Hermanson G. Bioconjugate Techniques, Second Edition

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Protocol

1. Prepare the protein or macromolecule to be thiolated in a non-amine containing buffer

at pH 8.0. For the modiﬁ cation of ribosomal proteins (often cited in the literature) use

50 mM triethanolamine hydrochloride, 1 mM MgCl

, 50 mM KCl, pH 8.0. The magne-

sium and potassium salts are for stabilization of some ribosomal proteins. If other pro-

teins are to be thiolated, the same buffer may be used without added salts for stabilization.

Alternatively, 50 mM sodium phosphate, 0.15 M NaCl, pH 8.0, or 0.1 M sodium borate,

pH 8.0 may be used. For the modiﬁ cation of polysaccharides, use 20 mM sodium borax,

pH 10, to produce reactivity toward carbohydrate hydroxyl residues. Dissolve the protein

to be modiﬁ ed at a concentration of 10 mg/ml in the reaction buffer of choice. Lower con-

centrations also may be used with a proportional scaling back of added 2-iminothiolane.

2. Dissolve the Traut ’s reagent (Thermo Fisher) in water at a concentration of 2 mg/ml

(makes a 14.5 mM stock solution). The solution should be used immediately. For the

modiﬁ cation of IgG at a concentration of 10 mg/ml using a 10-fold molar excess of

Traut ’s reagent, add 45.8  l of the stock solution to each ml of the protein solution.

3. React for 1 hour at room temperature (a 4 °C reaction temperature may be used success-

fully as well).

4. Purify the thiolated protein from unreacted Traut ’s reagent by gel ﬁ ltration using your

buffer of choice (i.e., 20 mM sodium phosphate, 0.15 M NaCl, 1 mM EDTA, pH 7.2). The

addition of EDTA to this buffer helps to prevent oxidation of the sulfhydryl groups and

the resultant disulﬁ de formation. After puriﬁ cation, use the thiolated protein immediately

70 1. Functional Targets

Figure 1.61 Traut ’s reagent can undergo side reactions after the modiﬁ cation of an amine-containing molecule.

The terminal thiol group can recyclize to create another iminothiolane derivative that effectively ties up the thiol.

in a conjugation reaction to avoid the recyclization of the free thiol with concomitant

decrease in thiol availability.

5. The degree of SH modiﬁ cation may be determined using the Ellman ’s assay (Section

4.1, this chapter).

When 2-iminothiolane is used to modify proteins in tandem with 4,4 -dipyridyl disulﬁ de, a

protected sulfhydryl can be introduced in a single step (King et al., 1978). The simultaneous

reaction between a protein, 2-iminothiolane and 4,4 -dipyridyl disulﬁ de yields a modiﬁ cation

containing pyridyl disulﬁ de groups. The pyridyl disulﬁ de subsequently may be reduced with

dithiothreitol (DTT) to yield a free sulfhydryl. Pyridyl disulﬁ des also are highly reactive toward

sulfhydryls through disulﬁ de interchange (Chapter 2, Section 2.6). The protocol is a modiﬁ ca-

tion of the method of King et al ., 1978.

Protocol

1. Dissolve 1–10 mg of a protein to be modiﬁ ed in 1.0 ml of 0.025 M sodium borate, pH 9.0.

2. Dissolve 2-iminothiolane in 0.025 M sodium borate to a concentration of 0.02 M.

3. Dissolve 4,4  -dipyridyl disulﬁ de at a concentration of 2 mg/ml in acetonitrile.

4. Add 0.2 ml of (3) and 1.0 ml of (2) to the protein solution.

5. React for 2 hours at room temperature.

6. Purify the modiﬁ ed protein by gel ﬁ ltration or dialysis.

Occasionally, a protein modiﬁ ed in this manner will begin to precipitate as the reaction pro-

ceeds. Stopping the reaction earlier or adding a smaller quantity of modifying reagents may

limit this effect.

Modiﬁ cation of Amines with SATA

A versatile reagent for introducing sulfhydryl groups into proteins is SATA, N-succinimidyl

S-acetylthioacetate (Duncan et al., 1983). The active NHS ester end of SATA reacts with amino

groups in proteins and other molecules to form a stable amide linkage ( Figure 1.62 ) (Chapter 2,

Section 1.4). The modiﬁ ed protein then contains a protected sulfhydryl that can be stored with-

out degradation and subsequently deprotected as needed with an excess of hydroxylamine ( Figure

1.63 ). Since the protecting group can be removed without adding disulﬁ de reducing agents like

DTT, disulﬁ des indigenous to the native protein will not be affected. This is an important consid-

eration if disulﬁ des are vital to activity, such as in the case of antibodies and some protein toxins.

4. Creating Speciﬁ c Functionalities 71

Figure 1.63 Deprotection with hydroxylamine of the acetylated thiol of SATA-modiﬁ ed proteins yields a free

sulfhydryl group.

72 1. Functional Targets

Figure 1.62 SATA can react with available amine groups in proteins and other molecules via its NHS ester

end to form protected sulfhydryl derivatives. The illustrated protein is glutathione- S-transferase (E.C.2.5.1.18)

(Ji et al ., 1995).

SATA is often used to form antibody–enzyme conjugates utilizing maleimide-containing

heterobifunctional crosslinking agents. Most polyclonal antibody molecules may be modiﬁ ed

to contain up to about 6 SATA molecules per immunoglobulin with minimal effect on anti-

gen binding activity. Some sensitive monoclonal antibodies, however, may be susceptible to

modiﬁ cation and should be tested on a case-by-case basis. The modiﬁ ed antibody then may be

deprotected and reacted with a maleimide-activated enzyme to form a conjugate useful in immu-

noassays (Chapter 20, Section 1.1). Conjugates formed using SATA are usually of low molecular

weight with very few high-molecular-weight oligomers. They also maintain a bivalent antibody

structure, assuring a conjugate containing two antigen binding sites. This is an advantage

over reduction schemes that break the antibody molecule into two heavy–light chain pairs to

create sulfhydryls, since disulﬁ de cleavage yields antibody fragments with only one antigen bind-

ing site.

SATA has been used to form conjugates with avidin or steptavidin with excellent retention

of activity (Chapter 23, Section 3.1). It also has been used in the formation of a therapeutically

useful toxin conjugate with recombinant CD4 (Ghetie et al., 1990), to study syntaxin proteins

(Amessou et al., 2007), to prepare bispeciﬁ c antibodies (Lindorfer et al., 2001), and to make a

unique polylysine conjugate as a vehicle for drug delivery (Sakharov et al ., 2001).

SATA is freely soluble in many organic solvents. In use, it is typically dissolved as a stock

solution in DMSO, DMF, or methylene chloride, and then an aliquot of this solution is added

to an aqueous reaction mixture containing the protein to be modiﬁ ed.

The thiolation method described below is generally applicable for the modiﬁ cation of proteins

with SATA, particularly for subsequent conjugation with a maleimide-activated secondary pro-

tein. The degree of modiﬁ cation described usually yields 3–4 moles of SH groups per mole pro-

tein when thiolating immunoglobulins. Other macromolecules containing primary amines may be

modiﬁ ed using a similar procedure. The degree of modiﬁ cation observed with other molecules may

vary depending on the number of available primary amines and their relative reactivity. The molar

ratio of SATA to immunoglobulin added to a reaction for the modiﬁ cation of rabbit polyclonal

IgG versus the degree of sulfhydryl incorporation is illustrated in Figure 1.64 (Sykaluk, 1994).

4. Creating Speciﬁ c Functionalities 73

Figure 1.64 SATA modiﬁ cation of rabbit polyclonal IgG with the resultant sulfhydryl incorporation level.

The following protocol represents a generalized method for protein thiolation using SATA.

For comparison purposes, contrast the variation of this SATA modiﬁ cation method as outlined

in Chapter 20, Section 1.1 for use in the preparation of antibody–enzyme conjugates.

Protocol

1. Dissolve the protein to be thiolated at a concentration of 1–5 mg/ml in 50 mM sodium

phosphate, pH 7.5, containing 1–10 mM EDTA. Other non-amine containing buffers

such as borate, HEPES, and bicarbonate also may be used as the reaction medium. The

effective pH for the NHS ester modiﬁ cation reaction is in the range of 7.0–9.0, but envi-

ronments closer to neutrality will limit the hydrolysis of the ester.

2. Dissolve the SATA reagent (Thermo Fisher) in DMSO at a concentration of 65 mM

(15 mg/ml). Note : DMSO should be handled in a fume hood.

3. Add 10  l of the SATA solution to each ml of protein solution.

4. Mix and react for 30 minutes at room temperature.

5. Separate modiﬁ ed protein from unreacted SATA and reaction by-products by dialysis

against 50 mM sodium phosphate, pH 7.5, containing 1 mM EDTA or by gel ﬁ ltration

on a Sephadex G-25 column (Pharmacia) using the same buffer.

6. Deprotect the acetylated SH groups as needed by adding 100 l of 0.5 M hydroxy-

lamine hydrochloride in 50 mM sodium phosphate, 25 mM EDTA, pH 7.5, to each ml of

the SATA-modiﬁ ed protein solution.

7. Mix and react for 2 hours at room temperature.

8. Purify the sulfhydryl-modiﬁ ed protein by dialysis against 50 mM sodium phosphate, 1 mM

EDTA, pH 7.5, or by gel ﬁ ltration on a Sephadex G-25 column using the same buffer.

The deacetylated protein should be used immediately to prevent loss of sulfhydryl content

through disulﬁ de formation. The degree of SH modiﬁ cation may be determined by performing

an Ellman ’s assay (Section 4.1, this chapter).

Modiﬁ cation of Amines with SATP

SATP, succinimidyl acetylthiopropionate, is an analog of SATA (Chapter 1, Section 4.1) contain-

ing one additional carbon atom in length (Fuji et al., 1985). The compound retains all the advan-

tages of a protected sulfhydryl, including stability of the modiﬁ ed protein and selective release of

the protecting group with hydroxylamine to free the sulfhydryl as needed ( Figure 1.65 ). SATP is

soluble in DMF and methylene chloride. It is usually ﬁ rst solubilized in organic solvent and an

aliquot added to an aqueous solution containing the macromolecule to be modiﬁ ed. It is particu-

larly useful in adding an N-terminal SH group at the completion of peptide synthesis.

74 1. Functional Targets

Protocol

1. Dissolve the protein or peptide to be thiolated at a concentration of 10 mg/ml in 50 mM

sodium phosphate, pH 7.5, containing 1 mM EDTA. Other non-amine containing buffers

such as borate, HEPES, and bicarbonate also may be used as the reaction medium. The

effective pH for the NHS ester modiﬁ cation reaction is in the range of 7.0–9.0. Conditions

closer to neutral pH will limit the degree of NHS ester hydrolysis during the reaction.

2. Dissolve the SATP reagent (Molecular Probes) in DMF at a concentration of 65 mM

(16 mg/ml). Note : DMF should be handled in a fume hood.

3. Add 10  l of the SATP solution to each ml of protein or peptide solution.

4. Mix and react for 30–60 minutes at room temperature (or 2–4 hours at 4 ° C).

5. Separate modiﬁ ed protein from unreacted SATP and reaction by-products by dialysis

against 50 mM sodium phosphate, pH 7.5, containing 1 mM EDTA or by gel ﬁ ltration

using a desalting column with the same buffer. If a peptide of low molecular weight is

being modiﬁ ed, careful gel ﬁ ltration using a matrix having a low exclusion limit will sep-

arate the peptide from the reaction by-products, but the separation should be done on an

automated system to accurately capture the peaks. In this case, use either Sephadex G-25

or Sephadex G-10 for the chromatography.

6. Deprotect the acetylated SH groups as needed by adding 100 l of 0.5 M hydroxy-

lamine hydrochloride in 50 mM sodium phosphate, 25 mM EDTA, pH 7.5, to each ml of

the SATP-modiﬁ ed protein solution.

7. Mix and react for 2 hours at room temperature.

8. Purify the sulfhydryl-modiﬁ ed protein by dialysis against 50 mM sodium phosphate,

1 mM EDTA, pH 7.5, or by gel ﬁ ltration on a Sephadex G-25 column using the same

buffer. Again, if a peptide of low molecular weight is being modiﬁ ed, use careful gel ﬁ l-

tration for puriﬁ cation.

The deacetylated protein should be used immediately to prevent loss of sulfhydryl content

through disulﬁ de formation. The degree of SH modiﬁ cation may be determined by perform-

ing an Ellman ’s assay (Section 4.1, this chapter).

4. Creating Speciﬁ c Functionalities 75

Figure 1.65 SATP reacts with amine-containing proteins or other molecules via its NHS ester end to create

protected sulfhydryl derivatives in a manner similar to that of SATA. Deprotection can be done with hydroxy-

lamine to free the thiol.

Modiﬁ cation of Amines with SPDP

SPDP, N-succinimidyl 3-(2-pyridyldithio)propionate, is one of the most popular heterob-

ifunctional crosslinking agents (Chapter 5, Section 1.1). The NHS ester end of SPDP reacts

with amine groups to form an amide linkage, while the 2-pyridyldithiol group at the other

end can react with sulfhydryl residues to form a disulﬁ de linkage (Carlsson et al., 1978).

The crosslinker is used extensively to form immunotoxin conjugates for in vivo administra-

tion (Chapter 21, Section 2.1). The reagent is also useful in creating sulfhydryls in proteins

and other molecules. Once modiﬁ ed with SPDP, a protein can be treated with DTT (or other

disulﬁ de reducing agents, see Section 4.1, this chapter) to release the pyridine-2-thione leaving

group and form the free sulfhydryl ( Figure 1.66 ). The terminal SH group then can be used

to conjugate with any crosslinking agents containing sulfhydryl-reactive groups, such as male-

imide or iodoacetyl functionalities (for covalent conjugation) or 2-pyridyldithiol groups (for

reversible conjugation).

There are three forms of SPDP analogs currently available commercially (Thermo Fisher):

the standard SPDP, a long-chain version designated LC-SPDP, and a water-soluble, sulfo-NHS

form also containing an extended chain, called sulfo-LC-SPDP (Chapter 5, Section 1.1). The

main disadvantage to using SPDP to create sulfhydryls is the necessity of using a reducing agent

to remove the pyridine-2-thione group. Reducing agents also will affect indigenous disulﬁ des

within a protein molecule, cleaving and reducing them. This method therefore works well for

proteins containing no sulfhydryls or no disulﬁ des that are critical to function, but it may cause

loss of activity or subunit breakdown in proteins containing essential disulﬁ des.

76 1. Functional Targets

Figure 1.66 SPDP-modiﬁ ed proteins can be reduced with DTT to yield free sulfhydryl groups for conjugation.

The following procedure is similar to the method of Cumber et al. (1985), but with some

modiﬁ cations.

Protocol

1. Dissolve the protein or macromolecule to be thiolated at a concentration of 10 mg/ml in

50 mM sodium phosphate, 0.15 M NaCl, pH 7.2. Other non-amine containing buffers

such as borate, HEPES, and bicarbonate also may be used in this reaction. The effective

pH for the NHS ester modiﬁ cation reaction is in the range of 7.0–9.0, but environments

closer to neutrality will limit ester hydrolysis.

2. Dissolve SPDP (Thermo Fisher) at a concentration of 6.2 mg/ml in DMSO (makes a 20 mM

stock solution). Alternatively, LC-SPDP may be used and dissolved at a concentration

of 8.5 mg/ml in DMSO (also makes a 20 mM solution). If the water-soluble sulfo-LC-

SPDP is used for the reaction, a stock solution in water may be prepared just prior to

adding an aliquot to the protein solution. In this case, prepare a 10 mM solution of sulfo-

LC-SPDP by dissolving 5.2 mg/ml in water. Since an aqueous solution of the crosslinker

will degrade by hydrolysis of the sulfo-NHS ester, it should be used quickly to prevent

signiﬁ cant loss of activity. If a sufﬁ ciently large amount of protein will be modiﬁ ed to

allow accurate weighing of sulfo-LC-SPDP, the solid may be added directly to the reac-

tion mixture without preparing a stock solution in water.

3. Add 25 l of the stock solution of either SPDP or LC-SPDP in DMSO to each ml of the

protein to be modiﬁ ed. If sulfo-LC-SPDP is used, add 50 l of the stock solution in water

to each ml of protein solution.

4. Mix and react for at least 30 minutes at room temperature. Longer reaction times, even

overnight, will not adversely affect the modiﬁ cation.

5. Purify the modiﬁ ed protein from reaction by-products by dialysis or gel ﬁ ltration using

50 mM sodium phosphate, 0.15 M NaCl, pH 7.2.

6. To release the pyridine-2-thione leaving group and form the free sulfhydryl, add DTT at

a concentration of 0.5 mg DTT per mg of modiﬁ ed protein. A stock solution of DTT may

be prepared to make it easier to add it to a small amount of protein solution. In this case,

dissolve 20 mg of DTT per ml of 0.1 M sodium acetate, 0.1 M NaCl, pH 4.5. Add 25  l

of this solution per mg of modiﬁ ed protein. Release of pyridine-2-thione can be followed

by its characteristic absorbance at 343 nm (   8.08  10

 1

7. Mix and react at room temperature for 30 minutes.

8. Purify the thiolated protein from excess DTT by dialysis or gel ﬁ ltration using 50 mM

sodium phosphate, 0.15 M NaCl, 1 mM EDTA, pH 7.2. The modiﬁ ed protein should be

used immediately in a conjugation reaction to prevent sulfhydryl oxidation and forma-

tion of disulﬁ de crosslinks.

Modiﬁ cation of Amines with SMPT

SMPT, succinimidyloxycarbonyl- -methyl--(2-pyridyldithio)toluene, contains an NHS ester

end and a pyridyl disulﬁ de end similar to SPDP, but its hindered disulﬁ de makes conjugates

formed with this reagent more stable (Thorpe et al., 1987) (Chapter 5, Section 1.2). The reagent

is especially useful in forming immunotoxin conjugates for in vivo administration (Chapter 21,

Section 2.1). A water-soluble analog of this crosslinker containing an extended spacer arm is

also commercially available as sulfo-LC-SMPT (Thermo Fisher).

4. Creating Speciﬁ c Functionalities 77

SMPT or sulfo-LC-SMPT may be used as thiolation reagents by ﬁ rst reacting its NHS ester

end with an amine-containing molecule and then releasing the pyridine-2-thione leaving group

with DTT to free the sulfhydryl ( Figure 1.67 ). The disadvantage of this approach is the neces-

sity of using a reducing agent to create the SH group modiﬁ cation. This method of thiolation

only should be used if there are no disulﬁ des in the target molecule that are critical to function.

If a reductant cannot be used, choose a thiolation method that does not need DTT treatment,

such as the use of Traut ’s reagent or SATA (Section 4.1, this chapter).

Since SMPT is not soluble in aqueous solutions it must be ﬁ rst dissolved in organic solvent

and an aliquot of this stock solution transferred to the reaction solution. The reagent is soluble

78 1. Functional Targets

Figure 1.67 SMPT can be used to modify the amine groups of proteins to form disulﬁ de intermediates. The

disulﬁ des can be reduced with DTT to create free thiols for subsequent conjugation purposes.

in DMF and DMSO, but is much more stable in solutions of acetonitrile. A stock solution of

SMPT in acetonitrile may be kept frozen without loss of activity. The NHS ester of SMPT also is

extraordinarily stable to hydrolysis in water. Even when an SMPT/acetonitrile aliquot is added

to an aqueous solution and stored at room temperature, SMPT will only lose about 5 percent

of its activity after 16 hours. By contrast, other NHS esters usually have half-lives measured in

minutes or hours in aqueous environments, depending on the pH.

Sulfo-LC-SMPT is not as stable as SMPT. The sulfo-NHS ester is more susceptible to hydrol-

ysis in aqueous solutions and the pyridyl disulﬁ de group is more easily reduced to the free

sulfhydryl. Stock solutions of sulfo-LC-SMPT may be prepared in water, but should be used

immediately to prevent loss of amine coupling ability.

Protocol

1. Dissolve the protein or macromolecule to be thiolated at a concentration of 10 mg/ml in

50 mM sodium phosphate, 0.15 M NaCl, pH 7.2. Other non-amine-containing buffers

such as borate, HEPES, and bicarbonate also may be used as the reaction medium. The

effective pH for the NHS ester modiﬁ cation reaction is in the range of 7.0–9.0, but con-

ditions close to neutrality will limit ester hydrolysis.

2. Dissolve SMPT (Thermo Fisher) at a concentration of 7.7 mg/ml in acetonitrile (makes

a 20 mM stock solution). Alternatively, the water-soluble sulfo-LC-SMPT may be used

and dissolved at a concentration of 6.0 mg/ml in water (makes a 10 mM solution). This

should be done just prior to adding an aliquot to the thiolation reaction. Since an aque-

ous solution of the crosslinker will degrade by hydrolysis of the sulfo-NHS ester, it

should be used quickly to prevent signiﬁ cant loss of activity. If a sufﬁ ciently large amount

of protein will be modiﬁ ed to allow accurate weighing of sulfo-LC-SMPT, the solid may

be added directly to the reaction mixture without preparing a stock solution in water,

but this is not recommended with most reactions.

3. Add 25 l of the stock solution of SMPT in acetonitrile to each ml of the protein to be

modiﬁ ed. If sulfo-LC-SMPT is used, add 50 l of the stock solution in water to each ml

of protein solution.

4. Mix and react for at least 30 minutes at room temperature. Longer reaction times, even

overnight, will not adversely affect the modiﬁ cation.

5. Purify the modiﬁ ed protein from reaction by-products by dialysis or gel ﬁ ltration using

50 mM sodium phosphate, 0.15 M NaCl, pH 7.2.

6. To release the pyridine-2-thione leaving group and form the free sulfhydryl, add DTT at

a concentration of 0.5 mg DTT per mg of modiﬁ ed protein. A stock solution of DTT may

be prepared to make it easier to add it to a small amount of protein solution. In this case,

dissolve 20 mg of DTT per ml of 0.1 M sodium acetate, 0.1 M NaCl, pH 4.5. Add 25  l

of this solution per mg of modiﬁ ed protein. Release of pyridine-2-thione can be followed

by its characteristic absorbance at 343 nm (   8.08  10

 1

7. Mix and react at room temperature for 30 minutes.

8. Purify the thiolated protein from excess DTT by dialysis or gel ﬁ ltration using 50 mM

sodium phosphate, 0.15 M NaCl, 1 mM EDTA, pH 7.2. The modiﬁ ed protein should be

used immediately in a conjugation reaction to prevent sulfhydryl oxidation and forma-

tion of disulﬁ de crosslinks.

4. Creating Speciﬁ c Functionalities 79