Hermanson G. Bioconjugate Techniques, Second Edition

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390 7. Dendrimers and Dendrons

Figure 7.26 Dendrimers made with a disulﬁ de-containing core can be reduced to produce dendrons having free

thiol groups for surface modiﬁ cation. Dative binding of these thiol-dendrons to gold or metallic surfaces can

provide a high density of amine groups for coupling proteins or other molecules.

carboxyl functionalities. In this method, 25 Ag (I) ions were entrapped within each dendrimer

and photoactivated by irradiation with UV light. This reduced the silver ions to Ag (0) metal

nanoclusters totaling 25 atoms per dendrimer. The resultant silver/dendrimer nanocompos-

ites were ﬂ uorescent with excitation in the range of 300–400 nm and emission in the range of

400–500 nm. Investigations into the use of the complexes as ﬂ uorescent detection reagents for

cell-based imaging proved positive, with the properties closely matching the expected charac-

teristics of the parent dendrimer for cell uptake and cytotoxicity.

391

Crosslinking and modiﬁ cation reagents may possess functionality other than their reactivity

toward certain chemical groups. In particular, the cross-bridge of the molecule can be designed

to contain constituents that allow cleavage of the reagent after use. Occasionally, the coupling

reaction itself provides linkages susceptible to subsequent cleavage. Why would you want to

break a conjugate apart after having formed it? The ability can be very important in studies

involving the biospeciﬁ c interactions between two molecules, especially if only one of the mol-

ecules is known or characterized. Cleavability allows the conjugation reaction to be veriﬁ ed

through identiﬁ cation of the crosslinked molecules after conjugation and puriﬁ cation of the

complex. Precise points of modiﬁ cation can be determined by mass spec analysis after cleav-

age. In some cases, puriﬁ cation of unknown target molecules is facilitated by the ability to

cleave the crosslinking bonds after isolation of the complex. For instance, a protein modiﬁ ed

near its binding site with a photoreactive heterobifunctional crosslinker can be incubated with

its receptor or speciﬁ c binding molecule, a covalent linkage then can be formed by exposure to

UV light, and the complex analyzed by subsequent cleavage of the crosslink.

In another example, ligands can be biotinylated with a cleavable biotinylation reagent and

then incubated with receptor molecules. The resulting complex can be isolated by afﬁ nity chro-

matography on immobilized (strept)avidin. Final puriﬁ cation of the ligand–receptor can be

accomplished by cleaving the biotin modiﬁ cation sites while the complex is still bound to the

support. The receptor complex thus can be eluted from the column without the usual harsh

conditions required to break the avidin–biotin interaction.

The ability to cleave a crosslink also can provide a means of transferring a label from one

protein to another. For instance, a photoreactive heterobifunctional crosslinker that is iodinat-

able and cleavable can be used to tag an unknown receptor molecule after conjugation. For

example, the crosslinker SASD (Chapter 5, Section 3.2) can be iodinated before it is employed

in a crosslink ing reaction. It is then used to modify a protein through its amine-reactive NHS

ester end and puriﬁ ed from excess crosslinker. After incubation of the modiﬁ ed protein with

speciﬁ c binding molecules (e.g., other proteins) and photoreactive crosslinking, the conjugate

can be broken by reduction of the disulﬁ de group within the cross-bridge of the reagent. Since

the radiolabeled part of the crosslinker now is attached to the unknown interacting molecule,

the tracer is effectively transferred from the initially modiﬁ ed molecule. Thus, unknown inter-

acting molecules can be tracked after their binding to an SASD-labeled substance.

Cleavable Reagent Systems

392 8. Cleavable Reagent Systems

Another crosslinker, SAED (Chapter 5, Section 3.9), can be used in a similar fashion, but

instead of transferring a radioactive label, it contains a ﬂ uorescent portion that is transferred to

a binding molecule after cleavage. Similarly, sulfo-SBED routinely is used to study protein inter-

action. Cleavage of a disulﬁ de bridge after capture of interacting proteins results in transfer of

a biotin label to the unknown prey protein (Chapter 28, Section 3.1). The biotin modiﬁ cation

then can be used to detect or isolate the unknown interactor for subsequent identiﬁ cation.

The ability to break a crosslink can be an important feature of a modiﬁ cation or conjuga-

tion reagent. This chemistry typically is built into the cross-bridge or reactive ends of a rea-

gent using disulﬁ des, glycol groups, diazo bonds, esters, sulfone groups, or acetal linkages. The

following sections describe these chemical characteristics and their respective cleavage

conditions.

1. Cleavage of Disulﬁ des by Reduction

The formation of a disulﬁ de linkage between crosslinked molecules is an important option

for many conjugation chemistries. Examples of reagents that have this capability include the

pyridyl disulﬁ de containing heterobifunctionals like SPDP (Chapter 5, Section 1.1) and SMPT

(Chapter 5, Section 1.2). Other non-sulfhydryl-reactive crosslinkers still may possess a disulﬁ de

group within their cross-bridge construction. The presence of such disulﬁ de groups, whether

designed in the crosslinker or created as a product of their reactions, allows for speciﬁ c cleav-

age of the complex or modiﬁ ed molecule after conjugation. Disulﬁ de bonds can be broken by a

number of methods (Chapter 1, Section 4.1), utilizing either direct hydrogenolysis by a strong

reductant such as sodium borohydride or through a disulﬁ de interchange process with a com-

pound containing one or more free sulfhydryls ( Figure 8.1 ).

Cleavage of disulﬁ de bonds is done easily by incubation with a reducing agent at a level

of 10–100 mM concentration. If the disulﬁ des in the crosslinks are the only ones present in

the complexed molecules, then reduction will yield unconjugated molecules—one or both of

Figure 8.1 Cleavage of disulﬁ de-containing crosslinking compounds can be done using a reducing agent such

as DTT. Reduction causes the conjugates to break apart into their original components with each component

containing a portion of the crosslinker that terminates in a thiol group.

which will contain a portion of the crosslinker, and on both molecules a free sulfhydryl will be

created. Caution should be used with this method of cleavage, however, if other disulﬁ des are

present in the conjugated molecules. Some protein disulﬁ des, for instance, also may be affected

by the reduction step. Complete cleavage of all disulﬁ des in crosslinked proteins by inclusion

of unfolding agents (i.e., guanidine) may yield additional protein fragments of lower molecular

weight due to subunit disassociation.

2. Periodate-Cleavable Glycols

Crosslinking agents can be designed to contain adjacent carbon atoms possessing hydroxyl

groups. Cross-bridges containing such diols or glycol residues can be constructed from the

inclusion of an internal tartaric acid spacer or similar compound in their synthesis (e.g., DST,

Chapter 4, Section 1.3). These groups can be easily cleaved by oxidation with sodium perio-

date (Chapter 1, Section 4.4). Treatment with 15 mM periodate at physiological pH will break

the carbon–carbon bond between the glycol portion, oxidizing each hydroxyl to an aldehyde,

and cleaving the associated crosslinked molecules ( Figure 8.2 ). Under these conditions, gly-

cosylated portions of glycoproteins or other carbohydrate-containing molecules also will be

affected, forming additional aldehyde groups. In some cases, the production of aldehyde resi-

dues may cause secondary reactions to occur, especially Schiff base formation with available

amine groups. To avoid unexpected crosslinks that form through such intermolecular Schiff

base formation, Tris or ethanolamine may be included to tie up the aldehydes as they form.

Sodium periodate also may affect tryptophan residues in some proteins. The oxidation of

tryptophan can result in activity losses if the amino acid is an essential component of the active

site. For instance, avidin and streptavidin may be severely inactivated by treatment with perio-

date, since tryptophan is important in forming the biotin-binding pocket. In addition, many

other amino acid residues are susceptible to oxidation by periodate (Chapter 1, Section 1.1).

Limiting the time of oxidation is important to restricting oxidation to diol groups while not

affecting other protein structures.

The use of periodate as a cleavage agent does have advantages, however. Unlike the use of

cleavable crosslinkers that contain disulﬁ de bonds which require a reductant to break the con-

jugate, cleavage of diol-containing crosslinks with periodate typically preserves the indigenous

disulﬁ de bonds and tertiary structure of proteins and other molecules. As a result, with most

proteins bioactivity usually remains unaffected after mild periodate treatment.

Figure 8.2 Crosslinkers containing a diol group in their cross-bridge design may be cleaved by oxidation with

sodium periodate.

2. Periodate-Cleavable Glycols 393

394 8. Cleavable Reagent Systems

Figure 8.4 Crosslinkers containing an ester group in their cross-bridge are susceptible to cleavage under alka-

line conditions using hydroxylamine.

Figure 8.3 Crosslinking agents that form diazo bonds may be cleaved using sodium dithionite.

3. Dithionite-Cleavable Bonds

Crosslinking compounds containing diazo bonds within their structures can be speciﬁ cally

cleaved with dithionite (Jaffe et al., 1980). In addition, crosslinks formed by the reaction of

a diazonium compound (Chapter 2, Section 6.1) with a tyrosine residue also can be broken

using this reagent. Sodium dithionite (also called sodium hydrosulﬁ te) reduces the diazo link-

age, breaking the bond between the nitrogens, and leaving a primary amine on both fragments

of the crosslinker ( Figure 8.3 ). The reaction usually is carried out in alkaline conditions; a

25-minute incubation with 0.1 M dithionite in 0.2 M sodium borate, pH 9, works well. As the

diazo bonds are broken, any color associated with the reagent will disappear.

Dithionite also is capable of cleaving oxime linkages that are formed as the result of the

reaction of an aldehyde and aminoxy group (Pojer, 1979). Thus, crosslinks formed between

two proteins or other molecules using this reaction strategy can be speciﬁ cally broken using

sodium dithionite.

4. Hydroxylamine Cleavable Esters

Hydroxylamine is a powerful nucleophile which, under alkaline conditions, is effective in

breaking ester bonds. Crosslinking agents containing esteriﬁ ed spacer components can be

cleaved after undergoing a conjugation reaction by incubation with 0.1 M hydroxylamine, pH

8.5, for 3–6 hours at 37°C (Abdella et al., 1979). The reaction results in the formation of an

amide derivative on one fragment of the cleaved crosslinker and a hydroxyl group on the other

fragment ( Figure 8.4 ). Thioester bonds also are susceptible to cleavage under these conditions.

Thioesters may be broken with the production of an amide and a sulfhydryl group on either

side of the crosslinker fragments.

An example of a hydroxylamine-cleavable reagent is EGS (Chapter 4, Section 1.5) which

contains two ester bonds made by the esteriﬁ cation of ethylene glycol with succinic acid.

Cleavage with hydroxylamine yields two fragments terminating with an amide bond and con-

comitant release of ethylene glycol. Mass spec evidence of the cleavage products from an EGS

crosslink indicates that the cleaved parts can undergo cyclization to form a succinimide group

on both fragments (Petrotchenko et al ., 2005).

5. Base Labile Sulfones

The presence of a sulfone group in a crosslinking reagent can allow for cleavage of a conjugate

through hydrolysis of the linkage under basic conditions. In 0.1 M sodium phosphate, adjusted

to pH 11.6 by addition of Tris base, containing 6 M urea, 0.1 percent SDS, and 2 mM DTT,

sulfone groups were successfully cleaved after incubation at 37°C for 2 hours (Zarling et al.,

1980). In that study, peptide antigens on the surface of lymphocyte receptors were crosslinked

with the homobifunctional, amine-reactive reagent BSOCOES (Chapter 4, Section 1.4), puriﬁ ed,

and cleaved for analysis. The presence of urea, SDS, and DTT were not absolutely necessary for

breaking the sulfone bond, rather they served to disrupt completely protein–peptide structure

for complete dissociation of the complex.

In addition to BSOCOES, the amine-reactive, bis- ﬂ uorobenzene reagent DFDNPS (Chapter 4,

Section 4.2) also contains an internal sulfone group that is easily cleaved under basic condi-

tions (Wold, 1961, 1972). Hydrolysis of the sulfone yields two crosslinker fragments, one ter-

minating in a sulfonic acid group and the other containing a hydroxyl group ( Figure 8.5 ).

Figure 8.5 Crosslinkers that have an internal sulfone group in their cross-bridge may be cleaved using base.

5. Base Labile Sulfones 395

396

Labels, tags, and probes are relatively small modifying agents that can be used to label pro-

teins, nucleic acids, and other molecules. These compounds often contain groups that provide

sensitive detectability by virtue of some intrinsic chemical or atomic property such as ﬂ uorescence,

visible chromogenic character, radioactivity, or bioafﬁ nity toward another protein. Most probes

can be designed to contain a reactive portion capable of coupling to the functional groups of

biomolecules. After modiﬁ cation of a protein via this reactive part, the probe becomes covalently

attached, thus permanently tagging it with a unique detectable property. Subsequent interactions

that the labeled protein is allowed to undergo can be followed through the tag ’s visibility.

Labeling molecules by adding a radioactive component was one of the ﬁ rst means of creating

highly sensitive detection capabilities. Covalent modiﬁ cation of activated aromatic rings with

125

131

I easily can be done through tyrosine side chains in proteins or by the use of a phenolic-

ring-containing modiﬁ cation agent such as the Bolton–Hunter reagent (Chapter 12, Section 5).

Other methods of introducing a radioactive isotope involve the intermediary use of metal-

chelating modiﬁ cation reagents such as diethylenetriamine pentaacetic acid (DTPA) (Chapter 10,

Section 1). Heavy metal isotopes may be held in coordination complexes on a protein or other

molecule and provide extreme sensitivity for in vivo diagnostic procedures involving the detec-

tion of malignancies. Such complexes coupled to monoclonal antibodies also are being used in

the treatment of cancer by their ability to cause cell death in proximity to the bound radiolabel.

Detection of probes or labels usually takes one of three general forms: spectrophotometric,

radiosensitive detectors, or indirectly through another labeled substance. Spectral probes can

be of two types, chromogenic or ﬂ uorescent. Chromogenic labels typically are reserved for non-

covalent staining of gross structural features within cells and tissues, as these are present at rela-

tively high concentration. The sensitivity of visible wavelength dyes often is not good enough to

provide detectability for low-concentration antigens or low-copy proteins. Even if a protein is

covalently modiﬁ ed with a chromogen, the number of associated dye molecules needed to detect

it just through its absorbance properties could be prohibitively large to make it viable.

Fluorescent labels, by contrast, can provide tremendous sensitivity due to their property of

discrete emission of light upon excitation. Proteins, nucleic acids, and other molecules can be

labeled with ﬂ uorescent probes to provide highly receptive reagents for numerous in vitro assay

procedures. For instance, ﬂ uorescently tagged antibodies can be used to probe cells and tis-

sues for the presence of particular antigens, and then detected through the use of ﬂ uorescence

microscopy techniques. Since each probe has its own ﬂ uorescence emission character, more

Fluorescent Probes

than one labeled molecule—each tagged with a different ﬂ uorophore—can be used at the same

time to detect two or more target molecules.

A ﬂ uorescent molecule has the ability to absorb photons of energy at one wavelength and sub-

sequently emit energy at another wavelength. The absorption process is also called excitation,

since the quantum energy levels of some of the compound ’s electrons increase with photon

uptake. The absorption band is not isolated at a discrete photon energy level, but spread out

over a range of wavelengths with at least one peak of maximal absorbance within this spectral

region. The extinction coefﬁ cient ( , expressed as M

 1

) at the absorbance peak maximum

is a unique characteristic of each ﬂ uorophore under a given environmental condition.

The excess energy of an excited ﬂ uorophore can be lost as heat or through collisions with adja-

cent molecules or released as photons of light as the electrons return to the lower, ground-state

energy level ( Figure 9.1 ). This process of light emission occurs in less than 10

 4

seconds after

Figure 9.1 Energy diagram showing the transition states involved in the absorption and decay of electro-

magnetic energy. Energy may be released through heat or internal collisions, transferred to other molecules,

or it may be emitted as photons of light. Fluorescence occurs from within the singlet system as light energy

is released, returning the electrons to the ground state. Phosphorescence occurs from the triplet system and

involves a longer emission process at lower energies than that of ﬂ uorescence.

9. Fluorescent Probes 397

398 9. Fluorescent Probes

excitation and is known as ﬂ uorescence. Fluorescence takes place from the lowest excited singlet

state. According to Stoke ’s Law, the emission wavelength is always longer and thus of lower energy

than the wavelength of excitation (Kawamura Jr., 1977). The ratio of total photon emission over

the entire range of ﬂ uorescence to the total photon absorption is called the quantum yield (QY).

QY values range from 0 to 1. The larger the QY value the more efﬁ cient the photon emission or

luminescence. For a particular ﬂ uorophore under ﬁ xed environmental conditions, both its extinc-

tion coefﬁ cient and its QY are fundamental characteristics of its photochemical behavior.

A ﬂ uorescent compound suitable for analytical studies should have not only a high QY, but

its ﬂ uorescence emission spectrum should be separated sufﬁ ciently from its excitation spec-

trum to assure good signal isolation. A ﬂ uorophore ’s Stoke ’s shift is a measure of the separa-

tion of its maximal absorbance wavelength from its emission wavelength maxima ( Figure 9.2 ).

The greater the Stoke ’s shift, the better the signal isolation and therefore less interference from

Rayleigh-scattered excitation light.

The majority of reported ﬂ uorophores of practical use in labeling biomolecules contain an

aromatic ring system as the generator of luminescence. In general, as the conjugated electron sys-

tem gets larger, the emission wavelength is shifted to the red. Also, the extinction coefﬁ cient and

QY of larger conjugated systems typically are greater than that of small aromatic compounds.

Aromatic ring constituents can have a pronounced effect on ﬂ uorescence. The presence of ring

activators or electron donating groups (e.g., ortho and para directors) generally increases QY,

while the presence of electron-withdrawing groups (e.g., meta directors) reduces QY. An example

of this effect can be found in the photoreactive heterobifunctional crosslinker SAED (Chapter 5,

Section 3.9). Before photoactivation, SAED possesses a photoreactive phenyl azide group on

its AMCA-derived end. This electron-withdrawing group quenches the ﬂ uorescence of the

compound so that the AMCA group does not behave as it characteristically does in the underi-

vatized state. Upon photolysis, however, the phenyl azide either gets coupled to a target molecule

Figure 9.2 Typical spectral scan of a ﬂ uorescent compound showing its absorbance peak or wavelengths of

most efﬁ cient excitation and its emission peak or wavelengths where light emission occurs. The Stoke ’s shift is

the distance in nanometers between the absorbance peak and the emission peak. The larger the Stoke ’s shift, the

less interference that will occur from excitation light when measuring ﬂ uorescence emission.

or breaks down to an amine. In either case, the presence of an amine (or its derivative) provides

enough ring-activating properties to restore the coumarin group ’s ﬂ uorescent character.

Another potential ring constituent having dramatic affect on ﬂ uorescence is the presence of

heavy atoms. Aromatic rings possessing heavy atoms diminish QY by enhancing the probabil-

ity of the excited singlet state going on to triplet transition. Energy decay from a triplet excited

state causes phosphorescence instead of ﬂ uorescence. The phosphorescent band is located at

longer wavelengths (and thus lower energies) relative to the ﬂ uorescent spectrum. The energy

transition to the triplet state therefore is in direct contention with ﬂ uorescence, and has the

effect of decreasing overall luminescence. In this regard, the relative ﬂ uorescent quenching

effect of halogen substitution on aromatic-ring-containing ﬂ uorophores is: I  Br  Cl  F.

In practice, the presence of a chloride atom on a ﬂ uorescent aromatic ring may still allow some

luminescence to occur; however, the presence of bromide or iodine atoms nearly guarantees

complete elimination of ﬂ uorescence. Thus, radioiodination of a ﬂ uorescent ring structure is

not possible without dramatically decreasing (or eliminating) its QY.

The aromatic ring systems of most of the following ﬂ uorophores consist of polycyclic struc-

tures. To maintain ﬂ uorescence in such compounds, it is important that the entire system be

coplanar or the rings be in the same dimensional plane. In fact, the differences between some non-

ﬂ uorescent chromogenic dyes and their corresponding, structurally similar ﬂ uorophoresare minor,

but the planer nature of the ﬂ uorophores gives them their luminescent properties. For instance,

Figure 9.3 illustrates the similarity of the dyes phenolphthalein and malachite green to the nearly

identical ﬂ uorescent molecules ﬂ uorescein and rhodamine, respectively. The only difference among

Figure 9.3 Fluorescent character in organic compounds is often determined by the presence of a planar aro-

matic ring system. The ﬂ uorescent compounds differ only in the closure of their central ring system, which pro-

duces the required constraints to create a planar triple-ring conﬁ guration.

9. Fluorescent Probes 399