Hermanson G. Bioconjugate Techniques, Second Edition

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460 9. Fluorescent Probes

(Chapter 27, Section 2.1). These derivatives can undergo transamination reactions with hydrazide-

or amine-containing probes to yield covalent bonds (Draper and Gold, 1980).

Lucifer Yellow CH is commonly used as a neuronal tracer by staining cells and then ﬁ xing

them with formaldehyde or glutaraldehyde. It also can be used to label glycoproteins or gly-

colipids on cell surfaces after periodate oxidation (Spiegel et al., 1983; Lee and Fortes, 1985).

The labeling of oxidized ribonucleotides and gangliosides can be done similarly (Spiegel et al .,

1985; Sun et al ., 1988).

This ﬂ uorophore has an excitation maximum at 428 nm and an emission maximum at

534 nm. The extinction coefﬁ cient of the molecule in aqueous solution is about 12,000 M

 1

Cascade Blue hydrazide and Lucifer Yellow CH derivatives can be excited simultaneously by

light of less than 400 nm, resulting in the possibility for two-color detection at 420 and 534 nm.

Figure 9.43 The hydrazide group of this Lucifer Yellow derivative can react with aldehyde-containing mol-

ecules to form hydrazone bonds.

Lucifer Yellow CH is soluble in aqueous solution, and it should be stable for awhile if pro-

tected from light. The reagent is available as three different salts of the sulfonate groups. The

ammonium salt of the ﬂ uorophore is soluble to a level of 9 percent in water, while the lithium

and potassium salts have a solubility of 5 and 1 percent, respectively. A concentrated stock

solution of the ﬂ uorophore may be prepared in water and an aliquot added to a buffered reac-

tion medium to facilitate the transfer of small quantities. For aqueous reactions, a pH range of

5–9 will result in efﬁ cient hydrazone formation with aldehyde or ketone residues.

7. Phycobiliprotein Derivatives

Phycobiliproteins are intensely ﬂ uorescent proteins that function as components in the photo-

synthetic apparatus of eukaryotic, blue-green algae and cyanobacteria (Glazer, 1981). The pro-

teins are found as aggregates in phycobilisome particles near the chlorophyll regions (Kronick,

1986). In the native state, phycobiliproteins do not ﬂ uoresce; rather excitation energy is

designed to be efﬁ ciently transferred to chlorophyll molecules for utilization in synthetic proc-

esses within the cell. Once puriﬁ ed, however, excitation energy is released from phycobilipro-

teins as strong luminosity. The ﬂ uorescent quantum efﬁ ciencies of these proteins can be as high

as 0.98, far better than most synthetic probes (Grabowski and Gantt, 1978). In addition, each

biliprotein contains multiple chromophoric bilin prosthetic groups, conferring extremely high

absorbance coefﬁ cients to each protein molecule. B-phycoerythrin, for example, typically con-

tains 34 chromophoric groups giving an effective, combined extinction coefﬁ cient at 545 nm of

2.4  10

 1

(Glazer and Hixson, 1977). The strong absorption bands are in the visible

region of the spectrum, extending from the green to the far red wavelengths. These absorption

spectra extend over a broad range of potential excitation wavelengths, allowing for versatility

in the excitation source employed and creating large Stoke ’s shifts, thus minimizing interference

from Rayleigh-scattered light (Loken et al., 1987).

Due to the presence of multiple ﬂ uorescent groups in each phycobiliprotein, conjugates of

these molecules form extraordinarily luminescent probes. Labeling of macromolecules with

phycobiliprotein derivatives can provide absorption coefﬁ cients 30-fold higher than labe-

ling with small, synthetic ﬂ uorophores. Their ability to be monitored by ﬂ uorescing in the

red region of the spectrum decreases potential interferences from indigenous biological ﬂ uo-

rescence. The protected bilin (tetra-pyrrole) prosthetic groups are not easily affected by their

external environment. They are not readily quenched by conjugation to another molecule or

affected by other components in solution. The prosthetic group orientation within the protein

molecules enables ﬂ uorescence to take place independent of pH or ionic strength. The excel-

lent solubility of phycobiliproteins in aqueous solution allows easy chemical manipulation for

modiﬁ cation or conjugation reactions, and their hydrophilic nature provides low nonspeciﬁ c

binding character in ﬂ uorescent detection applications.

There are three main classes of phycobiliproteins, differing in their protein structure,

bilin content, and ﬂ uorescent properties. These are phycoerythrin, phycocyanin, and allo-

phycocyanin (APC). There are two main forms of phycoerythrin proteins commonly in use:

B-phycoerythrin isolated from Porphyridium cruentum and R-phycoerythrin from

Gastroclonium coulteri. There also are three main forms of pigments found in these proteins:

phycoerythrobilin, phycourobilin, and phycocyanobilin (Glazer, 1985). The relative content

of these pigments in the phycobiliproteins determines their spectral properties. All of them,

7. Phycobiliprotein Derivatives 461

462 9. Fluorescent Probes

however, have extremely high absorption coefﬁ cients ranging from a magnitude of 10

–10

and excellent QY ranging from 0.51 up to 0.98.

The spectral properties of four major phycobiliproteins used as ﬂ uorescent labels can be

found in Tables 9.1 and 9.2 . The bilin content of these proteins ranges from a low of four

prosthetic groups in C-phycocyanin to the 34 groups of B- and R-phycoerythrin. Phycoerythrin

derivatives, therefore, can be used to create the most intensely ﬂ uorescent probes possible

using these proteins. The ﬂ uorescent yield of the most luminescent phycobiliprotein molecule

is equivalent to about 30 ﬂ uoresceins or 100 rhodamine molecules. Streptavidin–phycoerythrin

conjugates, for example, have been used to detect as little as 100 biotinylated antibodies bound

to receptor proteins per cell (Zola et al ., 1990).

Conjugation of phycobiliproteins to targeting components such as antibodies, avidin, biotin,

or other molecules preserves the binding or activity of the attached constituent and does not alter

the spectral characteristics of the bilin prosthetic groups. Common heterobifunctional crosslink-

ing agents can be used to create phycobiliprotein conjugates, including SPDP (Chapter 5,

Section 1.1), SMCC (Chapter 5, Section 1.3), and SMPB (Chapter 5, Section 1.6) (Oi et al.,

1982). These crosslinkers react with amine groups on the phycobiliproteins, producing activated

intermediates able to couple with sulfhydryl-containing molecules. Thiolating reagents such as

2-iminothiolane, N-Succinimidyl-S-acetylthioacetate (SATA), and SAMSA (Chapter 1, Section 4.1)

can be used to create thiols on the secondary molecule to effect the ﬁ nal coupling reaction.

Glazer and Stryer (1983) report on the preparation and use of tandem phycobiliprotein con-

jugates wherein B-phycoerythrin is crosslinked to APC to create an energy donor–acceptor pair.

The B-phycoerythrin component can be excited at 545 nm, emitting energy at 575 nm that can

be accepted by APC, which in turn emits light at 660 nm. The result is a large shift in the spec-

tral characteristics from that of the individual proteins, increasing the effective Stoke ’s shift to

Table 9.1 Properties of the Phycobiliproteins

Property B-phycoerythrin R-phycoerythrin C-phycocyanin Allophycocyanin

Source Porphyridium

cruentum

Gastroclonium

coulteri

Anabaena variabilis Anabaena variabilis

Subunit structure (  )

 (  )

(  )

Molecular weight 240,000 240,000 72,000 110,000

Pigment content

(bilin groups)

34 34 4 6

Absorbance

maximum

546 nm 566 nm 614 nm 650 nm

Molar extinction

coefﬁ cient

2.4  10

2.0  10

5.8  10

7.0  10

Emission maximum 575 nm 574 nm 643 nm 660 nm

Table 9.2 Spectral Properties of Phycobiliproteins

Phycobiliprotein

Molecular

weight

Absorption

maximum (nm) EC (cm

 1

)

Emission

maximum (nm) Fluorescence QY

B-phycoerythrin 240,000 546, 565 2,410,000 575 0.98

R-phycoerythrin 240,000 496, 546, 565 1,960,000 578 0.82

Allophycocyanin 104,000 650 700,000 660 0.68

EC  extinction coefﬁ cient; QY  quantum yield

over 100 nm. Conjugation of such tandem pairs to other proteins can create superior ﬂ uores-

cent reagents.

Phycobiliproteins also can be modiﬁ ed with organic ﬂ uorescent probes to produce tandem

dyes having modulated emission properties. In this sense, dyes having excitation wavelengths

that overlap with the emission wavelength of a phycobiliprotein can be used to extend the

emission of the tandem complex farther into the red region. For instance, a Cy5-type dye (see

Section 8, this chapter) can be used to modify R-phycoerythrin (RPE) to produce a conjugate

that can be excited at the normal wavelengths used for RPE, but emit light at the Cy5 range of

660–665 nm instead of the typical 575 nm emission for the phycobiliprotein itself. Fluorescence

resonance energy transfer (FRET) of excitation energy from the bilin units in RPE to the Cy5

dyes modifying the protein results in the red-shifted emission characteristics of the tandem

dye construct. In an optimized tandem dye conjugate, the ﬂ uorescence emission of the phyco-

biliprotein is almost entirely eliminated by the FRET signaling to the organic dye.

Preparation of a series of phycobiliprotein tandem dyes allows multiplexed analysis of differ-

ent targets in a sample. In addition, since RPE can be excited by the argon-ion laser at 488 nm,

a ﬂ uorescein-labeled probe can be used concurrently with RPE alone and RPE-tandem conju-

gates to create a multiplexed system of different ﬂ uorescent probes that can be used simultane-

ously. Table 9.3 shows the different combinations of dyes that can be used in this type of assay

with RPE and APC.

The following protocol is a generalized method for creating sulfhydryl-reactive phyco-

biliprotein reagents for coupling to  SH containing molecules. The procedure uses the heter-

obifunctional crosslinker, SPDP (Chapter 5, Section 1.1). Other amine- and sulfhydryl-reactive

crosslinking agents may be used in a similar manner.

Protocol

1. Dialyze the phycobiliprotein into 50 mM sodium borate, 0.3 M NaCl, pH 8.5 ( Note :

Commercial preparations of these proteins come as an ammonium sulfate suspension).

Table 9.3 Tandem Dye Fluorescence Properties

Excitation (nm)

Emission (nm)

488

Argon-ion laser (488)

568

Krypton-argon laser (568)

647/650

Krypton-argon (647) or

red diode laser (650)

519 DyLight 488 or

Alexa 488

575 R-phycoerythrin (RPE) R-phycoerythrin (RPE)

590 Lissamine rhodamine

615–630 RPE-Texas Red,

RPE-Alexa 610, or

RPE-DyLight 594

RPE-Texas Red, or

RPE-DyLight 594

660–665 RPE-Alexa 647 or

RPE-DyLight 649

RPE-DyLight 649 DyLight 649,

Allophycocyanin (APC)

702–709 RPE-Alexa-680 or

RPE-DyLight 680

RPE-Alexa 680 or

RPE-DyLight 680

APC-Alexa 680 or

APC-DyLight 680

719–735 RPE-Alexa 700 RPE-Alexa 700 APC-Alexa 700

770–780 RPE-Alexa 750 or

RPE-DyLight 750

RPE-Alexa 750 or

RPE-DyLight 750

APC-Alexa 750 or

APC-DyLight 750

7. Phycobiliprotein Derivatives 463

464 9. Fluorescent Probes

After dialysis, adjust the protein solution to a concentration of 1 mg/ml. Higher protein

concentrations may be used, but the amount of crosslinking reagent added to each ml

of the reaction should be proportionally scaled up, as well. Protect the protein solution

from undue exposure to light.

2. Dissolve SPDP at a concentration of 6.2 mg/ml in DMSO (makes a 20 mM stock solu-

tion). Alternatively, LC-SPDP may be used and dissolved at a concentration of 8.5 mg/ml

in DMSO (also makes a 20 mM solution). If the water soluble sulfo-LC-SPDP is used, a

stock solution in water may be prepared just prior to adding an aliquot to the reaction. In

this case, prepare a 10 mM solution of sulfo-LC-SPDP by dissolving 5.2 mg/ml in water.

Since an aqueous solution of the crosslinker will degrade by hydrolysis of the sulfo-NHS

ester, it should be used quickly to prevent signiﬁ cant loss of activity. If a sufﬁ ciently large

amount of phycobiliprotein will be modiﬁ ed, the solid may be added directly to the reac-

tion mixture without preparing a stock solution in water to allow accurate weighing of

sulfo-LC-SPDP.

3. Add 25 l of the stock solution of either SPDP or LC-SPDP in DMSO to each ml of the

protein solution. If sulfo-LC-SPDP is used, add 50 l of the stock solution in water to

each ml of protein solution.

4. Mix and react for at least 30 minutes at room temperature. Longer reaction times, even

overnight, will not adversely affect the modiﬁ cation.

5. Purify the modiﬁ ed protein from reaction by-products by dialysis using a membrane with

a low molecular weight cutoff or gel ﬁ ltration using desalting resin and a buffer consist-

ing of 50 mM sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2.

The SPDP-activated phycobiliprotein may be reacted with a sulfhydryl-containing protein to

create a ﬂ uorescent conjugate linked through disulﬁ de bonds.

8. Cyanine Dye Derivatives

One of the most popular ﬂ uorescent dye types for labeling biomolecules is built from two cati-

onic nitrogenous ring structures linked by a polymethine bridge. One of the rings must have a

quaternized nitrogen atom possessing a positive charge. The ring structure types can be highly

varied, from 5- or 6-membered heterocycles to multiple fused ring systems. The polymethine

bridge to a great extent determines the ﬂ uorescence character of the dye as well as contributing

to the naming convention that distinguishes each dye within a family of similar structures. Thus,

the general structure of all cyanine dyes can be represented by

XCHCH CHY()tt

where X and Y are the nitrogenous rings at both ends of the polymethine bridge and n can

vary from 0 to 3. The name of a cyanine dye often has a number following it, which reﬂ ects the

number of carbon atoms in the polymethine chain. Thus, if n  1, the dye is called a tricyanine

or Cy3, if n  2 it is a pentacyanine or Cy5, and if n  3 it is a heptacyanine or Cy7.

The longer the polymethine bridge in a cyanine dye, the higher the absorbance and emis-

sion wavelengths become. In general, for each incremental increase of n, the absorbance and

emission characteristics of the dye increase by about 100 nm. Thus, Cy3 dyes typically display

excitation and ﬂ uorescence in the mid-500 nm range, Cy5 has spectral properties in the mid-

600 nm range, and Cy7 in the mid-700 nm range.

Though the bridge structure affects signiﬁ cantly the spectral character of a cyanine dye,

the heterocyclic rings also can contribute heavily to its properties. For instance, the structure

of the ring systems can highly affect the absorptivity and brightness of a particular cyanine

dye. Some heterocycle structures, such as the commonly used indol rings of many commer-

cial cyanine compounds, have extremely high extinction coefﬁ cients and thus provide intensely

bright ﬂ uorescent labels. Other constituents on the heterocyclic rings can provide a blue shift

or a red shift in the spectral characteristics of a dye. This is the basis for creating interme-

diate Cy dyes having ﬂ uorescent properties between the standard ones. For instance, Cy5.5

falls between the emission wavelengths of Cy5 and Cy7, completely due to an alternative fused

ring structure (e.g., benzo-indolium groups, see Figure 9.44 ). The relative ﬂ uorescence effects

of different ring systems and numerous possible substitutions on these rings often can be pre-

dicted from decades of investigation and data (Gruber, 2002). Thus, many commercial sup-

pliers of these dyes have ﬁ ne-tuned their properties to make them more suitable for certain

applications.

The nonreactive base structures of cyanine dyes (or carbocyanines) have been used for

many years as components in photographic emulsions to increase the range and sensitivity

of ﬁ lm and also in CD-R and DVD-R optical disks to record digital information. A major

innovation came when Ernst et al. (1989) and Waggoner et al. (1993) recognized that cyanine

dyes would make excellent labels for ﬂ uorescence detection, and for this reason, they synthe-

sized reactive dye derivatives, which then could be covalently attached to proteins and other

molecules.

The nitrogenous ring structures at each end of a cyanine dye can be the same (symmetri-

cal structure) or different (unsymmetrical), depending on the heterocycle type and constituents

on the rings. Reactive cyanine dyes nearly always contain unsymmetrical structures due to the

presence of a reactive arm on one end to facilitate conjugation to biomolecules or the presence

of one or more negatively charged groups, which are designed to increase water solubility.

The earliest cyanine dye labels were relatively hydrophobic due to the lack of hydrophilic

groups or having only a minimal number of charged groups on the molecules. These dyes,

while intensely ﬂ uorescent, often cause aggregation or precipitation of labeled proteins, espe-

cially if more than just a few ﬂ uorescent molecules are attached to a single biomolecule.

Dye–dye interactions due to ring stacking or hydrophobic interactions also cause ﬂ uorescence

quenching, because energy transfer can take place between dye molecules, negating the emis-

sion of light.

To make cyanine dyes more biocompatible for protein labeling, sulfonate groups typically

are added to the ring systems or at the ends of short spacer arms protruding from the base

dye structure. The addition of sulfonate groups provides a negative charge character that helps

solubilize the cyanine dye in aqueous solution and prevents dye–dye interactions through like

charge repulsion. In general, the more sulfonates that a cyanine dye possesses the greater will be

its hydrophilicity and the lower its tendency to bind nonspeciﬁ cally to hydrophobic structures

on proteins or other molecules. Cyanine dyes of this type having from two to four sulfonate

groups are available commercially, with three or four sulfonates per dye giving the best results

for bioconjugation purposes in aqueous solution.

8. Cyanine Dye Derivatives 465

466 9. Fluorescent Probes

Figure 9.44

The common nitrogenous ring structures of cyanine-type dyes. Indolium-based dye derivatives are

the most frequently used due to the high extinction coefﬁ cient of the indol groups.

Cyanine dyes also are used as labels for oligonucleotide probes. Unlike the hydrophilic cya-

nine dyes valuable for protein labeling, the use of dye-phosphoramidite compounds to syn-

thesize DNA or RNA probes typically requires the use of more hydrophobic dye structures to

make them compatible with the solvents and reactions of oligonucleotide synthesis. Thus, indol

cyanines containing few or no sulfonates are used in these applications to label oligos for appli-

cations such as array detection, hybridization assays, and RT-PCR.

In addition, small unsymmetrical cyanine compounds have been designed that bind to the

minor groove of DNA to measure DNA concentration or stain DNA by ﬂ uorescence. These

derivatives often contain a positive charge character with short crescent shapes to wrap around

the helical structure of double-stranded oligonucleotides. As minor groove binding dyes inter-

act with DNA, they typically undergo a slight twist in their molecular structure, which dramat-

ically increases ﬂ uorescence QY and brightness. For example, the minor groove binding dye

BOXTO displays a 300-fold increase in ﬂ uorescence when binding to double-stranded DNA

(Karlsson et al. , 2003).

Most companies selling cyanine dyes do not reveal their exact structures. This likely is due

to each company keeping proprietary the small synthetic tweaks that create unique ﬂ uores-

cence properties for their dyes. However, some structures are available through published

documents, such as patents and early publications (Leung et al., 2005). Figure 9.45 illustrates

some of these structures, which may not reﬂ ect precisely what any one company actually offers

today, but it gives an idea of the types of modiﬁ cations that can be done to add water solubility

and reactivity.

Cyanine dyes are commercially available in a number of different structural conﬁ gurations

containing appropriate reactive groups to couple with many of the major functional groups

(Thermo Fisher, GE Healthcare, Invitrogen, Sigma, Dyomics, Atto-Tec, and Denovo Biolabels).

Most of these dyes contain sulfonate groups to increase their biocompatibility, but careful com-

parisons should be made, as most companies do not provide structures. The following sections

describe some of the reactive cyanine dye reagents and the protocols that are used to label pro-

teins and other molecules.

Amine-reactive Cyanine Dyes

Amine-reactive cyanine dyes typically contain an NHS ester group on the end of a short hydro-

carbon spacer for attachment to biomolecules. The NHS ester can react with amine groups on

proteins to form amide bond linkages ( Figure 9.46 ). This reaction is efﬁ cient at physiological

pH or under slightly more alkaline conditions. While poly-sulfonated dye molecules are very

water-soluble, it is best ﬁ rst to prepare a stock solution in organic solvent to prevent NHS ester

hydrolysis prior to adding a small aliquot to a reaction mixture.

The following protocol for protein labeling with an NHS ester-cyanine dye is based on the

Thermo Fisher instructions for use of DyLight dyes. When preparing a ﬂ uorescently labeled

protein the degree of modiﬁ cation should be optimized to provide maximal ﬂ uorescence signal

while not affecting the activity or binding potential of the protein for other molecules. In fact,

dye loadings of greater than about 8 ﬂ uorescent labels per protein will result in ﬂ uorescence

quenching due to energy transfer between dye molecules. Higher loadings will not result in

greater ﬂ uorescence signals, only greater potential to interfere with protein activity. Therefore,

limit the molar excess of dye over protein to a level that will provide less than about 8 substitu-

tions per protein molecule.

8. Cyanine Dye Derivatives 467

468 9. Fluorescent Probes

Figure 9.45 Typical Cy5 derivatives contain negatively charged sulfonate groups for water solubility and a side

chain terminating in a reactive group for covalent coupling. The most common reactive groups are an NHS ester

for labeling amines, a maleimide group for coupling to thiols, and a hydrazide group for labeling aldehydes.

Protocol

1. Dissolve a protein or antibody to be labeled with an NHS ester-cyanine dye in 0.1 M

sodium phosphate buffer, pH 7.2–7.5, at a concentration of 1–10 mg/ml. The higher

the concentration of protein, the greater will be the yield of the labeling reaction. Other

buffers also may be used, including borate buffer or carbonate buffer, but higher pH

reactions will result in greater hydrolysis of the NHS ester and may decrease labeling

Figure 9.46 An NHS ester-containing cyanine dye can be used to label amine-containing proteins or other mol-

ecules to form amide bonds.

8. Cyanine Dye Derivatives 469