Analytical Currents: Simple approach to IC50 - Analytical Chemistry

Analytical Currents: Simple approach to IC50. Anal. Chem. , 2004, 76 (1), pp 10 A–10 A. DOI: 10.1021/ac0414907. Publication Date (Web): January 1, 2...
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ANALYTICAL CURRENTS A bright idea for antigen detection Geoffrey Waldo, Andrew Bradbury, and colleagues at the Los Alamos National Laboratory, Baylor College of Medicine, and Lewis and Clark College have generated libraries of molecules that combine the specificity and affinity of antibodies with the real-time fluorescence of green fluorescent protein (GFP). The new molecules, called fluorobodies, exhibit many of the same properties as antibodies without the need for detection with labeled secondary antibodies. Previous attempts by other researchers to insert sequences into GFPs yielded molecules with severely compromised fluorescence. Thus, Waldo, Bradbury, and colleagues made insertions into a highly stable mutant form of GFP, reasoning that a more stable starting molecule would tolerate insertions better than the normal form. To get GFPs that would functionally mimic antibodies, the researchers placed looped antigen-binding

regions of antibodies into the loops of GFP molecules. Fluorobodies were selected by phage display, wherein one fluorobody is attached to a phage that infects bacteria. Sixtyfive percent of the bacterial clones were fluorescent, which indicates that the fluorobodies within the clones retained this property of GFP. The researchers screened the new molecules against five different antigens and found that ~20–80% of the fluorobodies tested specifically bound a particular antigen. Green bacterial colonies contain functional fluoroThose fluorobodies that were bodies. (Adapted with permission. Copyright 2003 fluorescent and specific were com- Nature Publishing Group.) pared to antibodies in a number of the researchers conclude that the fluotests, including flow cytometry, gel shift robodies function in a way similar to assays, protein arrays, and immunofluoantibodies. (Nat. Biotechnol. 2003, 21, rescent staining of cells. On the basis of 1473–1479) the data generated by these experiments,

An explosives detector with a bang J. D. Adams, T. Thundat, and co-workers at

TNT molecules easily stick to the un-

TNT

the Oak Ridge National Laboratory, the Uni-

coated cantilevers of the new device.

versity of Tennessee, the U.S. Department

As the molecules slowly desorb, the re-

of Homeland Security, and the University of

searchers apply a voltage to a piezoresis-

Nevada have developed a trinitrotoluene

tor in the cantilever, which increases its

(TNT) microsensor that is much smaller

temperature and causes a miniature explo-

than the bulky ion mobility spectrometers

sion. The cantilevers are robust and func-

(IMSs) currently used in airports. Although

tion normally after hundreds of explosions.

the sensor is still not optimized, it has a 11

Piezoresistive heater

The researchers also tested other sub-

100 M

TNT microsensor. (Adapted with permission. Copyright 2003 Nature Publishing Group.) was applied. The authors say that they have

detection resolution of 70 pg (1.9  10

stances that they thought might interfere

detected additional explosives with the de-

molecules) TNT, which researchers say is

with detection of TNT, such as water, ace-

vice that detonate at different deflagration

comparable to that of the most recently

tone, ethanol, and gasoline, but all of these

points and desorption times. (Nature 2003,

reported IMS.

desorbed before the deflagration voltage

425, 474)

© 2004 AMERICAN CHEMICAL SOCIETY

J A N U A R Y 1 , 2 0 0 4 / A N A LY T I C A L C H E M I S T R Y

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ANALYTICAL CURRENTS

Water-soluble Hg(II) sensor The sensor re-

of the Massachusetts Institute of Technolo-

sponse is not affected

gy have developed a fluorescent sensor

by the presence of

that is capable of detecting environmentally

most metal cations

relevant concentrations of Hg(II) in water.

found in the environ-

The sensor is highly selective for Hg(II),

ment, with the excep-

even in the presence of several other met-

tion of Cu(II). In addi-

als commonly found in the environment.

tion, the binding of

Unlike most other small-molecule Hg(II)

Hg(II) to the sensor

probes, it does not require the use of or-

molecule is reversible.

ganic solvents.

When the researchers

30 Fluorescence intensity

Stephen J. Lippard and Elizabeth M. Nolan

25 20 +Hg(II)

15 10 5 0 480

500

520 540 560 580 Wavelength (nm)

600

620

Fluorescence response of the mercuric ion sensor to the addition of Hg(II) in water at neutral pH and ionic strength of 100 mM. The concentration of the sensor molecule is 1 µM.

The new water-soluble sensor uses

added a heavy metal

fluorescein as the reporting group, and

ion chelator to a mix-

because Hg(II) ions have a high affinity for

ture containing the sen-

sulfur, a sulfur-containing moiety is incor-

sor molecule and Hg(II), they saw an imme-

set the standard for the maximum allowable

porated into the ligand framework. The un-

diate reduction in fluorescence to within

concentration of Hg(II) in drinking water at

bound sensor exhibits an emission maxi-

20% of the background level. Subsequent

2 ppb. When the researchers added the

mum at 524 nm and a low quantum yield of

addition of Hg(II) restored the fluorescence

new sensor to an aqueous solution contain-

0.04. When Hg(II) binds to the sensor, the

because of Hg(II) complexation.

ing 2 ppb Hg(II), they observed a fluores-

emission maximum red shifts to 528 nm and

Hg(II) is converted by bacteria into toxic

cence increase of about 11%, which is an

the quantum yield increases to 0.11. The re-

methylmercury in the marine environment,

indication that the sensor can detect Hg(II)

sulting enhancement in fluorescence inten-

where it bioaccumulates up the food chain,

at the low levels typically found in environ-

sity is due to the formation of a 1:1 complex

particularly affecting large edible fish. The

mental matrixes. (J. Am. Chem. Soc. 2003,

between Hg(II) and the sensor.

U.S. Environmental Protection Agency has

125, 14,270–14,271)

Photolabels to study Ras interactions Ras proteins—which are critical to cell growth and differentiation, the cell cycle, and apoptosis—need to be localized at the plasma membrane to work, and the posttranslational modifications that control this localization are still being studied. Now, Herbert Waldmann and his colleagues at the MaxPlanck-Institut für molekulare Physiologie and the Universität Dortmund (both in Germany) describe a “construction kit” for the modular assembly of “photoactivatable” Ras peptides to examine the protein–protein interac8 A

tions of this crucial signaling molecule. The new kit combines solid- and solution-phase syntheses to make peptides that incorporate benzophenone (BP) photolabels. BPs were chosen because they have previously been applied to protein detection, and their excitation wavelength is not harmful to proteins. The photolabel was placed within the farnesyl group because it is likely that this group is involved in membrane binding events. Different functional groups, such as biotin, can be incorporated at the N-terminal ends

A N A LY T I C A L C H E M I S T R Y / J A N U A R Y 1 , 2 0 0 4

of the peptides. In all, 24 peptides were synthesized. When two conjugates were attached to oncogenic N-RasG12V181 and the labeled proteins were inserted into cells, they retained their biological activity. In other experiments using photolabeled geranyl-BP-N-Ras and farnesyl-sensitive guanine nucleotide exchange factor, hSos1, the researchers looked at the role of the farnesyl group in Ras signaling, which is still being debated. (J. Am. Chem. Soc. 2003, 125, 12,749–12,758)

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Budding lipid membrane research Lipid bilayer membranes are a cell’s shape shifters, morphing to form buds and splitting into vesicles. Theoretical descriptions of this behavior have been difficult to validate, but Tobias Baumgart and Watt Webb at Cornell University and Samuel Hess at the National Institute of Child Health and Human Development have developed an optical method to test current theories. These analyses have led to experimental estimates of the boundary tension, which is thought to control membrane deformation. The researchers studied giant unilamellar vesicles formed from a mixture of the lipids sphingomyelin, cholesterol, and dioleoylphosphatidylcholine (DOPC). The lipids separate into two different liquid phases: Sphingomyelin and cholesterol exist in a liquid phase with shortrange order, while DOPC prefers a disordered liquid phase. The two phases were labeled with different dyes and stud-

ied with two-photon microscopy to relate the composition of the phases, or domains, to the mechanical properties of the vesicles, such as line and lateral tensions. The researchers demonstrated that line tension drives vesicles toward a “limit shape”—a large sphere with semicircular protrusions. When temperature is increased or osmotic pressure changes, a bud may form abruptly, which suggests that an energy barrier was overcome. They also observed long-range ordering, which appeared as locally parallel stripes and almost soccer ball-shaped hexagonal arrays of circular domains. When temperatures were increased to just below the point at which homogeneous membranes formed, circular domains began to undulate laterally and assume elliptical shapes. At even higher temperatures, a “stripe out” would occur—a phenomenon whereby numerous thin stripes with varying lengths

Bioactive aerogels for sensing Because aerogels are so porous, incorpo-

They propose that the outer protein

rating active proteins into them ought to

layer is damaged and unfolds dur-

yield biosensors that respond and regener-

ing aerogel processing. However,

ate much faster than sol–gel ones. Unfortu-

this sacrificial “skin” protects the

nately, proteins generally do not survive the

proteins underneath, which bind

harsh processing required to make aero-

NO in the same fashion as they

gels. Now, Debra Rolison and colleagues at

do in solution.

the U.S. Naval Research Laboratory intro-

Rolison and her colleagues

duce a method for preserving the activity of

also believe that this process may

cytochrome c during the formation of an

help shed some light on how life

aerogel and thereby produce a NO detector.

evolved. According to one prebiot-

The new aerogel technique uses small

ic evolutionary theory, single cells

(a)

(b)

(c)

(d)

A bilayer composed of two lipid phases (red and blue). (a) Separation of the phases at room temperature. (b) Formation of a bud. (c) Long-range ordering. (d) Stripes and an elliptical shape. (Adapted with permission. Copyright 2003 Nature Publishing Group.)

undulate rapidly, similar to the rippling instability of collapsing bubbles. (Nature 2003, 425, 821–824)

Adsorbed monolayer of cyt. c

Buffer-like internal environment

Au colloid Damaged (unfolded) layer of cyt. c

SiO2 aerogel environment ~102 nm

A suggested model of a gold particle with layers of active cytochrome c surrounded by a damaged outer layer.

amounts of colloidal gold that act as

began to form when precellular

nanoparticle seeds for the formation of self-

macromolecules were stabilized in

organized protein–protein superstructures

porous minerals in undersea thermal vents.

molecules is still possible. The researchers

that are captured during gelation of a silica

This, the researchers argue, is analogous

further suggest that an outer shell of sacri-

sol. When the nanoparticles are present,

to their bioaerogel, because in both cases,

ficial macromolecules may have isolated

>80% of the cytochrome c molecules remain

biomolecular structures are isolated in a

the interior units from damage in a prebiot-

viable after processing, the researchers say.

porous architecture, yet transport of small

ic system. (Nano Lett. 2003, 3, 1463–1467)

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ANALYTICAL CURRENTS hv2

Quantum dots illuminate DNA activities Itamar Willner and colleagues at the Hebrew University of Jerusalem (Israel) examine the modification of DNA with telomerase and polymerase using fluorescence resonance energy transfer (FRET) between CdSe–ZnS quantum dots and a dye. The quantum dots’ surfaces are modified and attached to a segment of DNA, which is then incubated with the DNA modification enzyme and a mixture of dATP, dCTP, dGTP, and Texas-red 14dUTP. During the progression of the telomerization or polymerization, the fluorescence emission shifts as the 560nm emission from the quantum dots decreases and the 610-nm emission of the dye increases (using 400-nm excitation). In the absence of the enzymes, the emis-

(a)

hv1'

sions don’t change. Thus Telomerase the researchers conclude that dUTP + dNTPs hv1 FRET between the quantum hv2 dots and the dye is responsible DNA M 1 3 for the shift in emission. Kleno  DNA hv1 w fra Telomeric DNA chains meagmen t dUTP sured with this technique and + dNT P s atomic force microscopy were (b) 1 nm high and 300 nm long, which corresponds to 1000 base hv1 units. Both telomerase and polymerase showed similar kinetics, Schematics of how fluorescence resonance energy reaching saturation after 60 min. transfer occurs between a quantum dot and dye The researchers suggest that incorporated into DNA during (a) telomerization and the technique may prove useful (b) polymerization. in the detection of malignant cence when and if hybridization and cells. It could also be applied to chipreplication proceed. (J. Am. Chem. Soc. based DNA sensors because it functions 2003, 125, 13,918–13,919) like a logic gate, only changing fluores-

Simple approach to IC50

P-pep

A remarkably simple NMR technique offers

mean that samples with numerous compo-

a rapid method for determining the inhibi-

nents present no problems. The protein

tion or activation of an enzyme—an impor-

concentration needed is determined by

tant factor in evaluating molecules as po-

the speed of the enzymatic reaction. sured as a function of the substrate. Alter-

19 strates with a –CF3 moiety and use F

natively, the speed of the reaction plotted

NMR to measure the binding constant

versus substrate concentration provides

(measured as KM) and the 50% mean inhi-

the same value. 19 A plot of the F signal of the starting

bition concentration (IC50) with protein con-

substrate or its product at various inhibitor

The approach is easily integrated into

concentrations against the inhibitor con-

high-throughput screening strategies.

centration yields the IC50. In the absence of

The new technique is called three

allosteric effects, a single point measure-

fluorine atoms for biochemical screening

ment yields an effective IC50. The authors

(3-FABS). By monitoring only the fluorine

report obtaining values in as little as 3 min

NMR spectrum, it offers a single, intense

(64 scans), which drops to a mere 12 s

peak to monitor; a chemical shift that is

when a cryoprobe is used. 3-FABS could also play a role in genomic

sensitive to minor structural changes; and

*

30 25 20

H89

15

IC50 = 0.72  0.05 M

10 5 0 0.1

centrations as low as a few nanomolars.

* 19F

[pep] in M

colleagues from Pharmacia (Italy) tag sub-

+ H89

pep

19 To determine KM, the F peak is mea-

tential drug leads. Claudio Dalvit and

+ 4 cpds + 4 cpds + H89

1

[H89] in M

10

Top: 19F NMR of substrate (pep) and the enzyme reaction product (P-pep) in the presence of four compounds and the inhibitor H89. The first and third spectra show product formation, which is halted whenever H89 is present. Bottom: Plot to determine IC50. test substrates can then be used to assign a

no spectral interferences. Even medium or

screening. A library of CF3-labeled substrates

protein or proteins of unknown function to

weak inhibitors can be detected, and clean

of known enzymes is created, and their rele-

a particular category, such as proteases.

spectra and low protein concentrations

vant chemical shifts are measured. These

(J. Am. Chem. Soc. 2003, 127, 14,620–14,625)

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