Alexa and Oregon Green dyes as fluorescence anisotropy probes for measuring protein–protein and protein–nucleic acid interactions

ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 308 (2002) 18–25 www.academicpress.com

Alexa and Oregon Green dyes as fluorescence anisotropy probes for measuring protein–protein and protein–nucleic acid interactions Elena Rusinova,a Vira Tretyachenko-Ladokhina,b,1 Oana E. Vele,a Donald F. Senear,b and J.B. Alexander Rossc,* b

a Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA c Department of Chemistry, The University of Montana, Missoula, MT 59812, USA

Received 27 November 2001

Abstract The fluorescence properties of Alexa 488, Oregon Green 488, and Oregon Green 514 (Molecular Probes (Eugene, OR)) are compared when conjugated to biomolecules and as model compounds free in solution. We show that these relatively new, green fluorescence probes are excellent probes for investigation of the thermodynamics of protein–protein and protein–nucleic acid interactions by fluorescence anisotropy. Unlike fluorescein, the emission of these dyes has minimal pH dependence near neutrality and is significantly less susceptible to photobleaching. Steady-state and time-resolved fluorescence anisotropy data are compared for two interacting proteins of different size and for the association of a transcription factor with a DNA oligonucleotide containing a specific binding site. The temperature dependence of the fluorescence lifetimes of the probes is reported, and the effects of molecular size and probe motion on steady-state anisotropy data are discussed. The critical interplay among correlation time, fluorescence lifetime, and the observed steady-state anisotropy is evaluated. Ó 2002 Elsevier Science (USA). All rights reserved.

Fluorescence anisotropy is widely used for measuring high-affinity protein–nucleic acid or protein–protein interactions (reviewed in [1,2]). The basis for the use of steady-state anisotropy to assay binding is that molecular rotations that occur during the lifetime of the excited state of a fluorophore will depolarize the fluorescence, thereby providing an observable that is sensitive to molecular size. To achieve the sensitivity necessary for determination of high-affinity equilibrium binding, covalently bound fluorescent dyes with large extinction coefficients and high quantum yields are used typically. The most commonly used probes, such as fluorescein and rhodamine, potentially can support observations at subnanomolar concentrations of reactants. A critical consideration, however, is the interplay between the fluorescence lifetime of the fluorescent probe *

Corresponding author. Fax: 406-243-6026. E-mail address: [email protected] (J.B. Alexander Ross). 1 Dr. Tretyachenko-Ladokhina holds a permanent appointment at the Institute of Biochemistry, National Academy of Sciences of Ukraine, Kiev, Ukraine.

and the differences in rotational correlation times of the macromolecule bearing the probe and the macromolecular complex that is the product of association. Since extinction and fluorescence lifetime tend to be inversely correlated, there is usually a tradeoff when choosing which dye is best to monitor a particular molecular association. This paper compares three, relatively new, green fluorescence probes: Alexa 488, Oregon Green 488, and Oregon Green 514 (Molecular Probes (Eugene, OR)). The Alexa and Oregon Green dyes are alternatives to fluorescein that offer additional useful properties. For example, these dyes feature high extinctions, comparable to fluorescein, but they are substantially less susceptible to photobleaching. Also unlike fluorescein, these dyes have minimal pH dependence near neutrality. To assess their utility as fluorescence probes to measure protein–protein and protein–DNA interactions, we have determined the limiting anisotropies of the Alexa and Oregon Green dyes, and we have considered the practical implications of the interplay between rotational correlation time and their fluorescence lifetimes. We

0003-2697/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 3 - 2 6 9 7 ( 0 2 ) 0 0 0 5 7 - X

E. Rusinova et al. / Analytical Biochemistry 308 (2002) 18–25

compare steady-state and time-resolved fluorescence anisotropy data for two interacting proteins of different size and for the association of a transcription factor with a DNA oligonucleotide containing a specific binding site. The effects of molecular size and probe motion on steady-state anisotropy data are discussed. Second, we report the temperature dependence of the fluorescence lifetimes of these probes. Changes in fluorescence lifetime can affect significantly the determination of enthalpy, entropy, and heat capacity changes from van’t Hoff analysis of anisotropy data as well as the interpretation of the thermodynamics. Our analysis of the fluorescence properties of these probes presents them as attractive and, in some cases, superior alternatives for the investigation of protein–protein and protein-nucleic acid interactions by fluorescence anisotropy.

Materials and methods Reagents. Alexa 488 maleimide, Oregon Green 488 iodoacetamide, and Oregon Green 514 carboxylic acid succinimidyl ester were obtained from Molecular Probes. The thioester tripeptide chloromethyl ketone, N a -[(acetylthio)acetyl]-D -Phe–Phe–Arg-CH2 Cl, was a gift from Dr. Paul Bock. All other chemicals for chemical modification were obtained from Sigma–Aldrich (St. Louis, MO) and used without further purification. Recombinant human Factor VIIa (VIIa)2 was purchased from Novo Nordisk (Denmark). Recombinant soluble human tissue factor (sTF) mutant sTF E84C and Escherichia coli (E. coli) cytidine repressor (CytR) were expressed and purified as described [3,4]. Genetic engineering of sTF E84C, which introduces a single free sulfhydryl group, is described elsewhere [5]. DNA oligonucleotides that were 50 -phosphorylated and unphosphorylated complementary strands were purchased from Oligo’s Etc. (Wilsonville, OR). Protein labeling. The active site of VIIa was covalently modified with the thioester tripeptide chloromethyl ketone according to a procedure described by Bock [6,7]. The free sulfhydryl group of the VIIa activesite label and the free Cys residue of sTF E84C were labeled with the thiol-specific fluorescence probes, Alexa 488 maleimide and Oregon Green 488 iodoacetamide, respectively, according to standard procedures described by Molecular Probes (www.probes.com). The efficiency of VIIa labeling varied between 40 and 60%, depending on the lot of VIIa. The labeling efficiency corresponded to the fraction of enzyme that was active, which was 2

Abbreviations used: bp, base-pair; VIIa, Factor VIIa; sTF, soluble tissue factor; CytR, cytidine repressor; Hepes, N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]; EDTA, ethylenediaminetetraacetic acid; Dansyl, 5-dimethylaminonaphthalene-1-sulfonyl, Mops, 4-morpholinepropanesulfonic acid.

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determined independently by a chromogenic assay that measures conversion of substrate Factor X to product Factor Xa [8]. The efficiency of sTF E84C labeling was 80–85%; the amount of disulfide-linked dimer present accounts for the unlabeled protein. The efficiency of labeling of both proteins was estimated from absorption spectra by making the assumption that the extinction coefficients of the probe at the lowest energy maxima are unaffected by conjugation. The lowest energy absorption maxima are near 492 nm, and the extinction coefficients are 68,000 and 72,000 cm1 M1 for Oregon Green 488 iodoacetamide and Alexa 488 maleimide, respectively (Molecular Probes). Mercaptoethanol adducts of these dyes were prepared in a 50 mM Hepes, pH 7.5, buffer containing 1 mM EDTA and 0.1 M NaCl. The absorption spectra of the adducts were found to be similar to those of the unreacted dyes. From the ratio of the lowest energy absorption maximum of a dye and its absorption at 280 nm, we determined that the extinction coefficients at 280 nm are about 20,200 and 11,600 cm1 M1 , respectively, for the mercaptoethanol adducts of Oregon Green 488 iodoacetamide and Alexa 488 maleimide. These values were used to account for the contribution of the dyes to the absorbance of the dye-conjugated proteins at 280 nm. A caution is that the lowest energy absorption band of the dyes is red-shifted 3–6 nm when conjugated with the proteins, but not with mercaptoethanol. This suggests that the high-energy absorption band might also be shifted, in which case the 280-nm extinctions of the protein-conjugated and mercaptoethanol-conjugated dyes will differ. A red or blue shift of 5 nm could change the 280 nm absorption by as much as 20%, causing a systematic error to the estimation of labeling efficiency. The significance of this error obviously depends upon the relative magnitude of the protein extinction coefficient. DNA labeling. The commercially available C6 or C12 linkers, generally recommended for labeling the 50 end of DNA with fluorescence probes, result in 8 or 14 atoms beyond the phosphorus atom, connected by single bonds. To reduce segmental motion of the dye, we generated a shorter linker between the fluorescent dye and the DNA by derivatization of the 50 -phosphate group with ethylenediamine using a carbodiimide-mediated reaction [9] to form a phosphoramide. This chemistry results in only four atoms beyond the phosphorus atom and introduces a strongly nucleophilic primary amine. Subsequent labeling, using a standard protocol for DNA conjugation with amine reactive probes (Molecular Probes), used the succinimidyl ester form of Oregon Green 514 to form a carboxamide. Unreacted dye was removed by ethanol precipitation of the DNA followed by exhaustive exchange with water using Centricon-3 concentrators (Amicon). The efficiency of dye conjugation was calculated using the same approach described above for the protein conjugates and assuming that the extinctions of the

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probe and DNA are additive at 260 nm. The extinction coefficients at 260 nm for the oligonucleotides were from Oligos Etc. The extinction coefficient of Oregon Green 514 succimidyl ester at 506 nm is 85; 000 cm1 M1 at pH 9 (Molecular Probes). Reaction of the Oregon Green 514 succimidyl ester with hydroxylamine to form the hydroxylamine carboxamide adduct was found to have no effect on the lowest energy absorption maximum, which is at 506 nm in pH 7.5 buffer. Assuming there is also no effect on the extinction at 506 nm, the hydroxylamine adduct has an extinction coefficient of 25,000 cm1 M1 at 260 nm. This value was used to calculate the contribution of the probe to the absorption of the DNA conjugate at 260 nm. Similar to the Alexa– protein conjugate, the lowest energy band of the Oregon Green–DNA conjugate shifts to 513 nm. However, a red (or blue) shift of 10 nm in 260 nm absorption band alters the 260-nm extinction coefficient by less than 10%. Since the dye absorption at 260 nm is substantially smaller than that of the DNA (< 5%), the systematic error due to such a spectral shift is negligible. Labeling efficiencies of about 80% were achieved by increasing the concentration of Oregon Green succinimidyl ester in the conjugation reactions to 20 mM, a fivefold higher concentration than recommended by vendor protocol. Double-stranded oligonucleotides were prepared as described [4]. Fluorescence anisotropy measurements. The steadystate anisotropy (r) is given by r¼

IVV  G  IVH ; IVV þ G  2IVH

ð1Þ

where IVV and IVH are the intensities measured with vertically polarized excitation, as indicated by the first subscript, and detected through vertically or horizontally oriented emission polarizers, respectively, as indicated by the second subscript. The factor G ¼ IHV =IHH , which is measured using horizontally polarized excitation, corrects for instrument polarization bias. Steady-state fluorescence measurements were made using either an SLM 8000c spectrofluorometer equipped with a double-grating excitation monochromator and a single-grating emission monochromator or an SLM 4800 spectrofluorometer equipped with single-grating excitation and emission monochromators and modified for single-photon counting. Time-resolved fluorescence measurements of the intensity and anisotropy decays were made by the time-correlated single-photon counting method as described [10]. Conditions for the steadystate or time-resolved measurements are given in the figure and table legends. Theoretical background. The usefulness of fluorescence anisotropy as an observable for binding depends upon the magnitude of the signal change resulting from molecular association. As discussed by Lakowicz [11], the observed value of the steady-state anisotropy

depends on three factors. First is the mean correlation time (/) due to rotational motions. This depends on size and shape of the macromolecule (or assembly) and probe linkage. Second is the fluorescence lifetime (s) of the probe. This depends upon the photophysics of the dye and interactions with the local environment. Third is the limiting anisotropy (r0 ), which is determined by the angle between the probe’s absorption and emission transition dipoles. The value of r0 can be determined by measuring the steady-state anisotropy of a probe in a highly viscous medium, for example, in glycerol at low temperature. The relationship between these parameters can be illustrated by the simplest case, in which fluorescence lifetime and rotational correlation time are single exponentials r0 r¼ ; ð2Þ 1 þ s=/ Eq. (2) shows that the maximum steady-state anisotropy is limited by the absolute value of r0 , and that the anisotropy change resulting from a macromolecular association reaction is determined by the s// ratio. The value of r0 ranges from 0.4 to )0.2 depending upon whether the absorption and emission transition dipole moments tend toward parallel or toward perpendicular to one another, respectively. Fluorescence generally occurs from the lowest vibrational level of the first excited singlet state (Kasha’s rule [12]), and excitation at the red edge of the lowest energy absorption band typically yields maximum, positive r0 values. For a given fluorescence lifetime, the rate of molecular motion governs s=/. In the limit of slow molecular motion (/ is much longer than s), the s=/ ratio approaches zero and r approaches r0 . In the limit of fast molecular motions (s is much longer than /), the s // ratio becomes very large and r approaches zero. Proteins and nucleic acids are not spheres. Consequently their time-dependent anisotropy is considerably more complicated, and the anisotropy decay may be represented by a sum of exponentials X rðtÞ ¼ bj et=/j ; ð3Þ j

P

where bj ¼ r0 [13,14]. Often the fluorescence lifetime of probes attached to proteins or DNA also is complex and may be represented as a sum of exponentials. As discussed by Chen et al. [15], under the condition where the lifetimes, si , are smaller than the rotational correlation times, Eq. (2) is well approximated by r0 hri ¼ ; ð4Þ 1 þ hsi=h/i where fluorescence lifetime is described by the intensityweighted average P ai s 2 hsi ¼ Pi i ; ð5Þ i ai s i

E. Rusinova et al. / Analytical Biochemistry 308 (2002) 18–25

where ai being the amplitude of the ith component lifetime, and the average rotational correlation time is described by a weighted harmonic mean "P #1 j bj =/j P h/i ¼ : ð6Þ j bj Time-resolved fluorescence anisotropy data are usually fit as sums of exponentials, so that the global motions are recovered as a ‘‘long’’ correlation time, corresponding to a harmonic mean correlation time, and faster probe motions resulting from rotation around the covalent bonds linking them to the macromolecule are recovered as a ‘‘short’’ correlation time rðtÞ ¼ r0 ½pshort et=/short þ plong et=/long ;

ð7Þ

where pshort ¼ c, plong ¼ 1  c, and c is a scaling factor with a value between 0 and 1. In addition, 1 1 /1 short ¼ /segmental þ /global ;

1 /1 long ¼ /global :

ð8Þ

Consequently, if the global rotations are slow compared to local probe motions, they make an essentially insignificant contribution to the short correlation time. This description presumes that every component resolved in the fluorescence intensity decay is associated with all global and local motions. While other kinetic models are possible, these require a more sophisticated level of analysis [10]. Identifying the correct kinetic model can provide considerable structural information about the dynamics of a macromolecule. Although such a detailed analysis can be highly informative for understanding the various physical behaviors that contribute to changes in the observable, it is not essential for using steady-state fluorescence anisotropy to measure binding interactions. Changes in interactions between the probe and the macromolecules involved in the assembly can alter the probe’s local motions. This can contribute to the change in anisotropy that occurs upon complex formation. But these interactions also can affect the probe lifetime, hence the intensity. An intensity change will affect the observed steady-state anisotropy, rtotal , according to the addition law Ftotal rtotal ¼ Ffree rfree þ Fbound rbound ;

ð9Þ

where the anisotropies of the bound and free forms of the labeled molecule, rbound and rfree , are weighted, respectively, by the fluorescence intensities Fbound and Ffree , thus assuring that the individual anisotropies are molar quantities.

Results and discussion The fluorescent green dyes Alexa 488, Oregon Green 488, and Oregon Green 514 offer several useful fluorescence properties for measuring thermodynamics of

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protein–protein and protein–DNA assemblies. In addition to very high extinction and high quantum yield, these include exceptional photostability, and no dependence of the fluorescence on pH in the neutral range. These properties provide high sensitivity and a stable signal that result in precise measurements of bindingdependent anisotropy changes. Popular probes such as fluorescein or rhodamine also have high extinction and quantum yields and also have maximum r0 values close to 0.4 [16]. Such a high r0 maximizes the opportunity to observe a change in anisotropy resulting from association. To determine the maximum anisotropy change that could occur upon binding to Alexa- and Oregon Greenconjugated biomolecules, we measured the limiting anisotropy of the Alexa and Oregon Green probe adducts at )10 °C in 100% glycerol. The absorption peaks of the lowest energy absorption band and corresponding limiting anisotropies are summarized in Table 1. The limiting anisotropies of the three probes are close to 0.4 and constant throughout the absorption band (Fig. 1). Because the r0 values are constant throughout the absorption band, a wide bandpass can be used for excitation to maximize the sensitivity of the fluorescence detection. Matching the fluorescence lifetime of the probe to the rates of the molecular motions of the macromolecular complex is a critical factor in measuring binding by fluorescence anisotropy. The probe lifetime constrains the changes in probe motion that can be observed and, therefore, the size of macromolecular reactants that can be investigated. For example, probes such as fluorescein or rhodamine typically have fluorescence lifetimes between 3 and 5 ns [16]. For comparison, the harmonic mean correlation time is about 25 ns for a 40 base-pair (bp) DNA oligonucleotide. If the probe has a lifetime of 4 ns, is immobile, and r0 ¼ 0:4, the expected steady-state anisotropy would be about 0.35. In this case, the maximum change that can result, due to changes in global motion when a protein–DNA complex is formed, is about 0.05. This difference is sufficient to monitor the interaction, but provides little sensitivity to the size of the complex, hence to its stoichiometry. For oligonucleotides much larger than 40 bp, sensitivity to protein– DNA association is lost, regardless of the size of the complex. Table 1 Absorption maxima (kmax ) and the limiting anisotropies of probe adductsa Sample

kmax (nm)

r0 b

Alexa 488-mercaptoethanol Oregon Green 488-mercaptoethanol Oregon Green 514-hydroxylamine

492 492 506

0.376 0.383 0.375

a Absorption spectra were of samples in pH 7.5 Hepes 10 mM buffer, and 0.14 M NaCl. b Samples in 100% glycerol at )10 °C. The measurement precision is

0:003.

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E. Rusinova et al. / Analytical Biochemistry 308 (2002) 18–25

Fig. 1. Limiting fluorescence excitation anisotropies of Alexa and Oregon Green dyes overlaid on the corresponding absorption spectra. Principle polarization spectra were collected for each dye in 100% glycerol at )10 °C. The emission wavelength was fixed at 560 nm for all probes. The bandpasses on excitation and emission were 4 nm.

Experimental steady-state anisotropies for similar length DNA oligonucleotides, however, tend to be much lower, with values in the range between 0.05 and 0.15. This has been attributed to local segmental motion of the dye [1]. The effect of such local motion is to decrease even further the change in anisotropy that results from changes in global motion. The length and flexibility of the covalent linkage between the dye and macromolecule and the tendency of the dye to interact with the macromolecule are important considerations because these determine the constraints on the local motions. Clegg and collaborators [17], in a classical study of DNA helical geometry using F€ orster resonance energy transfer, noted that rhodamine, which has a net positive charge at neutral pH, tends to interact strongly with the negatively charged DNA, thus limiting local motion.

While this increases sensitivity due to changes in global motion, it can also lead to complications due to quenching of the dye. By contrast, fluorescein, which has a negative charge, tends not to interact with the DNA, presumably due to electrostatic repulsion. The negative charge, however, might result in interaction with basic DNA-binding proteins. The Alexa and Oregon Green dyes are uncharged near neutral pH, thereby minimizing the possibility of interaction with any of the macromolecular reactants. To examine the relationships among molecular size and shape, fluorescence lifetime, and steady-state anisotropy, we compared the time-resolved fluorescence properties of Alexa 488 conjugated to Factor VIIa (kabs max ¼ 497 nm), Oregon Green 488 conjugated to sTF (kabs max ¼ 498 nm), and Oregon Green 514 conjugated to a DNA oligonucleotide (kabs max ¼ 513 nm). The molecular weights of sTF and VIIa are about 25 and 50 kDa, respectively, and both proteins are asymmetric [13]. sTF has two equal-sized immunoglobulinlike domains that join forming approximately a kinked  prolate shape with molecular axes of about 30 80 A [18,19], or nearly a 3:1 aspect ratio; the mean correlation time for a prolate shape of these dimensions would be about 15 ns at a viscosity of 1 cP. VIIa is composed of a light chain and a heavy chain. The heavy chain forms an essentially spherical serine protease domain of about 30 kDa. The isolated heavy chain would have a correlation time of about 13 ns at 1 cP. Previous time-resolved fluorescence anisotropy measurements of a Dansyl probe in the active site of VIIa yielded a global correlation time of about 100 ns and a shorter correlation time of about 14 ns [13]. The latter is similar to what would be expected for separate motion of the protease domain, whereas the very long global correlation time indicates that the whole 50-kDa protein must be highly asymmetric. These time-resolved spectroscopy results are consistent with the structure of VIIa, which is known only from a crystal structure of its complex with sTF [20]. In the complex, the light chain of VIIa is in an extended conformation. The DNA oligonucleotide is a 32-bp oligonucleotide of about 20 kDa, and the hydrodynamic shape might be approximated as a cylinder  long with a 27 A  diameter, yielding an aspect ratio 109 A of about 4:1. The mean correlation time for a prolate shape of these dimensions at a viscosity of 1 cP would be about 19 ns. When CytR is bound, the molecular weight increases to 96 kDa. A sphere with a volume equivalent to the sum of the volumes of the protein and the DNA would have a correlation time of about 50 ns, suggesting that in the absence of segmental probe motion, a significant increase in steady-state anisotropy would be expected, as discussed earlier. Table 2 lists the probe lifetimes, steady-state anisotropies, and rotational correlation times of the protein and DNA conjugates and of complexes with their spe-

E. Rusinova et al. / Analytical Biochemistry 308 (2002) 18–25

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Table 2 Fluorescence anisotropy properties of probe conjugates Samples

a

Alexa 488-VIIa Alexa 488-VIIaa complex with sTF Oregon 488-sTFa Oregon Green 488-sTFa complex with VIIa Oregon Green 514–DNAb Oregon Green 514–DNAb complex with CytR

Probe lifetime (ns)c

Steady-state anisotropyd

Time-resolved anisotropye bglobal

/global (ns)

bsegmental

/segmental (ns)

r0

3.9 4.0 3.8 3.7

0.235 0.238 0.165 0.243

0.251 0.248 0.191 0.238

24.7 30.5 13.6 38.7

0.083 0.084 0.115 0.107

1.1 1.1 1.0 1.2

0.334 0.332 0.306 0.345

3.9 3.9

0.117 0.141

0.110 0.122

11.8 23.7

0.160 0.164

0.9 0.9

0.270 0.286

a

Samples were in pH 7.5 Hepes 10 mM buffer, 0.14 M NaCl, 5 mM CaCl2 at 20 °C. Samples were in pH 7.1 Mops 10 mMP buffer, P 0.2 M NaCl, 1 mM EDTA, at 20 °C. c The mean lifetime is defined as hsi ¼ ai s2i = ai si and has a precision of 0:1 ns or better. d The steady-state anisotropy measurements have a precision of ( 0:003) or better. e The global rotational correlation time is a harmonic mean that Pdepends upon overall P shape [14]. When there is segmental flexibility, time-resolved anisotropy decay data analyzed as sums of exponentials, rðtÞ ¼ bj expðt=/j Þ and bj ¼ r0 may show a second, shorter component (/short ) that corresponds to the harmonic mean of the correlation times for whole molecule ð/long Þ and segmental ð/segmental Þ rotation. Accordingly, 1 1 /1 segmental ¼ /short  /long . Confidence limits are not given for the time-resolved anisotropy results because the parameters are highly correlated. b

cific protein ligands. In our previous study of VIIa [13], described above, the association with sTF generated only a small (0.02) change in the steady-state anisotropy of the Dansyl probe. This is not due to changes in global motion because the probe lifetime of about 11 ns is insufficient to discriminate between the relatively much slower global motions of VIIa (100 ns) alone and its complex with sTF. Instead, the small change in steadystate anisotropy is due to the loss of the jointed motion between the heavy and the light chains of VIIa that occurs upon binding sTF. In the present study, the VIIa heavy chain is labeled with Alexa 488, which has a much shorter lifetime than Dansyl. As a result, the steadystate anisotropy exhibits no sensitivity to sTF binding. By contrast, when the green probe (Oregon Green 488) is used on sTF, a substantial difference in steadystate anisotropy occurs upon formation of the complex with VIIa. The global correlation time for sTF alone is close to that expected, as discussed above. From these data, Oregon Green 488 or Alexa 488 can be used interchangeably because the conjugates have essentially the same lifetime. The smaller size of sTF yields a global correlation time that is only about threefold longer than the probe lifetime, and therefore much better determined than the global correlation time when the sTFconjugate is bound to VIIa. The global correlation time obtained for the complex, whether the green probe is on VIIa or sTF, is statistically no different from that observed for Dansyl-labeled VIIa. The important consequence of short probe lifetime and the long correlation time of the complex is a very large increase (0.078) in steady-state anisotropy of Oregon Green 488-conjugated sTF when it binds VIIa. Similarly, the short probe lifetime and the relatively long global correlation time of the DNA complex with CytR makes Oregon Green 514 a satisfactory aniso-

tropy probe for measuring CytR binding. The increase in steady-state anisotropy is smaller for the protein– DNA complex then for the sTF-VIIa complex. This appears to be due to a larger contribution from segmental motion of the probe (bseg ). Together, these results show that the steady-state anisotropy cannot be used as an observable for formation of complexes if the labeled macromolecule has a mean rotational correlation time that is more than 10-fold longer than the probe lifetime because the probe lifetime limits further change in the s=/ ratio. In practice, even a fivefold difference begins to limit the utility of the probe. Thus, the s=/ ratio is of critical importance in the interpretation of fluorescence anisotropy data in thermodynamic studies of higher order assemblies [2]. It is noteworthy, however, that when the lifetime of the probe is too short to be very sensitive to a change in global rotation, a significant change in steady-state anisotropy still might result from a change in segmental motion. But this is system-specific behavior and not predictable a priori. To determine enthalpies and heat capacities of assembly processes, the temperature dependence of the quantum yield (q) becomes an important consideration. The Alexa and Oregon Green dyes are characterized by fluorescence quantum yields similar to that of fluorescein (see Handbook of Fluorescent Probes and Research Products, Molecular Probes). The dianion form of fluorescein has a quantum yield of about 90% [21]. The high quantum yields of these green probes indicate that they should have relatively small temperature dependence near ambient temperature since q¼

kr ; kr þ knr

ð10Þ

where knr and kr are the nonradiative and radiative rate constants, respectively, and kr is temperature indepen-

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E. Rusinova et al. / Analytical Biochemistry 308 (2002) 18–25

dent. To determine whether there is significant temperature dependence of the lifetime, we measured the intensity decays of the green probe adducts over the range from 5 to 40 °C. Temperature-independent single exponential decays were observed for Alexa 488 (3.9 ns), Oregon Green 488 (3.8 ns), and Oregon Green 514 (4.1 ns). In a study of the temperature dependence of CytR binding to Oregon Green 514-labeled oligonucleotides [4], we observed less than 5% decrease in the mean fluorescence lifetime of the Oregon Green 514 probe over the range from 10 to 35 °C. The essentially temperature-independent fluorescence lifetime is a particularly useful property if the probe is to be used to investigate the thermodynamics of an assembly reaction, because it means that only the correlation time will be affecting the s=/ ratio. If a change in intensity is observed as a function of temperature when these probes are linked either to a protein or nucleic acid, it would indicate a temperaturedependent interaction between the probe and the macromolecule to which it is conjugated. This could suggest a conformational change in the macromolecule that alters either dynamic or static quenching interactions with the probe, and these in turn are likely to be sensitive to formation of a macromolecular complex. A detailed understanding of the quenching is not essential for using fluorescence anisotropy as an observable for binding, although appropriate correction must be made to account for the intensity change (cf. Eq. (2)). However, the thermodynamics of any interactions with the probe could complicate the interpretation of a van’t Hoff analysis. As discussed elsewhere [11,22], fluorescence lifetime measurements can resolve dynamic or static quenching mechanisms and this might be useful for interpretation of the thermodynamic parameters in molecular terms.

Conclusions Fluorescence anisotropy is rapidly gaining popularity as a highly sensitive method for measuring high-affinity protein–nucleic acid and protein–protein association equilibria. The sensitivity and applicability of the method is dependent on the photophysical properties of the fluorescent dyes that can be conjugated to protein or nucleic acids. The Alexa and Oregon Green dyes were developed recently as alternatives to fluorescein that offer the additional advantages that they resist photobleaching and have no pH dependence to their fluorescence properties near neutral pH. In investigating the utility of Alexa 488, Oregon Green 488, and Oregon Green 514 for anisotropy measurements, we have demonstrated several additional properties that are advantageous for investigation of macromolecular association reactions. These dyes have high limiting anisotropy,

which maximizes the possibility of observing change due to change in global motion upon association. The limiting anisotropy is constant across the absorption peak, which allows use of a wide excitation bandpass to maximize fluorescence intensity, which enhances sensitivity. We have shown that the fluorescence lifetime is essentially temperature independent at biologically important temperatures, making these dyes ideal for determination of thermodynamic parameters by van’t Hoff analysis. Finally, the practical implications of the interplay between rotational correlation time and the fluorescence lifetimes of the Alexa and Oregon Green dyes have been demonstrated by using these dyes to investigate several protein-protein and protein–DNA interactions. The results apply generally to the use of these or other fluorescent dyes in investigations of macromolecular associations.

Acknowledgments The work was supported by NSF Grants DBI9724330 (W.R. Laws, PI; J.B.A. Ross, Co-PI) and MCB-9728186 (D.F. Senear, PI), and by NIH Grants HL29019 (J.B.A Ross, PI) and CA63317 (R. Osman, PI; J.B.A. Ross, Co-PI).

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