Hermanson G. Bioconjugate Techniques, Second Edition

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430 9. Fluorescent Probes

appropriate for excitation by the 568 nm line produced by an argon–krypton mixed laser used

on some confocal devices. White light illumination also can be used to excite the dye, while

its emission is detected using an appropriate ﬁ lter. Compared to other rhodamine derivatives,

Texas Red ﬂ uorophores display low background in staining techniques and are among the

most photostable probes available.

Texas Red hydrazide is soluble in DMF and may be dissolved as a concentrated stock solu-

tion in this solvent prior to the addition of a small aliquot to an aqueous reaction medium. The

solid and all solutions made from it must be protected from light to avoid photo-decomposition.

Prepare the stock solution fresh immediately before use. A suggested protocol on the use of

this ﬂ uorescent probe may be obtained by following the method outlined for ﬂ uorescein-5-thi-

osemicarbazide in Section 1 of this chapter. Optimization may be necessary to achieve the best

level of ﬂ uorescent modiﬁ cation ( F / P ratio) for a particular application.

3. Coumarin Derivatives

Coumarin (2 H-1-benzopyran-2-one) is a naturally occurring substance found in tonka beans,

lavender oil, and in sweet clover (Merck Index 11: 2563) ( Figure 9.21 ). Many of its derivatives

are highly ﬂ uorescent compounds that are widely studied (Schimitschek et al., 1974; Ernsting

et al., 1982; Jones et al., 1984, 1985; Eschrich and Morgan, 1985; Baranowska-Kortylewicz

and Kassis, 1993a, b). The 7-amino-4-methylcoumarin derivatives have excellent ﬂ uorescent

properties useful for labeling biological molecules with a detectable tag (Uchino et al., 1979;

Bos, 1981). Particularly, the 3-acetic acid derivative of this molecule, known as AMCA, pro-

vides a carboxylate group from which to create easily reactive groups suitable for coupling to

proteins and other molecules.

Aminomethylcoumarin derivatives possess intense ﬂ uorescent properties within the blue

region of the visible spectrum. Their emission range is sufﬁ ciently removed from other common

ﬂ uorophores that they are excellent choices for double-labeling techniques. In fact, coumarin

ﬂ uorescent probes are very good donors for excited-state energy transfer to ﬂ uoresceins.

The following sections describe the most popular derivatives of aminomethylcoumarin used

to label proteins and other biological macromolecules.

Figure 9.20 Texas Red hydrazide reacts with aldehydes to create hydrazone bonds.

Amine-Reactive Coumarin Derivatives

Three main forms of amine-reactive AMCA probes are commonly available. One of them is

simply the free acid form of AMCA, which can be used to couple to amine-containing mol-

ecules using the carbodiimide reaction (Chapter 3, Section 1.1). The other two are active-ester

derivatives of AMCA—the water-insoluble NHS ester and the water-soluble sulfo-NHS ester

forms—both of which spontaneously react with amines to create stable amide linkages. All of

them react under mild conditions with primary amines in proteins and other molecules to form

highly ﬂ uorescent derivatives.

AMCA

AMCA, or 7-amino-4-methylcoumarin-3-acetic acid, is a ﬂ uorescent probe that exhibits a spec-

tacular blue ﬂ uorescence (Khalfan et al., 1986) (Thermo Fisher). AMCA absorbs light at a wave-

length of 345 nm and luminesces in the range of 440–460 nm. Its emission wavelength is in a

region that doesn ’t overlap with the emission spectra of other major ﬂ uorescent probes. This

makes double-staining techniques particularly effective with this ﬂ uorophore. AMCA also has

pronounced stability toward photobleaching, retaining its full ﬂ uorescence more than 3 times

longer than ﬂ uorescein-based probes. AMCA derivatives and labeled molecules ﬂ uoresce with

a bright blue color upon excitation. This color is easily visualized using ﬂ uorescent microscopes

or imagers. The blue emission color avoids problems of autoﬂ uorescence associated with high

background. In addition, the large Stoke ’s shift of the molecule minimizes interference from

Rayleigh light scatter effects during excitation. The ﬂ uorescent intensity of AMCA is not affected

by changes in pH in the range of 3–10. This is in marked contrast to other ﬂ uorescent probes,

such as ﬂ uorescein, which display considerable variability in their emission spectra with pH.

Figure 9.21 The basic structural characteristics of coumarin ﬂ uorophores.

3. Coumarin Derivatives 431

432 9. Fluorescent Probes

AMCA may be coupled to amine-containing molecules through the use of the carbodiimide

reaction using EDC (Chapter 3, Section 1.1). EDC will activate the carboxylate on AMCA

to a highly reactive o-acylisourea intermediate. Attack by a nucleophilic primary amine group

results in the formation of an amide bond ( Figure 9.22 ). Derivatization of AMCA off its car-

boxylate group causes no major effects on its ﬂ uorescent properties. Thus, proteins and other

macromolecules may be labeled with this intensely blue probe and easily detected by ﬂ uores-

cence microscopy and other techniques.

AMCA-NHS and AMCA-Sulfo-NHS

AMCA-NHS, succinimidyl-7-amino-4-methylcoumarin-3-acetic acid, is an amine-reactive

derivative of AMCA containing an NHS ester on its carboxylate group (Thermo Fisher). The

result is reactivity directed toward amine-containing molecules, forming amide linkages with

the AMCA ﬂ uorophore ( Figure 9.23 ). Proteins labeled with AMCA show little-to-no effect on

the isoelectric point of the molecule.

Reaction of AMCA-NHS with proteins proceeds efﬁ ciently in the pH range of 7–9. Avoid

buffers containing amines which can compete in the coupling reaction, such as Tris or glycine,

and avoid imidazole buffers since they promote hydrolysis of the NHS ester. AMCA-NHS is

relatively insoluble in aqueous buffers. The compound must be ﬁ rst dissolved in organic sol-

vent prior to adding a small aliquot to the reaction mixture. A concentrated stock solution may

be prepared in DMSO and stored up to 2 weeks refrigerated or frozen without loss of activity.

The solid and all solutions of AMCA-NHS should be protected from light to avoid photob-

leaching of the ﬂ uorophore.

AMCA-sulfo-NHS is an analog of AMCA-NHS which contains a sulfonate group on its

NHS ring (Thermo Fisher). The negative charge of this group provides enough polarity to

Figure 9.22 AMCA may be linked to amine-containing molecules through its carboxylate group using a carbo-

diimide reaction with EDC.

Figure 9.23 AMCA-NHS reacts with amines to form amide bonds.

3. Coumarin Derivatives 433

434 9. Fluorescent Probes

promote water solubility for the entire reagent. The reactivity and properties of AMCA-

sulfo-NHS are identical to those of AMCA-NHS.

Preparing an optimal ﬂ uorescent conjugate is largely dependent upon the degree of modiﬁ -

cation with the label. The following protocol is generalized for the labeling of a protein with

AMCA-NHS. For particular labeling experiments, it is often necessary to vary the amount of

ﬂ uorophore added to the reaction mixture to obtain the best combination of protein activity

and ﬂ uorescent intensity in the conjugate. Too much label and nonspeciﬁ c binding or ﬂ uores-

cent quenching may result; too little label and the complex will not possess enough ﬂ uorescent

intensity to be sufﬁ ciently detectable.

Protocol

1. Dissolve the protein to be modiﬁ ed in 50 mM sodium borate, pH 8.5, at a concentra-

tion of 10 mg/ml. Other buffers may be used for an NHS ester reaction, including 0.1 M

sodium phosphate, pH 7.5 (Chapter 2, Section 1.4).

2. Dissolve AMCA-NHS (Thermo Fisher) in DMSO at a concentration of 2.6 mg/ml.

Protect from light.

3. In subdued lighting conditions, slowly add 50–100 l of the AMCA-NHS stock solution

to each ml of the protein solution, with gentle mixing.

4. React for 1 hour at room temperature in the dark.

5. Remove excess reagent and reaction by-products by gel ﬁ ltration using a desalting resin.

The sample volume should be no more than about 5–8 percent of the column volume.

The F / P ratio of the puriﬁ ed, labeled protein may be determined by measuring the absorb-

ance at 345 and 280 nm. Ratios between 0.3 and 0.8 usually produce labeled molecules having

acceptable levels of ﬂ uorescent intensity and good retention of protein activity. AMCA-labeled

proteins may be lyophilized without signiﬁ cant loss of ﬂ uorescence. The addition of bovine

serum albumin (15 mg/ml) or another such stabilizer is often necessary to retain solubility of

the freeze-dried, labeled protein after reconstitution.

Sulfhydryl-Reactive Coumarin Derivatives

Two aminomethylcoumarin derivatives are available for labeling sulfhydryl-containing mole-

cules. The ability to label  SH groups in proteins provides a means of directing the modiﬁ cation

reaction to a limited number of sites, possibly avoiding active centers or binding regions better

than when using amine-reactive probes. The ﬁ rst sulfhydryl-reactive probe discussed in this sec-

tion makes use of a pyridyl disulﬁ de group on the AMCA derivative. The second probe is an

iodoacetyl compound made from a diethyl and aminophenyl derivative of the basic aminometh-

ylcoumarin structure.

AMCA-HPDP

AMCA-HPDP is N -[6-(7-amino-4-methylcoumarin-3-acetamido)hexyl]-3  -(2  -pyridyldithio)

propionamide. It is formed from AMCA plus a 1,6-diaminohexyl spacer off the carboxylate

that has been additionally modiﬁ ed at its terminal end with SPDP (Chapter 5, Section 1.1).

The result is a long spacer arm terminating in a pyridyl disulﬁ de group reactive toward free

sulfhydryl residues. The reaction of this group with a thiol creates a disulﬁ de bond between the

AMCA ﬂ uorophore and the molecule being modiﬁ ed. Thus, the ﬂ uorescent tag can be speciﬁ -

cally cleaved by reduction with DTT or other disulﬁ de reducing agents ( Figure 9.24 ).

Figure 9.24 AMCA-HPDP reacts with sulfhydryl groups through its pyridyl disulﬁ de end to form reversible

disulﬁ de bonds.

3. Coumarin Derivatives 435

436 9. Fluorescent Probes

The required sulfhydryl residues can be naturally occurring on a protein, created by reduc-

tion of cystine crosslinks or by thiolation (Chapter 1, Section 4.1). For the labeling of antibody

molecules, mild reduction with 2-mercaptoethylamine, DTT, or tris(2-carboxyethyl)phosphine

(TCEP) results in free sulfhydryl groups in the hinge region. Labeling in this area is advanta-

geous to direct the modiﬁ cation away from antigen binding regions. Sulfhydryl residues also

may be created on oligonucleotides without difﬁ culty (Chapter 27, Section 2.2).

Although the reaction of a sulfhydryl-containing molecule with AMCA-HPDP results in the

release of the chromogenic leaving group, pyridine-2-thione, using it to quantify the extent of

modiﬁ cation may be difﬁ cult, because it absorbs at 343 nm, which is in the same region as

AMCA itself (345 nm). The emission range of the AMCA probe is about 440–460 nm, in the

blue region of the spectrum.

The following protocol is a suggested method for labeling a protein with AMCA-HPDP. It

is assumed that the presence of a sulfhydryl on the protein has been documented or created.

The reaction conditions can be carried out in a variety of buffers between pH 6 and 9. Avoid

the presence of extraneous sulfhydryl-containing compounds (such as disulﬁ de reductants) that

will compete in the reaction. The inclusion of EDTA in the modiﬁ cation buffer prevents metal-

catalyzed sulfhydryl oxidation. Optimization for a particular labeling experiment should be

done to obtain the best level of ﬂ uorophore incorporation.

Protocol

1. Dissolve the sulfhydryl-containing protein to be labeled in 0.1 M sodium phosphate,

0.15 M NaCl, 10 mM EDTA, pH 7.2, at a concentration of 10 mg/ml.

2. Dissolve AMCA-HPDP in DMSO at a concentration of 0.5 mg/ml. Protect from light.

3. In subdued lighting conditions, add 50–100 l of the AMCA-HPDP stock solution to

each ml of sulfhydryl-containing protein solution. Mix.

4. React for 1 hour at room temperature in the dark with occasional mixing.

5. Remove excess ﬂ uorophore and reaction by-products by gel ﬁ ltration using a desalting

resin.

To determine the F / P ratio of the labeled protein, measure the absorbance of the puriﬁ ed

preparation at 345 nm and 280 nm. Ratios of 345 nm/280 nm within the range of 0.3–0.8

usually result in ﬂ uorescent conjugates with a good balance of high-intensity luminescence,

low nonspeciﬁ c binding, and excellent retention of biological activity within the protein

component.

DCIA

DCIA is 7-diethylamino-3-[(4 -(iodoacetyl)amino)phenyl]-4-methylcoumarin, a derivative of

the basic aminomethylcoumarin structure that contains a sulfhydryl-reactive iodoacetyl group

and a diethyl substitution on its amine. This particular coumarin derivative is among the most

ﬂ uorescent UV-excitable iodoacetamide probes available (Sippel, 1981) (Invitrogen).

The iodoacetyl group of DCIA reacts with sulfhydryls under slightly alkaline conditions to

yield stable thioether linkages ( Figure 9.25 ). They do not react with unreduced disulﬁ des in

cystine residues or with oxidized glutathione (Gorman et al ., 1987).

Care must be taken to protect iodoacetyl reagents from light, not only to maintain the

ﬂ uorescent yield of the coumarin component, but also to protect the iodoacetyl group from

light-catalyzed breakdown. DCIA is soluble in DMF and DMSO. Concentrated stock solutions

may be prepared in either solvent prior to addition of a small aliquot to a reaction mixture.

Protect solutions from light by wrapping vessels in aluminum foil and working in subdued light.

The spectral properties of this ﬂ uorophore are similar to those of other coumarin deriva-

tives. The excitation maximum occurs at about 382 nm and its emission peak at 472 nm,

producing light in the blue region of the spectrum. The extinction coefﬁ cient of DCIA at its

wavelength of maximum absorbance, 382 nm, is 33,000 M

 1

(in methanol).

Figure 9.25 DCIA can modify sulfhydryl groups through its iodoacetamide group to form thioether linkages.

3. Coumarin Derivatives 437

438 9. Fluorescent Probes

DCIA has been used to label numerous proteins and other biomolecules, including phosphol-

ipids (Silvius et al., 1987), to study the interaction of mRNA with the 30S ribosomal subunit

(Czworkowski et al., 1991), in the investigation of cellular thiol components by ﬂ ow cytometry

(Durand and Olive, 1983), in the detection of carboxylate compounds using peroxyoxalate chemi-

luminescence (Grayeski and DeVasto, 1987), and for general sulfhydryl labeling (Sippel, 1981).

A general protocol for the use of DCIA for ﬂ uorescently labeling proteins that contain

sulfhydryl residues may be obtained by following the method discussed for AMCA-HPDP

(previous section). After puriﬁ cation of the labeled protein, the F / P ratio of ﬂ uorophore incor-

poration may be determined by measuring its 382 nm/280 nm absorbance ratio.

Aldehyde- and Ketone-Reactive Coumarin Derivatives

Hydrazide groups can be coupled directly to aldehyde and ketone groups to form relatively stable

hydrazone linkages (Chapter 2, Section 5.1). One AMCA derivative is commonly available that

contains a hydrazine group modiﬁ cation on its carboxylate. Although most biomolecules don ’t

contain aldehyde or ketone groups in their native state, carbohydrates, glycoproteins, RNA, and

other molecules that contain sugar residues can be oxidized with sodium periodate to produce

reactive formyl groups. The use of modiﬁ cation reagents which generate aldehydes upon cou-

pling to a molecule also can be used to produce a hydrazide-reactive site (Chapter 1, Section 4.4).

DNA and RNA may be modiﬁ ed with hydrazide-reactive probes by reacting their cytosine

residues with bisulﬁ te to form reactive sulfone intermediates. These derivatives can undergo

transamination reactions with hydrazide- or amine-containing probes to yield covalent bonds

(Draper and Gold, 1980) (Chapter 27, Section 2.1).

AMCA-Hydrazide

AMCA-hydrazide is 7-amino-4-methylcoumarin-3-acetyl hydrazide, a hydrazine derivative off

the carboxyl group of the basic AMCA molecule (Thermo Fisher). AMCA-based ﬂ uorophores

are highly stable toward photobleaching. Molecules labeled with this probe retain their ﬂ uores-

cent intensity over 3 times longer than a ﬂ uorescein label when exposed to light. In addition,

AMCA derivatives exhibit a large Stoke ’s shift of over 100 nm, thus they are minimally affected

by Rayleigh scattering effects during excitation. The blue light emitted by these labels is in a

region of the spectrum well removed from the emission characteristics of other major ﬂ uores-

cent probes. This means that double-staining techniques easily can be used with an AMCA

label. AMCA also exhibits little luminescence dependency on pH over the range of 3–10.

The hydrazide derivative of AMCA can be used to modify aldehyde- or ketone-containing

molecules, including cytosine residues using the bisulﬁ te activation procedure described in

Chapter 27, Section 2.1. AMCA-hydrazide reacts with these target groups to form hydrazone

bonds ( Figure 9.26 ). Carbohydrates and glycoconjugates can be labeled speciﬁ cally at their

polysaccharide portion if the required aldehydes are ﬁ rst formed by periodate oxidation or

another such method (Chapter 1, Section 4.4).

AMCA-hydrazide has a maximal excitation wavelength of 345 nm and a maximum emis-

sion wavelength in the range of 440–460 nm. A solution of AMCA in PBS at a concentration of

16.7 ng/ml (71.61 nmoles/ml) gives an absorbance at 345 nm of about 1.28. This translates into

a molar extinction coefﬁ cient at this wavelength of about 13,900 M

 1

. Different solvents

and conditions may alter this value somewhat.

AMCA-hydrazide is soluble in DMSO or DMF and may be dissolved as a concentrated

stock solution in either of these solvents prior to the addition of a small aliquot to an aqueous

reaction medium. The solid and all solutions made from the ﬂ uorophore must be protected

from light to avoid photo-decomposition. Prepare the stock solution fresh immediately before

use. A suggested protocol on the use of this ﬂ uorescent probe may be obtained from the fol-

lowing method on the labeling of periodate-oxidized IgG. Optimization may be necessary to

achieve the best level of ﬂ uorescent modiﬁ cation ( F / P ratio) for a particular application.

Protocol

Oxidation of IgG Carbohydrate Residues with Sodium Periodate

1. Dissolve the antibody to be labeled in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.5, at

a concentration of at least 10 mg/ml. The immunoglobulin must be glycosylated to work

in this procedure.

Figure 9.26 AMCA-hydrazide can be used to label aldehyde-containing molecules, such as periodate-oxidized

carbohydrates.

3. Coumarin Derivatives 439