Hermanson G. Bioconjugate Techniques, Second Edition
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240 4. Homobifunctional Crosslinkers
chemotaxis in E. coli (Chelsky and Dahlquist, 1980), molecular identifi cation of receptors for
vasoactive intestinal peptide in rat intestinal epithelium (Laburthe et al., 1984), in studying the
crosslinking of the affi nity purifi ed CCAAT transcription, -CP1 (Kim and Sheffrey, 1990), and
to fi x tissue for immunostaining procedures (Xiang et al., 2004). The dithiol bridge in DSP also
has been used to activate metal surfaces or metal particles through dative bonding (Grubor
et al., 2004). The presence of the disulfi de group creates two dative bonds in each linker, which
signifi cantly increases the stability of the surface bonds. DSPs two NHS ester groups then can be
used to couple amine-containing molecules to the metallic surface, see Chapter 2, Section 2.8 for
additional information.
The sulfo-NHS version of DSP, dithio bis(sulfosuccinimidyl propionate) or DTSSP, is a
water-soluble analog of Lomant ’s reagent that can be added directly to aqueous reactions with-
out prior organic solvent dissolution (Staros, 1982). DTSSP still contains the disulfi de center
portion that is cleavable with the proper reducing agents, and the sulfo-NHS ends have virtu-
ally the same reactivity as DSP ( Figure 4.5 ). Due to its hydrophilicity, however, DTSSP will
not penetrate cellular membranes as does its more hydrophobic analog, DSP. It is therefore an
excellent choice for the crosslinking of cell surface components without affecting intracellular
substances.
DTSSP reportedly has been used to crosslink the extracytoplasmic domain of the anion
exchange channel in human erythrocytes (Staros and Kakkad, 1983), for the characterization
Figure 4.4 The reaction of DSP with amine-containing molecules yields amide bond crosslinks. The conjugates
may be cleaved by reduction of the disulfi de bond in the cross-bridge with DTT.
of a ribosomal complex in B. subtilis (Caufi eld et al., 1984), to investigate ascites hepatoma
cytokeratin fi laments (Knoller et al., 1991), for the study of the B-lymphocyte Fc receptor for
IgE (Waugh et al ., 1989), and to crosslink platelet glycoproteins (Jung and Moroi, 1983).
1.2. DSS and BS
3
Disuccinimidyl suberate (DSS) is an amine reactive, homobifunctional, NHS ester, crosslinking
reagent that produces an 8-atom bridge (11.4 Å) between conjugated molecules ( Figure 4.6 )
(Thermo Fisher). Its hydrocarbon chain is non-cleavable, so crosslinks formed are irreversible.
Many of the reported applications of DSS involve investigations of receptor–ligand binding
on cell surfaces using radiolabeled molecules. The crosslinker is hydrophobic and must be sol-
ubilized in organic solvent prior to addition to a conjugation reaction. Pre-dissolving in dry
dioxane, DMF, or DMSO may be done at higher concentration and then an aliquot added to
the aqueous reaction medium as needed. The fi nal concentration of the organic solvent in the
buffered reaction should not exceed 10 percent to avoid precipitation of biomolecules. Stock
solutions should be prepared fresh. DSS is membrane permeable and is therefore useful for
intracellular and intramembrane conjugations. The optimum conditions for the crosslinking
Figure 4.5 DTSSP can form crosslinks between two amine-containing molecules through amide linkages. The
conjugates may be cleaved by disulfi de reduction using DTT.
1. Homobifunctional NHS Esters 241
242 4. Homobifunctional Crosslinkers
reaction are a pH range of 7–9 using buffers and other salts which contain no amines. Avoid the
use of Tris or glycine. A phosphate buffer (PBS) at physiological pH works well, see Section 1
(this chapter) for additional information on NHS ester reactions.
Reported applications of DSS include crosslinking the A and B subunits of ricin (Montesano
et al., 1982), studying human somatotropin and the components of the lactogenic-binding sites
of rat liver (Caamano et al., 1983), crosslinking CSF-1 to its cell-surface receptor (Morgan
Figure 4.6 DSS reacts with two amine-containing molecules to form amide bond crosslinks. The cross-bridge is
non-cleavable.
and Stanley, 1984), studying angiotensin II interactions with its receptor (Petruzzelli et al .,
1985), crosslinking of vasoactive intestinal peptide to its receptor on human lymphoblasts
(Wood and O ’Dorisio, 1985), investigating insulin-dependent protein kinases (Petruzzelli et al .,
1985), identifying a cellular receptor for tumor necrosis factor (TNF) (Kull et al., 1985), affi n-
ity crosslinking of atrial natriuretic factor in aorta membranes (Vandelen et al., 1985), study-
ing the receptor for human interferon (Rashidbaigi et al., 1986), crosslinking of endorphin to
membranes rich in opioid receptors (Helmeste et al., 1986), immunoprecipitation studies of the
crosslinked complex of parathyroid hormone with its receptor (Wright et al., 1987), binding of
human interferon Y to its receptor (Novick et al., 1987), identifying the erythropoietin receptor
on Friend virus-infected erythroid cells (Sawyer et al., 1987), and binding of the p75 peptide to
an interleukin 2 receptor (Tsudo et al ., 1987).
Bis(sulfosuccinimidyl) subsrate (BS
3
) is an analog of DSS that contains sulfo-NHS esters
on both carboxylates. The affect of the negative charges provided by the sulfonate groups
lends water solubility to the compound. Prior organic solvent dissolution (before addition to
a reaction) is not necessary. The hydrophilicity of BS
3
also makes it membrane impermeable.
Therefore, cell labeling with BS
3
results in hydrophilic-region modifi cation and crosslinking,
targeting surface functionalities, whereas DSS is capable of targeting hydrophobic regions
within the membrane structure itself. As with DSS, BS
3
is non-cleavable, and thus all crosslinks
formed are irreversible. The reactivity of the sulfo-NHS esters is identical to NHS esters, being
highly reactive toward amines in the pH range of 7–9.
Reported applications of BS
3
include crosslinking of the -endorphin–calmodulin interac-
tion (Staros, 1982), crosslinking of the extracellular domain of intact human erythrocytes ’
anion exchange channel (Staros and Kakkad, 1983), crosslinking of hepatoma cytokeratin
fi laments (Ward et al., 1985), investigating the -lymphocyte Fc receptor for IgE (Lee and
Conrad, 1985; Staros et al., 1987), crosslinking of the tri-peptide Arg–gly–asp to an adhesion
receptor on platelets (Souza et al., 1988), crosslinking of the large and small subunits of cyto-
chrome b559 (Knoller et al., 1991), and for general receptor–ligand crosslinking (Waugh
et al., 1989). See also Dihazi and Sinz (2003), Koller et al. (2004), and Law et al. (2002)
for additional applications of BS
3
in proteomic applications. Also see Ishmael et al. (2005)
and Longshaw et al. (2004) for additional applications involving DSS in studying protein
interactions.
1.3. DST and Sulfo-DST
Disuccinimidyl tartarate (DST) is a homobifunctional NHS ester crosslinking reagent that
contains a central diol that is susceptible to cleavage with sodium periodate (Thermo Fisher).
DST forms amide linkages with -amines and -amines of proteins or other amine-containing
molecules ( Figure 4.7 ). The reagent is fairly insoluble in aqueous buffers, but it may be pre-
dissolved in THF, DMF, or DMSO prior to addition of an aliquot to a reaction. Optimal con-
ditions for reactivity include a pH range of 7–9 with no extraneous amines present that may
cross-react with the NHS esters. Avoid Tris, glycine, or imidazole buffers. Subsequent to conju-
gating proteins with DST, the crosslinks may be broken for analysis by treatment with 0.015 M
sodium periodate.
1. Homobifunctional NHS Esters 243
244 4. Homobifunctional Crosslinkers
Reported applications of DST include the crosslinking of ubiquinone cytochrome C reduc-
tase (Smith et al., 1978), characterization of the cell surface receptor for colony-stimulating
factor (Park et al., 1986), investigation of the Ca
2
-, Mg
2
-activated ATP of E. coli (Bragg and
Hou, 1980), and characterization of human properdin polymers (Farries and Atkinson, 1989).
Disulfosuccinimidyl tartarate (sulfo-DST) is an analog of DST that contains sulfo-NHS
esters. The negatively charged sulfonate groups contribute enough hydrophilicity to provide
water solubility for the reagent without the need for organic solvent dissolution before adding
it to a crosslinking reaction. The conditions for conjugation are otherwise identical to DST.
DST and sulfo-DST also have been used to study protein–lipid complexes (Predescu et al .,
2001), to investigate the binding protein of corticotropin-releasing factor (Jahn et al., 2002),
and the acidic C-terminal domain of rna1p (Haberland et al ., 1997).
1.4. BSOCOES and Sulfo-BSOCOES
BSOCOES [ bis[2-(succinimidyloxycarbonyloxy)ethyl]sulfone] is a water-insoluble, homo-
bifunctional NHS ester crosslinking reagent that contains a central sulfone group, which is
cleavable under alkaline conditions ( Figure 4.8 ) (Thermo Fisher). The two NHS ester ends
are reactive with amine groups in proteins and other molecules to form stable amide linkages.
Once proteins are crosslinked using this reagent, they may be dissociated for analysis by rais-
ing the pH to 11.6 and incubating for 2 hours at 37 °C. The sulfone group is base labile under
these conditions, and the conjugate cleaves at the center of the bridge.
BSOCOES is a hydrophobic crosslinker and therefore must be dissolved in organic solvent
prior to its addition to an aqueous reaction medium. Preparing a stock solution in DMF or
DMSO and then adding an aliquot to the crosslinking reaction is recommended. Do not exceed
Figure 4.7 DST may be used to crosslink amine-containing molecules, forming amide bond linkages. The cen-
tral diol of the cross-bridge is cleavable by treatment with sodium periodate.
Figure 4.8 BSOCOES reacts with amine-containing molecules to create amide bond crosslinks. The internal
sulfone group is cleavable under alkaline conditions.
246 4. Homobifunctional Crosslinkers
a concentration of more than 10 percent organic solvent in the buffered reaction to avoid pro-
tein precipitation.
Reported applications of BSOCOES include studying the polypeptide antigens on lymphocyte
cell surfaces (Zarling et al., 1980), crosslinking labeled -endorphin to its opioid receptors
(Howard et al., 1985), and isolation and characterization of calcitonin receptors in rat kidney
(Bouizar et al., 1986).
There also is a water-soluble version of this reagent available. Sulfo-BSOCOES [ bis[2-(sulfo-
succinimidooxycarbonyloxy)ethyl]sulfone] is built on the same chemical structure as BSOCOES,
but contains the negatively charged sulfonate groups on both of its succinimide rings. The pres-
ence of the sulfonates provides enough charge and hydrophilicity to lend water solubility to the
entire reagent. Thus, it may be added directly to aqueous reactions at concentrations of up to
10 mM. Prior dissolution in organic solvent, however, may provide solubility at greater concen-
trations in aqueous solutions.
Additional applications of BSOCOES and sulfo-BSOCOES include investigations of the cel-
lular and subcellular distribution of the type II vasopressin receptor (Fenton et al., 2007), TNF-
alpha (Grinberg et al., 2005), and studying mechanisms in the control of plasmid replication
(Das et al ., 2005).
1.5. EGS and Sulfo-EGS
Ethylene glycol bis(succinimidylsuccinate) (EGS) is a homobifunctional crosslinking agent that
contains NHS ester groups on both ends (Thermo Fisher). Its central bridge is constructed
from an ethylene glycol group esterifi ed on either side with succinic acid, the terminal car-
boxylates of which are activated by forming NHS esters. The two NHS esters are amine reac-
tive, forming stable amide bonds between crosslinked molecules within a pH range of about
7–9. Avoid amine-containing buffers such as Tris or glycine, since they will cross-react with
the NHS esters. Imidazole also should be avoided, because it has the effect of catalyzing the
hydrolysis of the NHS ester groups. The internal structure of EGS provides two cleavable
ester sites that may be broken at pH 8.5 by incubation with 1 M hydroxylamine for 3–6 hours
at 37 °C (Abdella et al., 1979) ( Figure 4.9 ). Thus, conjugates produced from the EGS cross-
linking of the specifi c interaction of proteins or other molecules subsequently may be cleaved
with hydroxylamine for analysis.
EGS is water-insoluble and must be dissolved in an organic solvent prior to its addition
to an aqueous reaction. Prepare a concentrated solution of EGS in DMF or DMSO and add
an aliquot of the stock solution to the reaction. Do not exceed a concentration of more than
about 10 percent organic solvent in the aqueous reaction buffer or precipitation of buffer salts
or protein may occur.
Reported applications of EGS include crosslinking studies of cytochrome P-450 (Baskin and
Yang, 1980a), conjugation of TNF with lymphotoxin (Browning and Ribolini, 1989), the con-
version of a gonadotropin-releasing hormone antagonist to an agonist (Conn et al., 1982a),
preparation of a conjugate of gonadotropin-releasing hormone with an agonist (Conn et al .,
1982b), covalent crosslinking of vasoactive peptide to its lymphoblast receptors (Wood and
O ’ Dorisio, 1985), and study of bombesin receptors in Swiss 3T3 cells (Millar and Rozengur,
1990).
A water-soluble analog of EGS also is available commercially (Thermo Fisher). Sulfo-EGS,
or ethylene glycol bis(sulfosuccinimidylsuccinate), contains negatively charged sulfonate groups
on its NHS rings. The resultant charge and hydrophilicity of this modifi cation provides water
solubility to the entire compound so that prior dissolution in organic solvent is not necessary.
EGS and sulfo-EGS also have been used to study the surface loop motion in FepA (Scott
et al., 2002), to characterize the high affi nity copper transporter in Saccharomyces cerevi-
siae (Peña et al., 2000), and in the study of protein interactions and large protein complexes
(Petrotchenko et al ., 2005{2005 a or b}).
1. Homobifunctional NHS Esters 247
248 4. Homobifunctional Crosslinkers
1.6. DSG
Disuccinimidyl glutarate (DSG) is a water-insoluble, homobifunctional crosslinker containing
amine-reactive NHS esters at both ends (Thermo Fisher). The active esters react with amino
groups in protein molecules in the pH range of 7–9 to form amide linkages. DSG is a non-cleav-
ablereagent, forming stable 5-carbon bridges between amine-containing molecules ( Figure 4.10 ).
DSG should be dissolved in an organic solvent prior to addition to an aqueous reaction
medium. Suitable solvents include DMF and DMSO. To initiate a reaction, add an aliquot
of the organic solution to the buffered medium containing the molecules to be crosslinked.
Reaction buffers should not contain any competing amine compounds such as Tris or glycine,
Figure 4.9 EGS reacts with amine-containing molecules to form amide linked conjugates. The ester groups
within its cross-bridge are cleavable under alkaline conditions using hydroxylamine.
as these will cross-react with the active esters. Avoid imidazole-containing buffers, also, since it
catalyzes the hydrolysis of NHS esters.
The reported applications of DSG include receptor–ligand studies by covalent crosslinking
of their complexes (Waugh et al., 1989), the capture of protein interactions on protein array
surfaces by DSG crosslinking (MacBeath, 2007), studying TorT, a member of a periplasmic-
binding protein family (Baraquet et al., 2006), and in the investigation of left-helical conforma-
tion of l -DNA for analyzing biomarkers (Hauser et al ., 2006).
1.7. DSC
N,N-Disuccinimidyl carbonate (DSC) is the smallest homobifunctional NHS ester crosslinking
reagent available (Aldrich). It is, in essence, merely a carbonyl group-containing two NHS esters.
The compound is highly reactive toward nucleophiles. In aqueous solutions, DSC rapidly will
hydrolyze to form two molecules of NHS with release of CO
2
. In nonaqueous environments,
it can react with two amine groups to form a substituted urea derivative with loss of two mol-
ecules of NHS. The reagent also can be used in anhydrous organic solvents to activate a hydroxyl
group to an amine-reactive succinimidyl carbonate derivative. This procedure is commonly used
Figure 4.10 DSG is a non-cleavable crosslinker that can react with two amine-containing molecules to form
amide bonds.
1. Homobifunctional NHS Esters 249