Hermanson G. Bioconjugate Techniques, Second Edition

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550 12. Iodination Reagents

always translate into a better radiolabeled probe. Chloramine-T, being a strong oxidant with

rapid reaction rates can easily over-label a target molecule or cause oxidative damage to sensi-

tive proteins (Lee and Grifﬁ ths, 1984). The reaction may be quenched with a reductant, usually

done by addition of sodium metabisulﬁ te. Although chloramine-T is still widely used, alterna-

tive iodination reagents that are insoluble or immobilized on insoluble supports provide milder

reaction conditions and are more controllable.

The following protocol is representative of those found in the literature for iodination of

protein molecules using chloramine-T.

Protocol

Caution: Handle all radioactive substances according to the radiation safety regulations insti-

tuted at each facility approved to handle such materials. Use adequate precautions to protect

personal safety and the environment. Dispose of radioactive waste only by following approved

guidelines.

1. Dissolve chloramine-T in 50 mM sodium phosphate, pH 7.0, at a concentration of 4 mg/ml.

Prepare fresh. Approximately 25 l of this solution (100 g) is required to iodinate 5  g

of a protein.

2. Dissolve sodium metabisulﬁ te in 50 mM sodium phosphate, pH 7.0, at a concentration

of 12.6 mM (240  g/100 l). Prepare fresh. Approximately 100 l of this solution is

required for a 5  g protein iodination.

3. Obtain fresh Na

125

I and adjust its concentration to approximately 0.5 mCi/ l; 2 l of

this solution are required to iodinate 5  g of protein.

4. Add to a suitable reaction vial, 25 l of a solution consisting of 5 g of a protein dis-

solved in 50 mM sodium phosphate, pH 7.0. Mix using a small magnetic stirring chip.

5. Add 2 l of the Na

125

I solution (about 1 mCi) to the protein in the vial. Seal the vial

using a screw-cap septum that can be penetrated with a syringe.

6. Using a syringe, add 25 l of the chloramine-T solution and continue to mix for at least

30 seconds. Longer reaction times can be done, but the solution-phase iodination usually

proceeds very rapidly.

7. Add 100 l of the sodium metabisulﬁ te solution to the iodination reaction to stop it. Stir

for 10 seconds.

8. Purify the iodinated protein from excess reactants by gel ﬁ ltration using a desalting resin.

The column may be pre-treated by passing a solution of bovine serum albumin (BSA)

through it to eliminate nonspeciﬁ c binding sites that could cause signiﬁ cant protein loss

in small-sample applications.

2. Iodobeads

Iodobeads (Thermo Fisher) is an immobilized preparation of a chloramine-T analog, con-

sisting of nonporous, polystyrene beads of diameter 1/8  that have been derivatized to contain

N-chlorobenzenesulfonamide groups (as the sodium salt) (refer to US patents 4,448,764 and

4,436,718). During the manufacturing process, the hydrophobic nature of the polystyrene is

changed to a rather hydrophilic surface due to the chlorosulfonamide modiﬁ cations. The surface

character results in excellent protein recoveries (typically greater than 90 percent). The oxidizing

capability of Iodobeads is limited to surface reactions on the outer shell of the nonporous poly-

styrene ball. The effect is to reduce the rate of iodine incorporation into macromolecules from

the extremely rapid 30-second reaction of soluble chloramine-T to a more relaxed pace of about

2–15 minutes. This also creates a milder oxidizing environment, thus minimizing the potential

for protein degradation or activity loss. A slower iodination process allows more control over

the level of iodine derivatization. Often, tyrosine iodination can be limited to mono-iodo forms,

avoiding the detrimental effects on solubility or activity that excessive modiﬁ cation can cause.

Markwell (1982) reported that the reaction mechanism for creating the electrophilic iodine

species may be somewhat different for Iodobeads than other oxidizing agents. It was dem-

onstrated that the active component remained at or near the surface of the beads during the

course of the iodination process. Markwell speculated that an intermediate reactive species,

N-iodobenzenesulfonamide, is formed from substitution of the chlorine atoms on the bead

(Figure 12.4 ). It is possibly that this intermediate is involved in the direct iodination of target

molecules that approach the bead surface.

Iodobeads may be used in a variety of buffer salts and in the presence of detergents or

denaturants without affecting iodination. The reagent, however, is susceptible to inactivation

by reducing agents such as disulﬁ de reductants, and it can be inactivated by moisture upon

storage. Also, avoid organic solvents that can dissolve or affect the surface characteristics of

polystyrene, such as dimethylformamide (DMF) or dimethyl sulfoxide (DMSO).

To determine the optimal reaction time for a particular radioiodination, 5 l aliquots of the

reaction medium can be removed every 30 seconds, diluted 1:20,000 with 20 mM Tris, 1 mM

ethylenediaminetetraacetic acid (EDTA), pH 7.4, containing 0.5 mg/ml BSA as a carrier protein.

Finally, precipitate a small amount of the diluted aliquot with trichloroacetic acid (TCA, 60 per-

cent), centrifuge to recover the pellet, wash the pellet once with TCA, and measure the amount

of radioactivity in the pellet and supernatant using a gamma counter. The reaction period repre-

senting optimal radiolabel incorporation should be used for subsequent radioiodinations.

Directing the iodination reaction toward histidine residues in proteins, as opposed to prin-

cipally tyrosine modiﬁ cation, is possible simply by increasing the pH of the Iodobeads reaction

from the manufacturer ’s recommended pH 7.0–8.2 (Tsomides et al., 1991). No reducing agent

is required to stop the iodination reaction as is the case with chloramine-T and other methods.

2. Iodobeads 551

552 12. Iodination Reagents

Simple removal of the bead(s) from the reaction is enough to eliminate the iodination process.

The mild nature of the Iodobeads iodination reaction can result in better recovery of active

protein than using soluble oxidants (Lee and Grifﬁ ths, 1984).

Each bead can iodinate up to 500 g of tyrosine-containing protein or peptide. This translates

into an oxidative capacity of about 0.55 mol per bead. The rate of reaction can be controlled

by changing the number of beads that are used and altering the sodium iodide concentration

added to the reaction. Reaction volumes of 100–1,000 l are possible per bead. The following

protocol is suggested for iodinating proteins. Optimization should be done to determine the best

incorporation level to obtain good radiolabel incorporation with retention of protein activity.

Protocol

Caution: Handle all radioactive substances according to the radiation safety regulations insti-

tuted at each facility approved to handle such materials. Use adequate precautions to protect

personal safety and the environment from contamination. Dispose of radioactive waste by fol-

lowing approved guidelines.

1. Wash one or more Iodobeads (Thermo Fisher) with the iodination buffer of choice.

Buffers containing 0.1 M sodium phosphate or 0.1 M Tris at slightly acidic to slightly

alkaline pH work well. A buffer consisting of 0.1 M sodium phosphate, pH 6.5, will give

the highest possible reaction rates and yields.

Figure 12.4 IODO-BEADS contains immobilized chloramine-T groups that can react with radioactive iodide in

aqueous solution to form a highly reactive intermediate. The active species may be an iodosulfonamide deriva-

tive, which then can iodinate tyrosine or histidine residues in proteins.

2. Add the desired number of beads to a solution of carrier-free Na

125

I in iodination buffer

at a concentration level of about 1 mCi per 100 g of protein to be modiﬁ ed. The total

reaction volume should be 100–1,000  l per bead.

3. Add from 5 g to 500 g of a tyrosine-containing peptide or protein dissolved in iodina-

tion buffer to the reaction mixture.

4. React for 2–15 minutes at room temperature. Reactions done at 4°C are possible, but

will result in slightly lower incorporation of iodine.

5. Stop the reaction by removing the solution from the beads. This can be done by simply

pipetting the solution away from the beads or by physically removing the beads. The

beads may be washed once with iodination buffer to assure complete recovery of protein.

Exact timing of the reaction is important to obtain reproducible results.

6. Remove excess

125

I from the iodinated protein by gel ﬁ ltration using a desalting resin.

3. Iodogen

Iodogen (Thermo Fisher), ﬁ rst described by Fraker and Speck in 1978, is 1,3,4,6-tetrachloro-

3 ,6 -diphenylglycouril, an N-haloamine derivative with oxidizing properties similar to those

of Iodobeads and chloramine-T. The compound is insoluble in aqueous solution, therefore

making it a type of solid-phase radioiodination reagent. However, unlike Iodobeads wherein

the oxidizing group is immobilized on another support material, Iodogen must be plated out

on the surface of a reaction vessel prior to doing an iodination. Due to the reagent ’s stability,

the plated reaction vessels can be prepared well in advance and stored in a desiccator until

needed (Markwell and Fox, 1978). Alternatively, pre-coated tubes now are available (Thermo

Fisher).

The reaction of Iodogen with iodide ion in solution results in oxidation with subsequent

formation of a reactive, mixed halogen species, ICl ( Figure 12.5 ). 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. In addition,

crosslinking or modiﬁ cation reagents possessing phenyl rings with activating groups present

(e.g., electron donating constituents: ––OH, ––NH

, etc.) can be iodinated using Iodogen. The

incidence of side reactions appears to be negligible.

3. Iodogen 553

554 12. Iodination Reagents

Since Iodogen is insoluble in aqueous solution and is plated on the surface of the vessel

during the iodination reaction, it is possible to stop the reaction simply by removing the aque-

ous phase. The plating technique is important for the successful use of this reagent. Failure to

plate properly the Iodogen reagent on the surface of the reaction vessel may cause the oxidizing

agent to become suspended in the reaction medium. However, even with well-plated vessels,

there is some potential that a portion of the iodinating reagent can break off in small pieces

and contaminate the aqueous phase. For this reason, it is not advisable to stop the iodination

reaction only by removing the supernatant as in the case of Iodobeads (Section 2, this chapter).

To be certain that the iodination has stopped, an aliquot of sodium metabisulﬁ te can be added

to assure complete cessation of the oxidative process. Alternatively, immediate separation of

the iodinated protein from the reactants by gel ﬁ ltration can be used to stop the reaction (any

suspended particles of Iodogen will be ﬁ ltered out on the top of the gel).

Speciﬁ c radioactivity of 1  10

cpm of

125

I per microgram of protein easily can be obtained

using Iodogen. Iodination efﬁ ciencies are typically 60 percent or better and may be controlled

by regulating the amount of I



concentration added to the reaction.

When iodinating intact cells, Iodogen can be used to radiolabel the outer cell surface proteins

or be directed more toward the inner membrane areas simply by modulating the reaction condi-

tions. Membrane proteins in hydrophobic regions can be labeled to a greater extent by includ-

ing a small excess of carrier iodide, using high salt conditions, or by employing detergents to

disrupt the membrane integrity. Cell surface hydrophilic proteins may be preferentially labeled

Figure 12.5 IODO-GEN is a water-insoluble oxidizing agent that can react with

125



to form a highly reactive

mixed halogen species,

125

ICl. This intermediate can add radioactive iodine atoms to tyrosine or histidine side

chain rings.

by not including components which increase cell permeability, by the use of carrier-free iodide,

and using short reaction times (Markwell and Fox, 1978).

The following protocol describes the use of Iodogen for the radioiodination of proteins and

peptides.

Protocol

Caution: Handle all radioactive substances according to the radiation safety regulations insti-

tuted at each facility approved to handle such materials. Use adequate precautions to protect

personal safety and the environment from contamination. Dispose of radioactive waste by fol-

lowing approved guidelines.

1. In a fume hood, dissolve 10–100 g of Iodogen (Thermo Fisher) in 100–500 l of chloro-

form, methylene chloride, or DMSO. The use of 10 g of Iodogen per 100 g of protein

or 107 cells to be iodinated will result in good incorporation yields.

2. Add the Iodogen solution to a clean, dry, glass reaction vessel in an amount needed for

the quantity of protein to be labeled. Slowly evaporate the solvent in the vessel using a

stream of dry nitrogen or other inert gas. Do not use a strong gas jet, since rapid evapo-

ration or turbulence in the solvent solution will cause uneven Iodogen distribution with

possible clumping. Do not merely leave the vessel to dry in a hood, since contaminants or

moisture may get into the reagent ﬁ lm. If done properly, the plating process should leave

a ﬁ lm of Iodogen on the inner surface of the vessel that is difﬁ cult to see—looking like

a slight clouding of the glass. After solvent evaporation, seal the container with nitrogen

and store in a desiccator until needed.

3. Dissolve the protein to be iodinated in a buffer compatible with its known biological

stability. Conditions ranging from pH 4.4 to 9 and temperatures from 0°C to 37°C can

be used with good results. The amount of protein to be labeled should be contained in a

volume of 100 l or less. The sample buffer should not contain reducing agents, antioxi-

dants, 2-mercaptoethanol, dithiothreitol (DTT), cysteine, glycerol, high detergent con-

centrations, or anything that may interfere with the iodination reaction or dislodge the

plated Iodogen.

4. Rinse the plated reaction vessel once with sample buffer to remove any loose particles of

Iodogen that may not be strongly adhered to the surface of the glass.

5. Add carrier-free Na

125

I to the reaction vessel in a ratio of about 500 Ci per 100 g protein.

6. React for 10–15 minutes at room temperature. Optimization of the reaction time and the

amount of

125

I added to the reaction may have to be done to obtain the best radioactivity

incorporation and retention of protein activity.

7. Remove the sample from the reaction vessel. This process should terminate the iodin ation

reaction, unless small Iodogen particles break off from the sides of the vessel. To assure safe

handling, carrier NaI may be added to the reaction mixture to a ﬁ nal concentration of 1 mM.

8. Remove excess reactants by gel ﬁ ltration using a desalting resin.

4. Lactoperoxidase-Catalyzed Iodination

An enzyme-catalyzed process also may be used to form reactive iodine species capable of iodin-

ating proteins and other molecules (Marcholonis, 1969; Morrison and Bayse, 1970). The

4. Lactoperoxidase-Catalyzed Iodination 555

556 12. Iodination Reagents

enzymatic approach utilizes lactoperoxidase in the presence of H

to oxidize

125



to I

. The

iodine thus formed may react with tyrosine or histidine sites within proteins, forming radiola-

beled complexes. Unlike the use of chemical oxidants for iodination, the enzymatic reaction is

very pH dependent—the optimum being between pH 6 and 7. If H

is directly added to the

reaction medium, it must be highly pure with no stabilizing agents such as metals, since they

inhibit the oxidation process.

An alternative to direct addition of H

is to form it in situ through the use of a second enzy-

matic reaction. Enzymobeads (originally from Bio-Rad, but no longer commercially available)

used immobilized lactoperoxidase along with immobilized glucose oxidase to create the neces-

sary oxidative environment. The glucose oxidase reaction transformed added glucose in the iodin-

ation medium to the required H

. As it was formed, the lactoperoxidase (coupled in tandem

to the same beads) would catalyze the formation of I

( Figure 12.6 ). The immobilized enzymes

create an iodination environment that is more oxidatively gentle than direct addition of a soluble

chemical oxidant like chloramine-T.

5. Iodinatable Modiﬁ cation and Crosslinking Agents

Radioiodination can be done by an indirect approach that utilizes a radiolabeled crosslin–

king or modiﬁ cation reagent which is then used to label the target molecule. One advantage of

indirect labeling over direct modiﬁ cation of tyrosine or histidine residues in proteins is to be

able to control the iodination to occur with functional groups other than just using indigenous

amino acids. In addition, the ability to add a radiolabeled modiﬁ cation agent to a molecule can

facilitate the radioactive tagging of substances that normally do not have radioiodinatable sites.

Another major advantage of using iodinatable modiﬁ cation agents is to eliminate the potential

for oxidative damage to sensitive biological molecules, as may occur when an oxidant is used

in direct iodination procedures.

For instance, an amine-reactive modiﬁ cation reagent can be radiolabeled and subsequently

used to couple with - and N-terminal amines on a protein molecule. The protein is not

exposed to oxidative conditions, and the level of radiolabeling can be discretely controlled by

the molar ratio of modiﬁ cation reagent addition. The use of iodinatable crosslinking reagents

can similarly provide radioactive tags incorporated into conjugates at the time of formation.

In addition, iodinatable bioconjugation reagents that react with groups such as sulfhydryls, alde-

hydes, or other functionalities of limited occurrence in proteins or other macromolecules, can

be used to direct the point of radiolabeling to areas away from active centers or binding sites,

thus better preserving biological activity. Finally, some photoreactive crosslinking agents can be

iodinated, used to label a targeting molecule, photolyzed at the point of binding to its target,

and the cross-bridge of the resulting complex chemically cleaved, resulting in the transfer the

radiolabel to the targeted component (Chapter 5, Section 3). This process can be used to fol-

low the targeted molecule in vivo or in cellular systems.

Direct iodination of proteins and other molecules does not provide the range of experi-

mental options available through indirect labeling. The main disadvantage of the indirect

labeling process is the additional steps needed to prepare the radiolabeled crosslinker or modi-

ﬁ cation reagent before iodination of the desired molecule. The following sections discuss some

of the major indirect iodination methods, including the reagents available for doing such

procedures.

5.1. Bolton–Hunter Reagent

Bolton and Hunter (1973) developed the reagent N-succinimidyl-3-(4-hydroxyphenyl)propionate

(SHPP) for the indirect radioiodination of proteins and other macromolecules (Thermo Fisher).

The NHS ester end of the molecule reacts with amine groups in target molecules to form sta-

ble amide bond derivatives (Chapter 2, Section 1.4). The other end of the reagent contains a

phenolic group that is ideally suited for modiﬁ cation with

125

I. Iodination of the phenol group

occurs ortho to the hydroxyl, thus accommodating either one or two iodine substitutions per

molecule ( Figure 12.7 ).

Figure 12.6 The immobilized glucose oxidase/lactoperoxidase system radioiodinates proteins through the inter-

mediate formation of hydrogen peroxide from the oxidation of glucose. H

then reacts with iodide anions to

form reactive iodine (I

). This efﬁ ciently drives the formation of the highly reactive H



species that is capa-

ble of iodinating tyrosine or histidine residues (see Figure 12.2).

5. Iodinatable Modiﬁ cation and Crosslinking Agents 557

558 12. Iodination Reagents

The use of the Bolton–Hunter reagent to incorporate radioactivity into proteins results

in at least as good incorporation of

125

I as direct labeling procedures using an oxidant. In many

cases, the degree of radioiodine labeling can be much greater than that possible by direct label-

ing of tyrosine residues, because the total number of amines in a protein (from N-terminal

Figure 12.7 The Bolton–Hunter reagent may be radioiodinated at its phenolic ring structure prior to reaction

with an amine-containing molecule to form an amide bond modiﬁ cation.

and lysine side chains) is typically signiﬁ cantly more than the number of tyrosines present.

The major advantage of the indirect approach, however, is that non-tyrosine containing pro-

teins also may be iodinated. In addition, substances sensitive to oxidant exposure can be labeled

without loss of activity or structural degradation. Ultimately, any molecule containing an avail-

able amino group can be radioiodinated with SHPP, even if it does not contain a strongly

activated aromatic ring system to allow direct iodine substitution. For reviews of protein modi-

ﬁ cation using radiolabeled Bolton–Hunter reagent (see Langone, 1980, 1981; Wilbur, 1992).

For its use in labeling cellular components (see Katz et al. (1982); Davies and Palek (1981)).

SHPP is relatively insoluble in aqueous environments and must be dissolved in an organic

solvent prior to addition to a reaction medium. Suggested solvents include dioxane and DMSO

that are low in water content to avoid hydrolysis of the NHS ester.

A water-soluble version of the original Bolton–Hunter reagent also is available, called sulfo-

SHPP (Thermo Fisher). This compound contains a negatively charged sulfonate group on the

NHS ring structure, which provides enough hydrophilicity to allow direct addition to aqueous

reaction mediums.

The following procedure describes the iodination process for the Bolton–Hunter reagent and

its subsequent use for the radiolabeling of protein molecules. Modiﬁ cation of other macromol-

ecules can be done using the same general method. For particular labeling applications, opti-

mization of the level of iodine incorporation may have to be done to obtain the best speciﬁ c

radioactivity with retention of biological activity.

Protocol

Caution: Handle all radioactive substances according to the radiation safety regulations insti-

tuted at each facility approved to handle such materials. Use adequate precautions to protect

personal safety and the environment from contamination. Dispose of radioactive waste by fol-

lowing approved guidelines.

1. Dissolve SHPP (Thermo Fisher) in dry dioxane or DMSO at a concentration of 0.5 mg/ml.

Prepare fresh. If sulfo-SHPP is used dissolution in organic solvent is unnecessary,

although it may facilitate the addition of a small quantity to an aqueous reaction.

2. Dissolve chloramine-T (Sigma) in 50 mM sodium phosphate, pH 7.5 (reaction buffer)

at a concentration of 100  g per 25–50  l of buffer. Prepare fresh.

3. Add 2 l of the SHPP solution to 50 l of the reaction buffer. Alternatively, dissolve

solid sulfo-SHPP into the reaction buffer to give a ﬁ nal concentration of 1  g/50 l. To

better facilitate measuring out sulfo-SHPP, a more concentrated solution may be pre-

pared in a greater volume and then immediately diluted to this concentration. Once the

Bolton–Hunter reagent is in an aqueous environment, the NHS ester end of the com-

pound will hydrolyze. Therefore, all aqueous handling of the reagent from this point on

should be done quickly to preserve enough amine-coupling activity to label the protein

after the iodination reaction.

4. Immediately add the chloramine-T to the SHPP solution. Mix well.

5. React for 15 seconds with mixing.

6. Add to the iodination reaction 5 l of DMF and 100 l of benzene. Mix to extract the

iodinated Bolton–Hunter reagent into the organic phase.

7. Remove the aqueous phase and transfer the organic phase into a clean glass vial or tube.

5. Iodinatable Modiﬁ cation and Crosslinking Agents 559