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2 Classification of Cross-Linking Procedures

2 Classification of Cross-Linking Procedures

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298



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation



This procedure has many applications. For example, in the study of ribosomal protein topography,

Hultin4 added sulfhydryl- and amino-group–directed bifunctional cross-linkers directly to a

suspension of ribosomes as described in the text box below. Similarly, Baskin and Yang5 added

dithiobis(succinimidylproprionate) directly to a suspension of microsomes for studying the protein

topography of rat liver microsomes. However, such one-step procedures are not desirable for nonassociating molecules because side reactions cross-linking the same molecules may occur, giving rise

to homopolymeric adducts with relatively few heterodimeric cross-links. This scenario would be

the case for the preparation of immunoconjugates where the proteins are free in solution.6 IgG, for

example, has a higher reactivity with 4,4′-difluoro-3,3′-dinitrodiphenyl sulfone than does horseradish peroxidase. This reagent thus preferentially reacts with IgG, giving rise to homopolymerization. The yield of conjugation between peroxidase and IgG is very low in the one-step procedure.

Modesto and Pesce7 have shown that in some instances the rate of reagent addition can influence

the conjugation yield. In general, slow addition of reagent over time, as opposed to addition of the

entire reagent at one time, leads to increased yields of coupled proteins. This slow addition protocol

is particularly appropriate in the case of large protein assemblies such as those found in membrane

preparations. Rapid addition procedures are less desirable since they may lead to the production of

both homopolymers and heteropolymers, particularly for nonreacting molecules. In this case, a twostep reaction procedure would be a better choice.

CROSS-LINKING OF PROTEIN CONTACT SITES IN MAMMALIAN

RIBOSOMES BY A ONE-STEP PROCEDURE4

Ribosomes (40–60 ODU/mL) were suspended in 20 mM triethanolamine/HCl buffer (pH 6.8)

containing 150 mM sucrose, 75 mM KCl, and 5 mM MgCl2. The cross-linking reagent (e.g.,

(2,5-dioxopyrrolidin-1-yl)-2-[2-(2,5-dioxopyrrol-1-yl)-4-hydroxy-phenyl]azobenzoate), preserved at −20°C as a 10 mM solution in dimethylsulfoxide, was added with stirring to a concentration of 0.1–0.4 mM. After incubation for 10 min at 35°C, the suspension was mixed

with 10 volumes of 75 mM KCl, 5 mM MgCl2, and 5 mM mercaptoethanol to abolish remaining maleimide functions. The diluted suspension was centrifuged and the pellet preserved

at −20°C for analysis.



9.2.2  Two-Step Cross-Linking Reactions

In this procedure, one of the components to be conjugated is first reacted with the cross-linker. The

modified molecule is then isolated or the unreacted reagent removed prior to addition of the second

component. This approach takes advantage of the differential reactivities of the functional groups

in heterobifunctional reagents as well as the differential selectivity of homobifunctional reagents

toward the molecules to be coupled.

Practically all heterobifunctional cross-linkers, particularly photosensitive reagents, are used

according to a two-step procedure.3 With N-hydroxysuccinimidyl (NHS)-ester-maleimido hetrobifunctional reagents, cross-linking is initiated with the NHS-ester reaction first to minimize hydrolysis of the NHS-ester in aqueous systems. The sulfhydryl then reacts with the maleimide group

in the second step since it is significantly more resistant to hydrolysis than the NHS-ester moiety.

For example, in the preparation of β-galactosidase–IgG conjugate with m-maleimidobenzoyl-Nhydroxysuccinimde (MBS), IgG (which does not have a free thiol group) is first labeled with the

reagent through an amino group reaction with the NHS-ester. After removal of excess reagent, by

either dialysis or gel filtration chromatography, β-galactosidase (which contains free thiol groups) is

added to react with the maleimide moiety of labeled IgG, resulting in the desired immunoconjugate

­product.6 This two-step procedure is described in the text box. Many other immunoconjugates are

prepared in a similar way.



General Approaches for Chemical Cross-Linking



299



A notable example of a homobifunctional reagent that shows differential reactivities usable in

a two-step coupling reaction is toluene-2,4-diisocyanate. The para-isocyante group is much more

reactive than that at the ortho-position owing to steric hindrance of the latter due to the methyl

group.8 As a first step in the reaction, the protein is mixed with the reagent at near 0°C where modification of the protein will take place with the para-isocyanate group. After this reaction, the second

protein to be cross-linked is added and the temperature is raised to 37°C, the temperature at which

the ortho-isocyanate group will react and cross-link the proteins.

CONJUGATION OF β-GALACTOSIDASE AND IGG

BY A TWO-STEP PROCEDURE6

Step 1: Purified donkey antisheep IgG antibodies were dissolved in 1.5 mL of 0.1 M phosphate buffer, pH 7.0, containing 50 mM NaCl to give an optical density of approximately 1.4

at 280 nm. A 15 μL aliquot of dioxan containing 0.32 mg of MBS was added to the antibody

solution, mixed, and maintained at 30°C for 1 h. The solution was applied to a Sephadex G-25

column (30 × 0.9 cm), equilibrated with 10 mM phosphate buffer, pH 7.0, containing 10 mM

MgCl2 and 50 mM NaCl, and eluted with the same buffer.

Step 2: A total of 3 mL of eluant having an optical density of 0.70 (equivalent to a total protein content of 1.5 mg) was pooled. One milliliter of phosphate buffer containing 1.5 mg of

β-galactosidase was immediately mixed with the antibody eluted from the column and maintained at 30°C for 1 h. The reaction was terminated by the addition of 1 M mercaptoethanol to

give a final concentration of 10 mM mercaptoethanol.



Similar differences in reactivity are seen in 1,5-difluoro-2,4-dinitrobenzene, bis(4-fluoro-3-niro)

sulfone, and 2,4-dichloro-6-methoxy-s-triazine probably due to electronic effects after nucleophilic

replacement. For 2,4-dichloro-6-methoxy-s-triazine, coupling occurs with tyrosine residues at

pH 7; however, the second chloro-group will only react with a tyrosine residue of another protein

under alkaline conditions.9 Similarly, under acid conditions, only one of the diazo groups of bisdiazotized o-dianisidine is reactive. For coupling of ferritin to rabbit gamma-globulin, the first step

of the reaction is carried out at pH 5 and the second at pH 9.4.10

Differential reactivity of a homobifunctional reagent toward different proteins has also been used

in two-step reactions. Glutaraldehyde, for example, reacts with γ-immunoglobulins much faster

than with horseradish peroxidase. Reaction of horseradish peroxidase with an excess of glutaraldehyde constitutes the first step of the reaction. After removal of excess reagent, the immunoglobulin

is added to generate the enzyme–immunoglobulin conjugate. Self-coupling of horseradish peroxidase is minimal due to the lack of available reactive groups.11 Ferritin has also been coupled to

γ-immunoglobulins under similar conditions.12

Another example of a two-step procedure is the cross-linking reaction with carbodiimides in

the presence NHS. Carbodiimides react with a carboxyl group to form an active o-acylisourea

intermediate, which is unstable in aqueous solutions and is therefore not useful for a two-step

conjugation procedure. However, the intermediate can be stabilized using NHS, converting the

protein carboxyls into succinimidyl esters that react with amino groups.13 A reaction of such a

procedure in cross-linking troponin C (TnC) and troponin T (TnT) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is described in the text box below. The advantage of this

two-step procedure over a one-step zero-length cross-linking is that only one component of the

complex is exposed to the cross-linker, which reduces the formation of cross-links among several proteins of a multicomponent complex. Furthermore, cross-links can be formed even in the

presence of other reagents, such as dithiothreitol and EDTA, which would interfere with direct

cross-linking with EDC.



300



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation



A TWO-STEP PROCEDURE CROSS-LINKING TNC AND TNT USING EDC13

TnC (1 mg/mL) was incubated for 15 min at 25°C in a solution containing 0.1 M Mes

(2-(N-morpholino)ethanesulfonic acid) buffer, pH 6.0, containing 0.5 M NaCl, 2 mM EDC,

and 5 mM NHS. The reaction was terminated by addition of 2-mercaptoethanol to a final

concentration 20 mM and the solution was further incubated at the same temperature for a few

minutes. The solution was passed through a Sephadex G-25 column (0.6 × 5 cm) equilibrated

with 0.5 M NaCl and 0.1 M Mes to remove low-molecular-weight components by centrifugation. The effluent was combined with TnT in a 1:l molar ratio in 0.5 M NaCl, 0.1 M Mes and

further incubated for 2 h. TnT can be added directly to the quenched solution without gel

filtration and incubated for 2 h. The cross-linked TnC–TnT adduct was analyzed by polyacrylamide gel electrophoresis.



9.2.3  Three-Step Cross-Linking Reactions

These procedures involve an additional step for the preparation of the proteins to be coupled, for

instance, the introduction of a thiol group as discussed in Chapter 2. Figure 9.1 shows an example

of a three-step coupling process for coupling IgG to albumin. In the first step, IgG is labeled with

pyrrole-α-acyl azide. The second protein, albumin, is reacted with the cross-linker, bis-diazotized

p-phenylenediamine, under acidic conditions. After isolation, the pyrrole-modified IgG and the

diazo-albumin are mixed and allowed to react at pH 6. Specific reaction occurs between the diazo

group and the pyrrole ring as shown in Figure 9.1. This method reduces the side reactions by adjusting the coupling conditions. At acidic pHs, the first diazotized group is reactive whereas the second

group is reactive at higher pH.14

Many immunoconjugates and immunotoxins are prepared by this three-step process. These reactions will be further discussed in Chapter 12.



9.2.4  Multistep Cross-Linking Reactions

More sophisticated cross-linking reactions involve multistep procedures. The process usually

involves several protein preparation steps before the final coupling reaction. Various multistep

cross-linking schemes are possible, depending on the chemical reagents and biological systems

used. A simple example is the use of N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) to prepare immunotoxins.15 Both immunoglobulin and toxin are separately reacted with SPDP. One of the

labeled proteins is then reduced to generate a free thiol group and then purified. The two modified



N3



IgG-NH2 +



N

H



IgG



N2+



O



N

H



N



N



H

N

N

H



O



O



Albumin + +N2



H

N



IgG



Albumin



N N



N N



Albumin



FIGURE 9.1  Conjugation of IgG and albumin by a three-step procedure.



N2+



General Approaches for Chemical Cross-Linking



301



proteins are then mixed to allow cross-linking to proceed, which provides an adduct with a cleavable disulfide bond. Using two different reagents, Rector et al.16 described a cross-linking procedure

for conjugating ricin and immunoglobulin as shown in the text box below. An iodoacetyl group was

introduced into the toxin with N-hydroxysuccinimidyl iodoacetate and the immunoglobulin was

thiolated with SPDP. Both modified proteins were isolated and mixed together to form a conjugate

with the structure: IgG-NH-CO-CH2CH2-S-CH-CO-NH-ricin.

Cross-linking effected by masked or disguised cross-linkers proceeds through several steps.

Disguised or masked reagents are compounds that can be easily converted to heterobifunctional

reagents in a simple reaction. These reagents are usually disguised as monofunctional agents. During

the procedure, extra steps are necessary to generate the functional group needed for cross-linking

process. There are relatively few published reports on these cross-linkers. Figure 9.2 shows the reactions of three of these compounds. 3-Amino-4-methoxyphenylvinyl sulfone (Figure 9.2A) has been

used to couple the enzymes catalase, trypsin, chymotrypsin, and ribonuclease to cellulose.17 The first

step involves a Michael addition reaction of a hydroxyl group to the vinyl sulfone. Treatment of the

label with sodium nitrite in acid converts the amino group on the benzene ring to a diazonium compound, which will react with tyrosine residues of the proteins to complete the cross-linking process.

A MULTISTEP CONJUGATION OF RICIN AND IMMUNOGLUBULIN16

Step 1: A solution of the N-hydroxysuccinimidyl iodoacetic acid (0.3 mg) in 200 μL dried

dimethylformamide was added to 1.55 mL solution of ricin (20 mg) in borate buffer. After

stirring for 30 min at room temperature, the reaction mixture was applied to a column

(22 × 1.6 cm) of Sephadex G-25 pre-equilibrated with phosphate buffer to isolate iodoacetylated ricin in about 12 mL of eluate. The number of iodoacetyl groups introduced into each

molecule of ricin was 1.5, as determined spectrophotometrically after reacting the modified

protein with 3-carboxy-4-nitrothiophenol.

Step 2: To a solution of immunoglobulin (43.6 mg) in borate buffer (4.3 mL) was added a solution

of SPDP reagent (0.218 mg) in dry dimethylformamide (70 μL). After stirring at room temperature

for 30 min, the mixture was applied to a column (20 × 1.6 cm) of Sephadex G-25 equilibrated with

acetate buffer. Elution with the same buffer isolated the substituted protein in 11 mL. The eluate

(10 mL) was concentrated to 3 mL in an Amicon ultrafiltration cell with a PMIO membrane.

Step 3: To the concentrate was added dithiothreitol (22 mg) in acetate buffer (0.5 mL). After

stirring for 30 min at room temperature the mixture was applied to a column (20 × 1.6 cm) of

Sephadex G-25 that had been equilibrated in nitrogen-flushed phosphate buffer.

Step 4: The protein solution, removed by elution in the same buffer, was added directly into

the iodoacetylated ricin solution prepared above. The mixture was then concentrated to 5 mL

by ultrafiltration. After stirring for 18 h at room temperature, the mixture was treated with

N-ethylmaleimide (1 mg) dissolved in dimethylformamide (100 μL). An hour later the solution

was applied to a column (82 × 3.2 cm) of Sephadex G-200 equilibrated with borate buffer and

eluted with the same buffer solution to isolate the conjugated product.



Similarly, ethyl N-(carbamoylcyanomethyl)acetimidate (Figure 9.2B), under mild conditions, reacts

with protein amino groups and cyclizes to form an aminoimidazoyl derivative. The resulting imidazole-derivatized amino group can be diazotized and then reacts with the tyrosyl residue of another

protein to form a cross-linked product. By this method, antigens have been coupled to antihuman

group O erythrocyte IgG.18

Another masked reagent, N-(2,2-dimethoxyethyl)-5-(hydrazidecarbonyl)pentanamide (Figure

9.2C), has been used to couple glycopeptides to proteins.19 The reagent is first converted to acylazide



302



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation

O



(A)



O



S

S



+



Cellulose-OH



O

O



O



Cellulose



CH3



O

O



CH3



NH2



NH2

O

S

NaNO2/H+



Cellulose



Protein-Tyr



O

O



CH3



O



N



N

Tyr-Protein



(B)



O



Protein1-NH2



+



O



CH3



O



N



N

H3C



H3C



N



N



Protein1



NH2



N



Protein1-NH



NH2



N



H3C



CH3



CH3CH2OH



H 2N



Protein2-Tyr



O



N



N



NH2



N



N



Protein1



NaNO2/H+



O



NH2



Protein2-Tyr



(C)

H3C



O



H

N



O

O



CH3



H3C



N

H



O



O CH

3

Protein-NH2



O



H

N



O



NH2 N2O4



N

H



O



Protein



N



Protein



O



O

N

H



Glycopeptide



NH2-Glycopeptide



O



H

N



H

O



H

N



N3



O



CH3



Glycopeptide 50% TFA



O

H

N



H3C



O



H

N



O



O



N

H



Glycopeptide



Pyridine

Borane



O



H

N

O



N

H



Glycopeptide



FIGURE 9.2  Cross-linking reactions of disguised reagents. (A) 3-Amino-4-methoxyphenylvinyl ­sulfone;

(B) Ethyl N-(carbamoylcyanomethyl)acetimidate; (C) N-(2,2-dimethoxyethyl)-5-(hydrazide carbonyl)

pentanamide.



on treatment with dinitrogen tetraoxide. After reaction with an amino-containing compound, the

blocked methyl acetal is removed with 50% trifluoroacetic acid to generate an aldehyde group.

Cross-linking is achieved between the aldehyde and a protein amino group by reductive alkylation.

The examples above demonstrated the versatility of the multistep process. Even more complex

procedures involve applications to protein–oligonucleotide and DNA–DNA cross-linking. Ghosh

et al.20 described two methods for the synthesis of oligonucleotide–enzyme conjugates. 5′-Thiolated

­oligonucleotide was synthesized in two steps using cystamine and EDC followed by dithiothreitol.



General Approaches for Chemical Cross-Linking



303



In the first approach, heterobifunctional N-succinimidyl-6-maleimidohexanoate (SMH) was used to

modify an amino group of calf intestine alkaline phosphatase and then coupled to 5′-thiolated oligonucleotide to give a 1:1 conjugate. In the second strategy, homobifunctional N,N′-(1,2-phenylene)

bismaleimide was used to activate the thiol group of the 5′-thiolated oligonucleotide. In the mean time,

horseradish peroxidase or beta-galactosidase was thiolated with 2-iminothiolane and then reacted with

the modified oligonucleotide to give an enzyme–oligonucleotide conjugate. Another complex reaction

is shown by Tona and Haner21 in interstrand cross-linking of DNA. In this case, 1,3-butadiene-containing building blocks were integrated into DNA oligomers such that they are in opposite positions

to each other in the double helix. Cross-linking between the diene-modified duplexes was achieved by

bifunctional dienophiles such as homobifunctional bismaleimide, N,N′-ethylenedimaleimide through

a double Diels–Alder reaction. The reaction resulted in clean cross-linking of the two DNA strands.



9.3  GENERAL CONDITIONS FOR CROSS-LINKING

Optimal cross-linking conditions are highly dependent on the type of reagent used and the particular system under investigation.3,22 For example, reaction times may range from minutes to hours and

the reagent concentration varies with its relative reactivity as well as the stability of each reagent.

Some reagents are readily hydrolyzed and consequently may have to be used in excess. Crosslinking can generally be carried out in buffers such as phosphate-buffered saline (PBS) or isotonic

phosphate. Since reagent type dictates the reaction conditions, the following discussion represents

generalized parameters for the most frequently used reagents only. For specific applications, the

reader is referred to the specific literature.



9.3.1  Choice of Reaction Medium

Conjugations should be carried out in a well-buffered system at a pH that maintains the integrity

of the biological macromolecules and is optimal for the reaction. The ionic strength should be in

the range of 25–100 mM in most cases. For thiol and α-amino groups, modification at pH 7.0–7.5

in phosphate buffers is ideal. More basic ε-amine of lysine requires more alkaline pH, in the range

of 8.0–9.5, where carbonate/bicarbonate or borate buffers are satisfactory. The optimal pH also

depends on the reagents used. Reactions with NHS esters are best carried out in pH 8.2 bicarbonate

buffer, and isothiocyanates at pH 9.0–9.5, provided by carbonate or borate buffers. It should be noted

that for these reactions, the buffer should not contain any free amino groups such as in Tris buffer.

For reagents that are poorly soluble in water or highly reactive with water, a water-miscible

cosolvent must be employed to dissolve the reagent before adding to the conjugation medium. Some

cosolvents are methanol, ethanol, 2-propanol, 2-methoxyethanol, dioxane, dimethylformamide

(DMF), and dimethylsulfoxide (DMSO). The most versatile of these are DMF and DMSO because

they are miscible with water in all proportions, are inert to many reactive reagents, and are compatible with most aqueous protein solutions even at up to 30% v/v ratios. For sulfonyl chlorides, DMF

is the solvent of choice since they react with DMSO. The cosolvent should be carefully dried and

stored over a drying agent to prevent competing hydrolysis of the reactive reagent.



9.3.2  Choice of Reaction Temperature and Time

As a general rule of thumb, conjugation reactions should be done at or below room temperature

since most reactions are rapid. Also, low temperatures tend to increase selectivity of the reaction,

resulting in fewer side reactions. To avoid overreaction, a high temperature should be accompanied

with a short reaction time and vice versa. It should be noted that the higher the temperature, the

faster will be the reaction. A convenient procedure is to add the reagent to a gently stirred buffered

solution of the macromolecules to be cross-linked in an ice-bath and then allow the bath to warm

up to room temperature over a period of about 2 h, although many published procedures specify



304



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation



overnight reaction times. Generally, 1–2 h is sufficient for conjugation reactions to go to completion.

However, depending on the reagent, longer reaction times may be necessary. The more reactive the

reagent, the shorter the recommended reaction time.



9.3.3  Choice of Reactant Concentrations

The degree of intermolecular and intramolecular cross-linking depends on the concentrations of the

macromolecules to be cross-linked. The higher the concentration, the higher will be the degree of

intermolecular cross-linking. For nonassociating proteins, concentrations above 10 μM are strongly

recommended with an optimum in the range of 50–100 μM. For associated macromolecular complexes, a dilute solution will decrease intercomplex cross-linking. The concentration of the crosslinker is also important. The higher the concentration of reagent, the faster will be the reaction and

the more cross-linking will take place. The degree of cross-linking is generally limited by the ratio

of the reagent to the biological substance. The choice of molar ratio depends on the available reactive moieties of the macromolecules, such as reactive amino acid side chains of the proteins. Low to

moderate ratios will decrease the side reactions. If there is no prior knowledge on the reaction, trial

and error may be necessary to get the desired products. The cross-linking reagent should be added

dropwise as slowly as possible to a slowly stirred solution. Stirring can be done with a magnetic stirbar at a slow speed to avoid denaturation of proteins and other biological molecules.



9.4  CROSS-LINKING PROTOCOLS FOR COMMONLY USED REAGENTS

In the study of protein–protein interactions by chemical cross-linking, the selection of a reagent is

critical. For each reagent to achieve a cross-linking goal, different reaction protocols are to be established. The following sections describe some general reaction conditions for the different classes

of cross-linkers and provide examples of protocols for some common reagents in each class. Many

other reaction procedures can be found in Niemeyer’s edited bioconjugation protocols.1 Specific protocols may be found in publications of commercial companies that provide the cross-linking reagent.



9.4.1  Examples for Zero-Length Cross-Linker

Carbodiimides are probably the most commonly used zero-length cross-linkers. Cross-linking is

usually performed at a pH between 4.5 and 5 where the reaction rate is rapid, requiring only a few

minutes for many applications. Carbodiimides are subject to hydrolysis and should be stored desiccated. 4-Morpholine ethanesulfonic acid (MES) is a good carbodiimide reaction buffer. Tris, glycine, acetate, and phosphate buffers can react with the cross-linkers and should be avoided. Below

are protocols for general applications. Other examples are discussed above.

9.4.1.1  Cross-Linking a Peptide and a Protein Using EDC

a. Prepare the protein at 10 mg/mL in 0.1 M MES buffer, pH 5. (The protein should be free of

other buffers such as Tris or interfering substances such as thios, amines, acetate, DTT.)

b.Prepare peptide (10 mg/mL) in the same MES buffer.

c.Prepare EDC at 10 mg/mL in distilled water (should be used immediately).

d.Mix 2 mg peptide and 2 mg protein (ratio may be optimize depending on the desired

­coupled ratio).

e.Add 0.5–1 mg EDC to the mixture (0.05–0.4 mg for each mg of total protein, usually

0.5 mg EDC for 1 mg BSA) with stirring.

f.Incubate for 2–3 h at room temperature.

g.Desalt by dialysis or gel filtration.



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