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6 Conditions for Cleavage of Cross-Linked Complexes

6 Conditions for Cleavage of Cross-Linked Complexes

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Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation

9.6.5  Ester and Thioester Bonds

Theoretically esters and thioesters can be hydrolyzed under both acidic and alkaline conditions.

They are most conveniently cleaved by 1 M hydroxylamine, in 50 mM Tris, pH 7.5–8.5, 25 mM

CaCl2 and 1 mM benzamidine for 3–6 h 25°C–37°C.88

9.6.6  Acetals, Ketals, and Orthoesters

These acetal, ketal, and orthoester bonds are acid labile and base stable.89 Cross-linked products are

stable at basic conditions and can be manipulated at pH 8–9.90 They are approximately 100 times

more stable at pH 7.4 and 1000 times more stable at pH of 8.4. They can be cleaved at pH 5.4 in

minutes to hours, orthoesters being the fastest, acetals the slowest, and ketals intermediate.


9.7.1  General Considerations

Cross-linking of two different proteins with a bifunctional reagent can give rise to intramolecular and intermolecular products. Intermolecularly, a range of products including the desired

1:1-conjugate and the undesired polyconjugates and polymers of each of the reactant proteins

can occur. Other possible products include aggregates of the various newly formed dimers,

oligomers, and polymers. These side reactions may lead to the loss of catalytic or immunologic

activities of the original reactants. Intrachain cross-linking may give rise to a variety of complications, including change of structure, which may lead to different mobility on polyacrylamide

gel electrophoresis. To address some of these problems, cleavable reagents are particularly

helpful. Upon cleavage, the molecule should return to its original molecular state, including

electrophoretic mobility. In most of the applications of cross-linking reagents a parallel experiment using the analogous monofunctional reagent should be carried out. These experiments

may elucidate whether chemical modification itself is the cause of the problem, such as loss of

the biological activity or immunogenicity. If such is the case a different reagent may be chosen.

Alternatively, protection of enzyme-active sites or immunological activities may be carried out

with inhibitors,91 substrates,92 or antigens.93,94

While sulfhydryl cleavable reagents have many advantages, there are also several disadvantages. Major advantages include (1) rapid cleavage of the disulfide bond under mild conditions,

(2) quantitative completion of the cleavage reaction, (3) ability to be cleaved both before and after

electrophoresis, and (4) the specificity of the reduction reaction. The disadvantages of the use of

these reagents are the following: (1) they are susceptible to disulfide exchange with the possibility

of linking noninteracting molecules; (2) their use precludes the application of reducing agents for

the isolation of cross-linked complexes; and (3) they cannot be used in a system that is sensitive to

oxidation and which would normally be kept under reducing conditions. Disulfide exchange usually involves the presence of free sulfhydryl groups that must be present in significant excess over

disulfides.95 This reaction can be decreased by lowering the pH of the reaction below the pKa of

sulfhydryl groups.96,97

For other cleavable reagents, there are also advantages and disadvantages. The major disadvantages with the use of glycol reagents include (1) the reduced rate of cleavage relative to

that achieved with disulfides, (2) the difficulty in achieving complete cleavage, (3) the lack of

specificity of the cleavage reaction, namely, the carbohydrate portions of glycoproteins can also

be disrupted, and (4) the oxidative side reaction of the carbohydrates may lead to potential formation of Schiff bases with the protein amino groups. A cross-linking reagent will have to be

carefully chosen to suit the particular system under investigation to reduce the complications to

a minimum.


General Approaches for Chemical Cross-Linking

9.7.2  Immunogenicity

In the application of cross-linking reagents to prepare antigen-carrier conjugates, the major

concern is the effect of the reagent on the immunogenicity of the antigen. Peeters et al.98 have

systematically investigated the problem with four cross-linking reagents. In a model system

using angiotensin and tetanus toxoid, it was found that cross-linking did not affect the immunogenicity of either the protein or the carrier in inducing antibody production. Antibodies were

also induced against the cross-linking reagent. However, flexible nonaromatic linkers of succinimidyl 6-(N-maleimido)-n-hexanoate and succinimidyl 3-(2-pyridyldithio)propionate showed

the least immunogenicity. It seems reasonable to assume that cross-linking does not affect the

antigenicity or immunogenicity of an antigen and that flexible cross-linkers are the best choices

for this application.

9.7.3  Stability

The stability of immunoconjugates and immunotoxins is of paramount importance in the application of these conjugated proteins. In general, immobilization of proteins tends to increase their

stability toward both mechanical and thermal denaturation.99 The same observation is also reported

for cross-linked proteins.100–103 In fact, higher activity of β-galactosidase was obtained after crosslinking with glutaraldehyde and dimethyladipimidate. Glutaraldehyde cross-linking of enzyme

crystals and polyethylene glycol modification of enzyme surface amino groups also enhance biocatalyst stability.

Some sense of the stability of a conjugate may be obtained from consideration of the Arrhenius

equation (Equation 9.1):

ln k =

A − Ea



When the natural logarithm of the rate constant of inactivation, k, is plotted against the reciprocal

of the absolute temperature, T, the slope of the line gives the value of Ea /R where R is the gas constant. The intercept is then equal to A, the Arrhenius constant. The magnitude of Ea, the activation

energy of the process, provides information about the stability of the conjugate toward denaturation.

The larger this value, the more stable the conjugate, since more energy is required for the inactivation. For horseradish peroxidase, peroxidase–IgG conjugate, and peroxidase–jacalin conjugate

cross-linked with gluteraldehyde, the values of Ea are shown in Table 9.1.104 These values indicate

that conjugated horseradish peroxidase is more stable than the free enzyme.

From the Arrhenius plot, it is also possible to predict the half-life of a conjugate at a certain temperature. The rate constant of inactivation at that temperature is determined by extrapolation of the

plot. The relationship of the half-life to the rate constant is given by the following equation:

T1/ 2 =





Heat of Inactivation of Horseradish

Peroxidase Conjugates


Horseradish peroxidase (free)

Horseradish peroxidase-IgG

Horseradish peroxidase-Jacalin

Ea (kcal/mol)





Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation

For horseradish peroxidase at the conditions where the rate constants are determined, the half-life at

4°C is found to be 21 years, This mechanism may be useful for predicting the stability of an immunoconjugate or immunotoxin at a particular temperature.


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Analysis of Cross-Linked



After cross-linking has been accomplished, the next step is usually purification and characterization

of the product(s) of the reaction. The target product must first be isolated from excess or unreacted

reagent. In many cases, simple dialysis may suffice to remove unreacted reagent from the reaction

solution. Typically, the solution is placed in a dialysis bag made of a semipermeable material usually

based on cellulose. The dialysis tubing has pores that will allow smaller molecules to pass through

while retaining larger species, that is, macromolecules. Some type of chromatography, for example,

size-exclusion chromatography (SEC) (discussed below), may also be used to either remove excess

reagent or isolate and characterize the cross-linked product. The isolated cross-linked protein may

then be further characterized by biochemical or biophysical techniques. In the following sections,

various analytical methodologies and examples of their application to protein cross-linking will

be described. Once the product has been purified, it may be subjected to many different types

of studies including spectroscopic (e.g., fluorescence, NMR, EPR, and Raman), immunochemical, biochemical, and enzymatic, and numerous examples of these type of studies have been given

throughout this book. In this chapter, however, we shall focus on methods to purify and characterize

the cross-linked product.


10.2.1  Size-Exclusion Chromatography

Perhaps the most common analytical method used to separate unused cross-linking reagents from

reacted products as well as to separate different reaction products is SEC, sometimes known as gelfiltration chromatography, when aqueous phases are used or gel-permeation chromatography in the

case of organic solvents. The method, originally developed in the 1950s, allows for the separation of

molecules based on their molecular size. SEC utilizes a stationary phase material, typically a polymer, which is subjected to varying extents of cross-linking to create pores, which allow molecules

below a certain size to enter the polymer matrix while excluding larger molecules (Figure 10.1). The

large excluded molecules elute from the gel matrix in the so-called void volume, before the smaller

size molecules. Stationary phase materials are available with different pore sizes, and, hence, different classes of SEC products should be used depending on the size of the target molecule. One

of the most popular SEC matrix is Sephadex, a trade name for a cross-linked dextran, in bead

form, originally produced by the Pharmacia company (now a part of GE Healthcare). The name

Sephadex is an acronym from Separation Phamacia Dextran. Sephadex resins are listed according

to their separation-size range, for example, Sephadex G-100 is useful for the separation of globular

proteins in the range of approximately 4,000–150,000 Da, whereas G-25 is useful for the range

of 1,000–5,000 Da. The resin is typically packed inside a column, and the solution containing the

cross-linked material is loaded onto the column, which may be run using gravity or via a pump

system such as fast protein liquid chromatography (FPLC) or high-performance liquid chromatography (HPLC). Each column must be calibrated with molecular weight standards covering the




Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation


FIGURE 10.1  (See color insert.) Depiction of a SEC experiment. As molecules flow down the column,

the larger ones move fastest since they cannot enter the resin matrix.

Log molecular weight

range of interest; for  example, a typical mix of molecular weight protein standards may include

thyroglobulin (67 kDa), IgG (156 kDa), BSA (66 kDa), ovalbumin (43 kDa), peroxidase (40 kDa),

myoglobin (17 kDa), and cytochrome c (12.4 kDa). In the case of HPLC columns, resins that can

mechanically withstand the elevated pressures are required. Such resins are often composed of

silica-based polymers, for example, the TSK-GEL SW-type packings, from Tosoh Bioscience LLC,

comprising rigid spherical silica gel particles chemically derivatized with diol-containing ligands.

This particular product line includes three pore sizes: 125 Å pore size for the analysis of small

proteins and peptides, 250 Å pore size for most protein samples, and 450 Å pore size for very large

proteins and nucleic acids. An example of a standardization curve is depicted in Figure 10.2.

SEC separation depends approximately upon differences in the hydrodynamic volumes of the

molecules, which in turn are related to the molecular weights as well as the molecular shapes. We

note that SEC cannot only be used to separate unreacted reagent from target molecules but may cannot also be used to separate a limited range of intermolecularly linked target molecules. For example,

if the cross-linking results in dimers, trimers, et cetera of a target protein the proper gel filtration





Retention time (min)



FIGURE 10.2  A generalized plot of the log of the molecular weight versus retention time for a SEC experiment such as that depicted in Figure 10.1.

Analysis of Cross-Linked Products


resin may be able to separate some or all of these products, which may then be subjected to more

detailed characterization. Numerous examples of the use of SEC are given throughout this book.

10.2.2  Electrophoresis

Electrophoresis refers to the movement of particles in an electric field. The most common electrophoresis technique used in the characterization of cross-linked molecules is gel electrophoresis in which the stationary phase is a cross-linked gel polymer, typically based on acrylamide,

which has different effective pore sizes achieved via cross-linking of the acrylamide into a

three-dimensional polyacrylamide matrix. Agarose is also sometimes used as a gel matrix.

The pore size within a gel matrix depends upon the concentration of both the acrylamide solution and the cross-linking solution. A common cross-linker is N,N′-methylenebisacrylamide

(Bis). The gel polymerization process is usually initiated by the addition of N,N,N′,N′tetramethylethylenediamine (TEMED) and ammonium persulfate. Pore sizes decrease with

increasing percentages of cross-linker. Nowadays, precast gels can be purchased with pore sizes

to match the particular application. For sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) (see below), a standard percentage of cross-linker to monomer is 37.5:1

(2.6%) while for denaturing DNA/RNA, electrophoresis ratios of 19:1 (5%) are common while

native DNA/RNA gels are typically 29:1 (3.3%). Most often, the gel matrix is in the shape of a

relatively thin rectangular sheet. Typically, two chambers are utilized, each containing different buffer solutions, namely, the anode buffer and the cathode buffer, with the cathode buffer

covering the gel in the upper negative electrode chamber, and the anode buffer covering the gel

in the lower positive electrode chamber. An electric field is applied across two buffer solutions.

The sample is loaded onto one end of the gel, typically the cathode end, and the molecules move

within the gel matrix toward the anode at a rate, which depends upon their charge-to-mass ratio.

After the electrophoresis is run for sufficient time to allow separation of the target materials

[often judged by running the gel until the fast moving dye bromophenol blue (3′,3″,5′,5″-tetrabromophenolsulfonphthalein) is near the bottom of the gel], the bands corresponding to the

different cross-linked products can be visualized by staining the gel with an appropriate dye.

For proteins, common dyes are Coomassie Blue (note there are different forms such as R-250

and G-250) or the more sensitive Silver stain. Ethidium bromide or the newer SYBR® dye (this

so-called safe dye is less carcinogenic than ethidium bromide but also much more expensive) is

often used to visualize DNA. SDS–PAGE is commonly used to separate proteins based on their

molecular weight. In this method, the protein solution is first mixed with SDS and heated to at

least 60°C. SDS is an anionic detergent, which binds to the proteins and helps denature them

while conferring a negative charge to each protein in proportion to the number of SDS molecules bound, which in turn is proportional to the size or molecular mass of the protein. When

the gel is run, a mixture of molecular weight standards is often used to determine the approximate position of different size products in the gel matrix. If cross-linking leads to oligomeric

products such a dimers, trimers, and tetramers., then the gel lane containing the cross-linked

material should show a “ladder” of proteins (Figure 10.3). One notes that the lower molecular

weight species run farther into the gel than the larger molecules. The amount of protein in

each band can be quantified using a gel scanner or densitometer. In 1970, Ulrich K. Laemmli

published a paper1 refining the SDS–PAGE method, and this paper has become one of the most

cited papers in science.

10.2.3  Light Scattering

Light scattering may also be used in connection with column chromatography (e.g., SEC) to

determine the extent of intermolecular cross-linking in a protein mixture. For example, Figure

10.4 depicts the light scattering trace and one might observe from a solution of cross-linked BSA


Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation

250 kDa

Direction of protein


150 kDa

110 kDa

50 kDa

25 kDa

10 kDa




cross-linking cross-linking

FIGURE 10.3  An idealized SDS–PAGE experiment showing molecular weight standards (left lane), a

30 kDa protein before cross-linking (middle lane), and the product after cross-linking (right lane).



Abs 220 nm






FIGURE 10.4  (See color insert.) Depiction of a SEC experiment on a commercial preparation of BSA that

has developed cross-linked products. The output from the column was monitored by absorbance at 220 nm as

well as by light scattering.

molecules eluting from a SEC column (adapted from Varian Application Note SI-02008). We note

that commercially available lyophilized preparations of albumins typically have 10%–15% of crosslinked proteins due to rearrangement of disulfide bonds, and, for some purposes, the monomeric

species may have to be purified from higher oligomers. Also shown in Figure 10.4 is the absorbance trace one might observe at 220 nm. The amount of protein in each band is approximated

by the 220 nm absorbance trace while the light scattering trace emphasizes the larger oligomers

disproportionately to their concentrations. This disproportionate weighting is due to the fact that

the scattered light signal is proportional to the square of the molecular weight of the particle—at

least in the range of Rayleigh scatter. Light scattering is preferably used in conjunction with a SEC

chromatography system, that is, in direct connection with the column output, since flow through the

column will eliminate particles, such as dust or bubbles, which can result in significant scattering.

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6 Conditions for Cleavage of Cross-Linked Complexes

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