other molecules: (1) direct labeling of the desired protein or other target molecule

in the presence of an oxidizing agent or (2) indirect labeling of the desired molecule by ﬁ rst

labeling an intermediate compound which is then used to perform the ﬁ nal modiﬁ cation. Direct

labeling methods are by far the most common, and the chemistries used in this process have

been reviewed (Regoeczi, 1984).

The prevailing procedures for direct coupling of

125

I to a protein or other molecule are

through the use of oxidizing agents. The in situ preparation of an electrophilic radioiodine

Figure 12.1 Iodide anion in aqueous solution undergoes an equilibrium reaction process to form the reactive



species.

Figure 12.2 The iodination of tyrosine or histidine residues in proteins by H



12. Iodination Reagents 547

548 12. Iodination Reagents

species is fundamental to the ability to modify certain reactive sites within the desired

molecules. The most common oxidizing compounds are N-haloamine derivatives, such as

N-chlorotoluenesulfonamide (chloramine-T) or 1,3,4,6-tetrachloro-3 ,6-diphenylglycouril

(Iodogen). In most instances, such compounds do not harm the proteins being labeled, although

careful control over reaction times should be done to prevent over-labeling or oxidative dam-

age. A secondary method of producing an oxidative effect is to use an enzyme-driven system.

The glucose oxidase/lactoperoxidase reaction creates reactive iodine through the production of

hydrogen peroxide from glucose with the subsequent action of peroxidase to form I

from I



Formation of the electrophilic halogen species leads to the potential for rapid reaction

with compounds containing strongly activating groups, such as in activated aryl compounds.

Particularly, substances containing aromatic ring structures that have substituents on the

ring which are electron donating can sufﬁ ciently activate the carbons on the ring to undergo

electrophilic substitution reactions. Therefore, phenols, aniline derivatives, or alkyl anilines

that contain OH, NH

, or NHR constituents respectively, are very susceptible to being iodin–

ated. In proteins, this translates into tyrosine side chain phenolic groups and histidine side

chain imidazole groups. Crosslinking compounds or modiﬁ cation reagents containing ring-

activated groups also are capable of being iodinated.

The addition of a radioactive iodine atom to a protein molecule typically has little effect on

the resultant protein activity, unless the active center is modiﬁ ed in the process. The size of an

iodine atom is relatively small and does not result in many steric problems with large mole-

cules. The sites of potential protein modiﬁ cation are tyrosine and histidine side chains. Tyrosine

may be modiﬁ ed with a total of two iodine atoms per phenolate group, whereas histidine can

incorporate one iodine. Sulfhydryl modiﬁ cation at cysteine residues is typically unstable.

The result of iodination at tyrosine groups can alter the spectral characteristics of the protein

in solution (Hughes, 1950). The typical protein absorbency at 280 nm can shift to a maximum

at about 305–315 nm due to the addition of iodine atoms to the phenolate ring of tyrosine.

The degree of absorbance shift is dependent on how many iodine atoms are incorporated into

the protein and whether they result in mainly mono- or di-iodotyrosine formation. In addition,

as the level of iodination increases, the solubility of a protein in aqueous solution can dramati-

cally decrease until complete insolubility results in proteins with high numbers of tyrosines.

Thus, controlling the degree of iodination is an important consideration both in choosing

the oxidant used and in controlling the time of reaction. Typically, most radiohalogenations

are done in a time period of 30 seconds to as long as 30 minutes. Optimization may have to be

done to determine the correct time to use for a particular modiﬁ cation reaction. Termination

of the iodination reaction may be done through addition of a reducing agent, such as sodium

metabisulﬁ te. Bisulﬁ te reduces the electrophilic iodine species to unreactive iodide, effectively

stopping the modiﬁ cation process.

The following sections discuss the major radioiodination reagents available for direct labeling

as well as the main crosslinkers or modiﬁ cation reagents used for indirect labeling techniques.

1. Chloramine-T

Chloramine-T, or N-chlorotoluenesulfonamide, has been one of the most popular oxidizing

reagents used for radioiodination techniques since its introduction by Greenwood et al. in 1963

(Sigma). It has strong oxidizing properties that readily lead to the formation of the required

electrophilic halogen species that result in iodine incorporation into target molecules. The reac-

tions of chloramine-T are well documented, being suitable for both macromolecular protein

iodination and small-molecule modiﬁ cation (Wilbur, 1992). It also can be used to modify mol-

ecules with other radioactive halogen elements, such as isotopes of bromine and astatine (Hadi

et al ., 1979; Mazaitis et al ., 1981).

The reaction of chloramine-T with iodide ion in solution results in oxidation with subse-

quent formation of a reactive, mixed halogen species, ICl ( Figure 12.3 ). Either

125

I or

131

I can

be used in this reaction. The ICl then rapidly reacts with any sites within target molecules that

can undergo electrophilic substitution reactions. Within proteins, any tyrosine and histidine

side chain groups can be modiﬁ ed with iodine within 30 seconds to 30 minutes. Since chlo-

ramine-T is a water-soluble reagent and the reaction is done completely in the solution phase,

higher incorporation of radioactive iodine can be obtained than using insoluble or immobilized

oxidants (see subsequent sections). However, a greater yield of speciﬁ c radioactivity does not

Figure 12.3 The strong oxidant chloramine-T can react with iodide anion in aqueous solution to form a highly

reactive mixed halogen species.

125

ICl then can modify tyrosine and histidine groups in proteins to form radi-

olabeled products.

1. Chloramine-T 549