Hermanson G. Bioconjugate Techniques, Second Edition

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250 4. Homobifunctional Crosslinkers

to activate poly(ethylene glycol) for conjugation with proteins and other molecules (Chapter 25,

Section 1.2). In this sense, DSC-activated hydroxylic compounds can be used to conjugate with

an amine-containing molecule to form a stable derivative ( Figure 4.11 ). The linkage created from

this reaction is a urethane derivative or a carbamate bond, displaying excellent stability.

Activation of hydroxyl groups with DSC can be done in acetone or dioxane by reacting for

4–6 hours at room temperature. Subsequent conjugation with amine-containing molecules is

done in organic or aqueous solutions. For buffered reactions, the optimal conditions include

a pH range of 7–9 using common buffer salts (avoid amine-containing components, such as

Tris). React for at least 4 hours at room temperature or up to overnight at 4 °C.

DSC also is used to activate hydroxylic particles for coupling to amine-containing ligands

(Miron and Wilchek, 1993). For methods involving particle conjugation using this homobi-

functional compound, see Chapter 14.

2. Homobifunctional Imidoesters

Crosslinking compounds-containing imidoesters at both ends are among the oldest homo-

bifunctional reagents used for protein conjugation (Hartman and Wold, 1966). The imidoester

(or imidate) functionality is one of the most speciﬁ c acylating groups available for the modiﬁ -

cation of primary amines, with minimal cross-reactivity toward other nucleophilic groups in

proteins. The -amines and -amines of proteins may be targeted and crosslinked by reacting

with homobifunctional imidoesters at a pH of 7–10 (optimal pH 8–9). The product of this

reaction, an imidoamide (or amidine) ( Figure 4.12 ), is protonated and thus carries a positive

Figure 4.11 DSC can react with hydroxyl groups to create a succinimidyl carbonate intermediate that is highly

reactive toward nucleophiles. In the presence of an amine-containing molecule, the active species can form sta-

ble carbamate linkages.

charge at physiological pH (Kiehm and Ji, 1977; Liu et al., 1977; Ji, 1979; Wilbur, 1992). The

result is no alteration of the charge characteristics of the crosslinked proteins, since the amines

being modiﬁ ed were themselves protonated and originally contributed to the overall positive

charge of the molecule. Imidoesters therefore can preserve the microenvironment within the

vicinity of the crosslink bridge, possibly retaining native structure and activity better than rea-

gents that modulate the net charge of a protein.

The amidine bond is quite stable at acid pH; however, it is susceptible to hydrolysis and

cleavage at alkaline pH. Derivatized proteins may be assayed by amino acid analysis after acid

hydrolysis without loss of imidate modiﬁ cations.

Imidoester crosslinkers are highly water-soluble, but undergo continuous degradation due to

hydrolysis. The half-life of the imidate functionality is typically less than 30 minutes, especially

in the alkaline conditions of the reaction medium (Hunter and Ludwig, 1962; Browne and

Kent, 1975). Concentrated stock solutions may be prepared before addition of a small amount

to a conjugation reaction, but they should be dissolved rapidly and used immediately.

The following list of homobifunctional imidoesters represent compounds that are commonly

used for protein crosslinking and are currently available from commercial sources.

2.1. DMA

Dimethyl adipimidate (DMA) is a short-chain, homobifunctional crosslinking agent-containing

imidoesters at both ends (Thermo Fisher). After reaction with amine groups on target mol-

ecules, the compound creates a non-cleavable, 6-atom bridge with terminal amidine bonds

(Figure 4.13 ). DMA is water-soluble and may be added directly to a crosslinking reaction or

pre-dissolved at higher concentration before addition of an aliquot to the reaction medium.

Stock solutions should be used immediately to prevent breakdown by hydrolysis. Reaction

buffers having a pH of 8–9 are optimal. Avoid buffers-containing primary amines (glycine and

Figure 4.12 Imidoesters react with amine groups to form amidine bonds, which are positively charged at physi-

ological pH.

2. Homobifunctional Imidoesters 251

252 4. Homobifunctional Crosslinkers

Tris), since these will cross-react with the imidoester groups. Borate or bicarbonate buff-

ers adjusted to the optimum pH range work well. The addition of (or the exclusive

use of) 0.1–0.2 M triethanolamine is often done to help catalyze the coupling reaction.

Reported applications of DMA include the crosslinking of bovine pancreatic ribonuclease A

(Hartman and Wold, 1967), treatment of erythrocyte membranes to reduce the effects of sickle

cell anemia (Waterman et al., 1975), conjugation and analysis of the outer membrane proteins of

Neisseria gonorrhoeae (Newhall et al., 1980), protein structural studies of bovine  -crystalline

(Siezen et al., 1980), crosslinking of hemoglobin S (Pennathur-Das et al., 1982), and form-

ing S-carbomethoxyvaleramidine during hydrolysis of DMA (Mentzer et al., 1982). The com-

pound also has been used to study uranyl–antibody interactions by atomic force microscopy

(Odorico et al., 2007), to produce antibody–drug conjugates for the treatment of non-Hodgkin

lymphoma (Polson et al., 2007), and to investigate the subcellular distribution of the type II

vasopressin receptor in kidney (Fenton et al ., 2007).

2.2. DMP

Dimethyl pimelimidate (DMP) is a homobifunctional crosslinking agent that has imidoester

groups on either end (Thermo Fisher). The imidoesters are amine reactive to give stable amidine

linkages with target molecules. The 7-atom bridge created by DMP crosslinks is non-cleavable and

Figure 4.13 DMA can crosslink two amine-containing molecules to form charged amidine linkages.

positively charged at physiological pH due to the protonated amidine bonds ( Figure 4.14 ). The

reagent is water-soluble and may be reacted with proteins or other amine-containing macro-

molecules at a pH of 8–9 in aqueous media. Use buffers that contain no amine groups that

may cross-react with the imidoesters. Avoid glycine or Tris buffers.

In protein crosslinking studies, DMP has been used to examine the subunit structure of mus-

cle pyruvate kinase (Davies and Kaplan, 1972), for the crosslinking of lactose synthetase (Brew

et al., 1975), and to conjugate a ﬂ uorescent derivative of -lactalbumin to glactosyltransferase

(O ’ Keefe et al., 1980). The reagent also has found use in the immobilization of antibody mol-

ecules to insoluble supports containing bound protein A (Schneider et al., 1982). The antibody

molecules are ﬁ rst allowed to interact with the coupled protein A, orienting them with their

antigen-binding sites facing away from the matrix. DMP is then added to covalently anchor

the antibodies to the protein A, forming a permanent immunoafﬁ nity matrix.

DMP also has been used to study the interaction between the endoplasmic reticulum and

microtubules (Ogawa-Goto et al., 2007), the conformational changes in the outer arm dynein

of Chlamydomonas in response to calcium (Sakato et al., 2007), and the secretion of the

adipocyte-speciﬁ c secretory protein adiponectin (Wang et al ., 2007).

2.3. DMS

Dimethyl suberimidate, DMS, is a homobifunctional crosslinking agent-containing amine-

reactive imidoester groups on both ends. The compound is reactive toward the -amine groups

Figure 4.14 DMP reacts with amine-containing compounds to form amidine bonds.

2. Homobifunctional Imidoesters 253

254 4. Homobifunctional Crosslinkers

of lysine residues and N-terminal -amines in the pH range of 7–10 (pH 8–9 is optimal). The

resulting amidine linkages are positively charged at physiological pH, thus maintaining the

positive charge contribution of the original amine. DMS creates 8-atom bridges between conju-

gated molecules that are not cleavable ( Figure 4.15 ).

DMS reportedly has been used as a tissue ﬁ xative for light and electron microscopy (Hassell

and Hand, 1974), for the study of the subunit structure of oligomeric proteins (Davies and

Stark, 1970), in the investigation of ATPase activity (Adolfson and Moudrianokis, 1976),

crosslinking ribonuclease A (Wang et al., 1976), binding studies of nerve growth factor to

its receptor (Pulliam et al., 1975), in the study of red cell shape (Mentzer and Lubin, 1979),

crosslinking of glycogen phosphorylase b (Hajdu et al., 1979), crosslinking of apo low den-

sity lipoproteins (Ikai and Yanagita, 1980), studying the mechanism of binding of multivalent

immune complexes to Fc receptors (Dower et al., 1981), investigating the quaternary struc-

ture of the pyruvate dehydrogenase multi-enzyme complex of Bacillus stearothermophilus

(Packman and Perham, 1982), crosslinking studies of the protein topography of rat liver micro-

somes (Baskin and Yang, 1982), afﬁ nity crosslinking studies of the protein topography of rat

liver microsomes (Pfeuffer et al., 1985), and the quantitative chemical crosslinking of CAD

protein (Lee et al ., 1985).

DMS also has been used to study the interaction between the Helicobacter pylori accessory

proteins HypA and UreE (Benoit et al., 2007) as well as to identify the interaction between the

2

-binding protein S100A11 and the Ca

2

- and phospholipid-binding protein annexin A6

(Chang et al ., 2007).

2.4. DTBP

Dimethyl 3,3-dithiobispropionimidate (DTBP) is a homobifunctional, reversible crosslinking

agent-containing imidoester groups on both ends (Thermo Fisher). The compound, commonly

called Wang and Richards ’ reagent, is water-soluble, and reacts with amines in the pH range

of 7–10 (optimum 8–9) to produce amidine linkages (Wang and Richards, 1974). Conjugated

Figure 4.15 The reaction of DMS with amine-containing molecules yields amidine linkages.

Figure 4.16 DTBP reacts with amine-containing molecules to form charged amidine bonds. The internal

disulﬁ de group can be cleaved with DTT to release the conjugate.

molecules subsequently may be cleaved by reduction of the internal disulﬁ de group of the 8-

atom bridge ( Figure 4.16 ). Crosslinked molecules may be analyzed by one- or two-dimensional

electrophoresis, making use of the easy reversibility of the disulﬁ de bond.

Reported applications include studying protein–protein interactions with paramyxoviruses

(Markwell and Fox, 1980), inhibiting adenylate cyclase activity (Young, 1979), studying red

cell shape (Mentzer and Lubin, 1979), crosslinking phytochrome to its putative receptor (Yu

and Schweinberger, 1979), investigating the subunit structure of Na, K-ATPase (dePont, 1979),

Newcastle disease virus proteins (Nagai et al., 1978), thylakoid membrane proteins (Novak-

Hofer and Siegenthaler, 1978), glutamate dehydrogenase–amino transferase complexes (Fahien

et al., 1978), vesicular stomatitis virus proteins (Mudd and Swanson, 1978), heamaggluti-

nin of inﬂ uenza virus (Wiley et al., 1977), pig heart lactate dehydrogenase crystals (Bayne and

Ottesen, 1977), beef heart mitochondrial coupling factor 1 (Baird and Hammes, 1977), chloro-

plast coupling factor 1 (Baird and Hammes, 1976), rabbit muscle skeletal sarcoplasmic reticulum

2. Homobifunctional Imidoesters 255

256 4. Homobifunctional Crosslinkers

protein (Louis et al., 1977), human hemoglobin and erythrocyte membrane proteins (Wang and

Richards, 1974, 1975; Miyakawa et al., 1978), subunit interface of the E. coli ribosome (Cover

et al., 1981), rat liver 60S ribosomal subunits (Uchiumi et al., 1980), proteins in avian sarcoma

and leukemia viruses (Pepinsky et al., 1980), outer membrane proteins of Neisseria gonorrhoeae

(Newhall et al., 1980), monooxygenase enzymes (Baskin and Yang, 1980b), cytochrome P-450

and reduced NAD phosphate-cytochrome P-450 reductase (Baskin and Yang, 1980b), crosslink-

ing initiation factor IF2 to proteins in 70S ribosomes of E. coli (Heinmark et al., 1976), study-

ing sheep red blood cell membranes (Brandon, 1980), conjugation of F-actin to skeletal muscle

myosin subfragment-1 (Labbe et al., 1982), studying decreased staining of proteins after electro-

phoresis (Leffak, 1983), and identifying molecular association of IA antigens after T- and B-cell

interaction (Shivdasani and Thomas, 1988).

DTBP also has been used to investigate the dimerization and actin bundling properties of vil-

lin (George et al., 2007), the interaction of the Mre11 complex with RPA (Olson et al., 2007),

the study of gamma-secretase complex assembly (Spasic et al., 2007), and the multi-protein

assembly of Kv4.2, KChIP3 and DPP10 (Jerng et al ., 2005).

3. Homobifunctional Sulfhydryl-Reactive Crosslinkers

Crosslinking agents that contain homobifunctional sulfhydryl-reactive groups at either end fall

into two general categories: those that form permanent bonds with available sulfhydryls and

those that create reversible linkages. Reactive groups yielding permanent links with sulfhydryls

usually form thioether bonds that are quite stable. Those that result in disulﬁ de bonds can be

cleaved with the use of a disulﬁ de reducing agent like DTT (Chapter 1, Section 4.1). Mercurial-

based coupling groups also can be reversed with reducing agents.

Many varieties of homobifunctional, sulfhydryl-reactive crosslinkers have been synthesized and

described in the literature. Some have been based on bis-mercurial salts (Edelhoch et al., 1953;

Edsall et al., 1954; Kay and Edsall, 1956; Singer et al., 1960; Mandy et al., 1961). Such mer-

curial-reactive groups also have been used in reversible covalent chromatography applications to

purify thiol-containing proteins (Cuatrecasas, 1970, 1972; Ruiz-Carrillo and Allfrey, 1973). Other

homobifunctional sulfhydryl-reactive reagents have been based on forming a mixed disulﬁ de active

group with TNB (5-thio-2-nitrobenzoic acid). Reaction of the TNB active group with a sulfhydryl-

containing macromolecule results in a reversible disulﬁ de linkage (Gaffney et al., 1983; Willingham

and Gaffney, 1983). Active groups consisting of bis-thiosulfonates also have been used to create––

SH-reactive crosslinkers (Bloxham and Sharma, 1979; Bloxham and Cooper, 1982). The thiosul-

fonate groups react with available sulfhydryls to form disulﬁ de linkages, in this case with loss of

the sulfonate groups. All of these disulﬁ de crosslinks are cleavable with disulﬁ de reducing agents.

A number of bis-alkyl halide-reactive groups have been used to create homobifunctional

sulfhydryl-reactive crosslinkers (Ozawa, 1967; Husain and Lowe, 1968; Wilchek and Givol,

1977). These react with sulfhydryls to create stable, nonreversible thioether bonds. Similar

thioether bond formation has been realized using various bis-maleimide derivatives (Moore

and Ward, 1956; Kovacic and Hem, 1959; Tawney et al., 1961; Fasold et al., 1963; Simon

and Konigsberg, 1966; Zahn and Lumper, 1968; Freedberg and Hardman, 1971; Chang and

Flaks, 1972; Wells et al., 1980; Heilmann and Holzner, 1981; Sato and Nakao, 1981; Moroney

et al., 1982; Partis et al., 1983; Chantler and Bower, 1988; Srinivasachar and Neville, 1989).

Sulfhydryls add to the double bond of the maleimide to create a thioether linkage.

The differences within these families of reagents generally relate to the length of the spacer

or bridging portion of the molecule. Occasionally, the bridging portion itself is designed to be

cleavable by one of a number of methods (Chapter 8). The great majority of homobifunctional,

sulfhydryl-reactive crosslinkers mentioned in the literature are not readily available from com-

mercial sources and would have to be synthesized to make use of them. The ones listed in this

section are obtainable from Thermo Fisher.

3.1. DPDPB

DPDPB, or 1,4-di-[3-(2-pyridyldithio)propionamido]butane, is a homobifunctional crosslink-

ing agent that contains sulfhydryl-reactive dithiopyridyl groups on both ends. These coupling

groups are identical to the sulfhydryl-reactive end of the popular heterobifunctional crosslinking

agent, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (Chapter 5, Section 1.1). Available

thiols on proteins and other molecules can react with the pyridyl disulﬁ de groups to form

disulﬁ de linkages with release of pyridine-2-thione ( Figure 4.17 ). Conjugation of two macromol-

ecules with DPDPB results in a 14-atom spacer of approximately 16 Å in length. Release of two

molecules of pyridine-2-thione during the crosslinking reaction may be followed by their char-

acteristic absorbance at 343 nm (   8.08  0.3  10

1

) (Stuchbury et al., 1975).

DPDPB is insoluble in aqueous solutions and should be initially dissolved in an organic solvent

prior to addition of a small aliquot to a buffered reaction medium. Preparation of a stock solution

in DMSO at a concentration of 25 mM DPDPB works well. The addition of an aliquot of this stock

solution to the conjugation reaction should not result in more than about 10 percent organic sol-

vent by volume in the buffered mixture or protein precipitation may occur.

DPDPB has two absorbance maxima, one peak at 237 nm (   1.2  104 M

1

)

and another at 287 nm (   8.8  10

1

) (Traut et al., 1989; Zecherle, 1990). Reduction

of the pyridyldithio groups causes a shift in the absorbance characteristics of the molecule, such

that the peak at 237 nm is shifted to 272 nm and the peak at 287 nm is shifted to 343 nm. This

absorbance shift correlates to the release of the pyridine-2-thione groups.

DPDPB has been used to study the endocytosis of cadherin from intracellular junctions

(Troyanovsky et al., 2006), the subunit arrangement in the ﬂ agellar rotor assembly (Lowder

et al., 2005), and the disease-associated mutations in myelin proteolipid protein in the endo-

plasmic reticulum (ER) (Swanton et al., 2005). DPDPB can be used to conjugate reduced anti-

body molecules to  -

D-galactosidase using essentially the same protocol as that described by

O ’ Sullivan et al . (1979).

3. Homobifunctional Sulfhydryl-Reactive Crosslinkers 257

258 4. Homobifunctional Crosslinkers

3.2. BMH

Homobifunctional crosslinking compounds-containing maleimide groups on both ends have

been used for quite some time (Moore and Ward, 1956; Kovacic and Hem, 1959; Tawney

et al., 1961; Fasold et al., 1963; Simon and Konigsberg, 1966; Zahn and Lumper, 1968;

Freedberg and Hardman, 1971; Chang and Flaks, 1972; Wells et al., 1980; Heilmann and

Holzner, 1981; Sato and Nakao, 1981; Moroney et al., 1982; Partis et al., 1983; Chantler and

Bower, 1988; Srinivasachar and Neville, 1989).

Bis-maleimidohexane (BMH) is a homobifunctional reagent containing a non-cleavable,

6-atom spacer between terminal maleimides (Thermo Fisher). The maleimide groups can react

Figure 4.17 DPDPB is a sulfhydryl-reactive crosslinker that forms disulﬁ de bonds with thiol-containing mol-

ecules. The conjugates may be disrupted using a disulﬁ de reducing agent such as DTT.

Figure 4.18 BMH contains two maleimide groups speciﬁ c for crosslinking sulfhydryl-containing molecules.

The thioether bonds that are formed are stable.

with sulfhydryls to form stable thioether linkages ( Figure 4.18 ). Crosslinks formed with this

reagent create a 16.1-Å bridge between conjugated macromolecules. The reaction takes place

optimally from pH 6.5–7.5. Within this pH range, the reaction is very speciﬁ c for sulfhydryls.

At higher pH values, cross-reactivity with amino groups may occur (see Chapter 2, Section 2.2).

4 . D i ﬂ uorobenzene Derivatives

Diﬂ uorobenzene derivatives are small homobifunctional crosslinkers that react with amine

groups. Conjugation using these compounds results in bridges of only about 3 Å in length,

potentially providing information concerning very close interactions between macromolecules.

4.1. DFDNB

DFDNB is the acronym for an aryl halide-containing compound having the structural names,

1,5-diﬂ uoro-2,4-dinitrobenzene or 1,3-diﬂ uoro-4,6-dinitrobenzene (Thermo Fisher). The reagent

4. Diﬂ uorobenzene Derivatives 259