A covalent fluorescence probe based on excited-state proton transfer

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 193, No. 1, March, pp. 88-100, 1979

A Covalent Fluorescence Probe Based on Excited-State Proton Transfer1 WILLIAM Biology

R. LAWS,

GARY

H. POSNER,

Department and McCollum-Pratt The Johns Hopkins University, Received

August

AND

Institute, Baltimore,

2, 1978; revised

LUDWIG

BRAND2

and Chemistry Department, Maryland 21218

October

11, 1978

The la&one (I) of 2-hydroxy-1-naphthaleneacetic acid was developed as a reagent for novel, highly efficient, covalent attachment of an excited-state proton transfer fluorescence probe (i.e. 2-naphthol) to protein amino groups. The lactone (I) was shown to react with amines faster than it was hydrolyzed. Reaction of the la&one (I) with bovine serum albumin (BSA) was faster than its reaction with corresponding concentrations of small organic amines, which suggested that the la&one (I) iirst adsorbed to BSA and subsequently reacted covalently; data are presented which suggest that this covalent binding occurs at a unique, single site on BSA (i.e., affinity labeling). Equilibrium studies involving instantaneous and timedependent fluorescence changes were interpreted in terms of a 1:l 1actone:BSA labeling ratio at neutral pH. Steady-state fluorescence spectroscopy of the 1:l 1actone:BSA conjugate and of the conjugate of the lactone with glycine ethyl ester were recorded, compared and interpreted in terms of the microenvironment of the protein-bound fluorescence probe. Applications are suggested for use of this new and powerful procedure for study of the conformational changes and molecular interactions in other proteins.

Fluorescent probes, such as dansyl chloride (1, 2) and others, that can be covalently conjugated to macromolecules have proved useful in studies of conformational changes and molecular interactions. The utility of a fluorescent probe can be enhanced if the effect of environment on the emission can be understood in terms of a known excited-state process. It is profitable to design new probes based on excitedstate reactions such as exciplex and excimer formation, solvent relaxation, or excitedstate proton transfer. Excited-state proton transfer is a particularly well-understood process (3; Laws and Brand, J. Phys. Chem., in press). Naphthols have a lower pKa in the excited state and will lose a proton when excited at a rate dependent on the solvent, pH, temper-

ature, and the presence of proton acceptors. This process is diagrammed in Fig. 1, where A is naphthol, B is naphtholate, and the rate constants indicated can be obtained from either steady-state (3) or lifetime (4; Laws and Brand, J. Phys. Chem., in press) data. As this is a two-state, reversible system the decay of both species can be described as a double exponential (Eqs. 1 and 2), with the creation of B* in the excited state leading to a negative pre-exponential term in the decay of B*. [A*lt = (yle-th [p],

-e-t/”

+ e--f’T2)

The lifetimes, 71 and TV, will be the same for both naphthol and naphtholate and, along with the pre-exponential terms czl, CQ, and & are functions of the rate constants and [H+]. Naphthol can, however, decay as a single exponential under conditions where the reaction is either irreversible (pH 2 5) or not favored (pH < 1). Thus, based on these well-defined decay characteristics of

1 Supported by NIH Grant No. GM 11632. Contribution No. 9% from the McCollum-Pratt Institute. * L. B. was supported by NIH Career Development Award Grant No. GM 10245, W. R. L. was supported by NIH training Grant No. GM 57, and G. H. P. was supported by NIH Grant No. CA 12658. 0003-9861/79/030088-13$02.00/O Copyright Q 1979 by Academic Press, Inc. AU rights of reproduction in any form reserved.

= p(

+ (yfi-t/~z

88

EXCITED-STATE

km

PROTON TRANSFER

B* + H+ b ‘> B

+ H+

FIG. 1. The kinetic scheme for excited-state proton transfer where A is naphthol and B is naphtholate.

A and B, and the ability to extract the rate parameters, naphthols have great potential in studying biological systems. Additional experiments that can be done with proton transfer probes include the determination of the availability and the pK, of proton acceptors and donors (Laws and Brand, J. Phys. Chem., in press) as well as energy transfer and polarization measurements. The aim of the present paper is to describe a convenient procedure for covalently attaching naphthols to primary amines. The reactive intermediate used is the la&one of

removed by roto-evaporation. The resulting lactone (I) was then recrystallized several times from nhexane, once with activated charcoal, and was shown to move as a single spot on the tic system described above. (The tic systems were visualized by shining uv light onto commercial silica gel plates carrying PF,,, indicator.) The uncorrected melting point was 102-103°C compared to 103-104°C in the literature (6-9). Formation of I was confirmed by IR analysis: The lactone spectrum contained no OH stretch, and the carbonyl band had moved from 1708 cm-’ for HNAA to 1810 cm-* for the la&one. Both I and HNAA were found to be heat-, air-, and lightsensitive. Moisture was found to slowly convert I back to HNAA. Consequently the la&one (I) is stored in the dark under argon at -2O”C, with reactions carried out under argon whenever possible. DCC lactonization is a convenient and high-yield modern method which does not involve use of mineral acids to promote

FLUORESCENCE

PROBE

89

2-hydroxy-1-naphthaleneacetic acid. This fluorescent probe has been reacted with bovine serum albumin, and both steadystate and nanosecond decay fluorometry have been used to monitor the titration of a proton-accepting (perhaps carboxyl) group near the dye molecule. The adduct between the la&one intermediate and glycine ethyl ester was also prepared to provide a reference compound for studying the effect of the solvent environment on the fluorescence of a naphthol covalently attached to an amino group. EXPERIMENTAL The starting material, 2-hydroxyl-l-naphthaleneacetic acid (HNAA),” was purchased from Aldrich Chemical Co., Inc., Milwaukee, Wisconsin, and purified by repeated recrystallizations from 20% ethanol/H,O (v/v), once with activated charcoal (5). The white flakes were pure by tic using a benzene: dioxane:acetic acid, 90:25:4 (v:v:v) solvent system on silica gel. Naptho[Z,l-blfuran-2(w)-one (I) was prepared at room temperature by dissolving purified HNAA in ethyl acetate and adding an equal molar amount of dicyclohexylcarbodiimide (DCC, Aldrich). Although the reaction is quite rapid, the solution was allowed to mix for several hours. The precipitated urea was filtered off, and the ethyl acetate was

lactonization. The insoluble by-product of this reaction, dicyclohexyl urea, is easily and simply separated from the soluble la&one product by filtration. The lactone prepared this way can be purified by fractional recrystallization without the need for any chromatographic purification. The lactone (I) is relatively insoluble in water. When introduced into aqueous systems by means of an organic solvent, the maximum concentration that can be attained with less than 1% organic solvent is approximately 5 x 10e5 M. The lactone (I) is quite soluble in 3 Abbreviations used: HNAA, 2-hydroxy-l-naphthaleneacetic acid; I, naphtho[2,1-blfuran-2-(m)one; II, 1-(2-hydroxynaphthyl)-acetamidoacetic acid, ethyl ester; DCC, dicyclohexylcarbodiimide; tic, thinlayer chromatography; BSA, bovine serum albumin.

90

LAWS, POSNER, AND BRAND

chloroform, dioxane, or ethanol. In the latter solvent it was found to be slowly cleaved over a period of several hours. For this reason stock solutions were made using dioxane as the solvent and stored in the dark. No apparent degradation was observed over a 24-h period as judged by fluorescence intensity or spectral shifts, or by thin-layer chromatography. 1-(2-HydroxynaphthyB-acetamidoacetic acid, ethyl ester (II) was prepared as follows: 180 mg I, 700 mg glycine ethyl ester hydrochloride (Nutritional Biochemicals Corp., Cleveland, Ohio), and 25 ~1 triethylamine were reacted in 20 ml ethanol for 5 h at 24°C. The reaction mixture was then applied to a silica gel 60 (E. Merck, Darmstadt, Germany) column, 60 x 3.2 cm, and eluted with the toluene:ethanol:ammonium hydroxide (29%), 200:31:1 (v:v:v) solvent. Fractions (10 ml) were collected and checked by tic for content. Fractions 12-16 contained a small amount of unreacted I, while 30-60 showed a single spot on tic that moved between HNAA and I and was assumed to be the adduct. Excess glycine ethyl ester hydrochloride and any HNAA formed by hydrolysis of I were eluted when the column was washed with methanol. The fractions containing the product were pooled, taken down to dryness, and purified by repeated recrystallizations, once with activated charcoal, from hot water with a trace of ethanol to complete solubilization. Purity was checked on tic using the above two solvent systems. Melting point: 102.5-104°C. Anal. Calcd for C,,H,,NO,: C, 66.9; H, 5.9; N, 4.9. Found: C, 66.85; H, 5.84; N, 4.62. Crystalline bovine serum albumin (BSA), lot Y4013, was purchased from Schwartz/Mann, Orangeburg,

Rates of hydrolysis, rates of reaction, and binding studies were done in similar ways. Lactone (I) in a dioxane stock solution was added in lo-p1 aliquots to 3 ml of a BSA or buffer solution. Fluorescence intensities were then obtained as a function of time for the rate experiments, or within 5 s after mixing for the binding studies. Steady-state fluorescent measurements, including the rate and binding experiments, were done using a thermostated Perkin-Elmer MPF-4. Excitation spectra were corrected using a rhodamine quantum counter while emission spectra were corrected based on quinine bisulfate and 2-aminopyridine references. Absorption measurements were taken on a Cary 14 recording spectrophotometer or a Beckman DU. For fluorescence studies, optical densities at the exciting

New York, and used without further purification. BSA at 20 mg/ml in buffer was labeled covalently with the addition of an aliquot of I dissolved in dioxane, equal to -1% the volume of the protein solution. The reaction mixture was then covered with argon, sealed, and stirred in the dark room at room temperature for 2 h. To remove any remaining I and HNAA produced by hydrolysis, the sample was then applied to a fresh Sephadex G-25 medium column and eluted with 0.01 M phosphate buffer, pH 7.6, at room temperature. Both HNAA and I stick tightly to the Sephadex and are not completely eluted, even with extensive flushing. For this reason a new Sephadex column was used for each experiment. The mole ratio of covalently reacted la&one (I) was determined spectrophotometrically making use of the extinction coefficient determined for II ( l s50= 3.3 x 103 M-* cm-’ in 0.01 M NaOH). This method was found to be unreliable due to low optical densities, protein absorption, and scattered light. More reliable results were obtained with the use of radioactive lactone (I). Carbon-14 was introduced into the carboxyl position of HNAA by reacting -1.5 g l-dimethylaminomethyl-2-naphthol (III) (Aldrich) in 90% ethanol with a twofold excess of NaCN containing 50 PCi K14CN (ICN Pharmaceuticals, Inc., Irvine, Calif.) in a sealed tube at 180-200°C overnight (10). The reaction mixture was then evaporated to dryness. The resulting salt was washed with ethanol and dissolved in aqueous base, from which the radioactive HNAA was precipitated with HCl. Lactonization and purification was carried out as indicated above yielding I with a specific activity of 2520 dpmlpmol.

wavelength were kept below 0.1 when possible. Inner filter corrections did have to be applied at the higher BSA and la&one (I) concentrations in the binding studies. Organic solvents used for spectral or lifetime work were of spectroquality grade. All other solvents and reagents were of reagent grade. Fluorescence decay curves were obtained using a single-photon counting instrument which has been described elsewhere in great detail (11). Excitation was through a Baird-Atomic 330-nm interference filter. The emission was observed at right angles through a Bausch and Lomb 0.5-m monochromator with a bandpass of 6.6 nm, or 13.2 nm for the protein conjugate decay experiments. The larger bandpass was needed to increase the intensity since a double Polacoat

EXCITED-STATE

PROTON TRANSFER

polarizer oriented at 55” was used on the emission side to avoid errors introduced by nanosecond timedependent polarization changes (12). Methods of analysis and “goodness of fit” criteria for the decay have also been published (11, 13). RESULTS

As shown in curve D of Fig. 2, the lactone (I) has little fluorescence in water. It is fluorescent in dioxane (curve B) showing vibrational structure and similar characteristics to HNAA in dioxane (curve A), although it does emit more to the blue. For comparison HNAA in water is also given (curve C), showing not only that it is fluorescent in aqueous solution, but also is able to undergo excited-state proton transfer. The peak around 360 nm is associated with the protonated species, A* in Fig. 1, while the ionized form, B*, emits with a maximum at 430 nm. The lactone (I) is expected to react with various side groups on proteins and also to be hydrolyzed in aqueous solutions to reform HNAA. In order to determine the best conditions for conjugating I with proteins, the rates of hydrolysis were studied under various pH, temperature, and salt conditions. The pH effect on the hydrolysis is shown in Fig. 3A. Since I is essentially nonfluorescent in water, any increase in fluorescence will be directly related to the appearance of HNAA (Fig. 2). The fluorescence intensity of totally hydrolyzed lac-

WAVELENGTH

FLUORESCENCE

PROBE

91

tone (I) was obtained from identical samples, heated at -60°C for 30 min, and then read later at the temperature of interest. Thinlayer chromatography was used to show that HNAA was the product of the hydrolysis. Hydrolysis appears to be fairly slow in the neutral pH range, with approximately 20% being converted in 30 min at pH 8. At higher pH values, the hydrolysis is more rapid until in 10 mM bicarbonate, pH 10, hydrolysis was essentially complete at 20°C in 20 min. Figure 3B shows the temperature dependence of lactone hydrolysis, comparing the rates at pH 8.05. As expected, an increase in temperature causes I to hydrolyze much faster. Hydrolysis at 5°C is slowed down enough that in 10 mM bicarbonate at pH 10 only 60% was converted in 30 min. Although sodium chloride or sodium acetate (0.1 M) had no effect on the rate of hydrolysis, 0.1 M ammonium chloride caused a dramatic increase in intensity which is attributed to amide formation. To investigate the reactivity of I with additional amines and other possible reactive biological groups, a study was done to see if any of these groups could cause an increased rate of appearance of fluorescence of the ionized species over that of hydrolysis. As seen in Fig. 4, methylamine hydrochloride caused faster formation of the ionized form. Even with the increased rate of hydrolysis, samples at pH 10 in 10 mM bicarbonate reacted 5-10 times faster than

(NM)

FIG. 2. Corrected emission spectra of HNAA in dioxane (A), I in dioxane (B), HNAA in water, pH 6.5 (C), and I in water, pH 6.5 (D). Curves are normalized to equal optical density at the exciting wavelength of 325 nm.

92

LAWS,P(lSNER,ANDBRAND 2s

r

0

A

5

10

TIME

15

( MIN

20

25

30

1

FIG. 3A. Aqueous hydrolysis of -5 X lo+ M lactone as a function of pH. Hydrolysis was carried out at 20°C in 10 mM phosphate buffer, with excitation at 325 nm and emission at 360 nm. Percentage hydrolysis was determined as explained in the text.

r

60

70 60 50 40 30 20 10 0

TIME

FIG. 3B. Temperature

( MIN

1

dependence of la&one (I) hydrolysis. Conditions as in Fig. 3A, pH 8.05.

at pH 7.6 at 20°C. That amide formation was that per amino group lysine is the same as occurring rather than amine-facilitated hy- glycine. To insure that the hydroxyl group drolysis was shown by tic using the toluene: of tyrosine would not react, even though ethanol:ammonium hydroxide solvent sys- tyrosine itself reacted slower than the other tem. A variety of possible reactive groups amino acids, phenol was added to I and were then tested under similar conditions found not to increase the rate of appearance (ZO’C, pH 7.6, 10 mM phosphate, [I] = 5 of 420-nm intensity. Cysteine, however, reX lo-’ M, [reactant] = 5 X lop3 M) to see if acted even faster than lysine. This might they could also cause an increase at 420 nm. indicate that the sulphydryl group is also As shown in Table I, methylamine hydro- capable of reacting at neutral pH; howchloride was able to react four times faster ever, it is unlikely that binding of the probe than glycine or histidine, which were ar- occurs to a free -SH group for two reasons. First of all, mercaptoethanol did not react bitrarily assigned as the base reaction rates. Lysine had twice the base rate, indicating with I in either water or ethanol, and

EXCITED-STATE

PROTON TRANSFER

TIME

FLUORESCENCE

( MIN

93

PROBE

1

FIG. 4. Rate of appearance of fluorescence intensity of the ionized species as a function of methylamine hydrochloride concentration. A represents a lo-fold excess of amine to 1, B a RIO-fold excess, and C a ZOO-fold excess. Curve labeled BSA is the rate of increase due to -1.5 x 10e5 M bovine serum albumin. The reaction was carried out at 20°C in 10 mM phosphate buffer, pH 7.6, with excitation at 325 nm. Hydrolysis was assumed to occur independently of the amide formation and was subtracted out.

second, hydroxy thioesters, such as A, would be expected to lactonize spontaneously.

Thus amines seem to be the only group able to react with the active ester at neutral pH. Also shown in Fig. 4 is the apparent rate of reaction of I in the presence of bovine serum albumin (BSA). An interesting feature is that although the BSA concentration is less than one-third of the la&one concentration, the rate is between 10 and 100 times that found for the methylamine hydrochloride experiments. There are close to 60 amino residues per BSA molecule. Even if some of these are assumed to be unreactive, the reaction rate of lactone (I) is still faster with BSA than with free amines. Since the reaction conditions (pH, temperature) were the same for both BSA and CH,NH, (therefore all amino functional groups should be equally protonated) and since amines do react, but not-SH groups, at neutral pH, this is a valid study of the relative rate of BSA vs CH,NH, binding with

the probe. A possible explanation for the rapid reaction of la&one (I) with BSA is that the dye rapidly adsorbs to BSA noncovalently near a reactive amine, enhancing the encounter frequency of the reacting groups and thus enhancing amide formation. In order to obtain more direct evidence that there is an interaction between the lactone (I) and BSA, equilibrium binding studies were performed. Since la&one (I) is relatively nonfluorescent in water but does have appreciable quantum yield in an organic solvent (Fig. Z), its adsorption to BSA could

TABLE

I

RELATIVE RATESOF REACTIONOF 1" Reactant

Relative rate

Glycine CH,NH, Histidine Lysine Tyrosine Cysteine

1 4 1 2 0.5 2.5

’ Reaction conditions: 20°C; pH 7.6, 10 mM phosphate; [I] = 5 X lo+ M; [reactant] = 5 x 10e3 M; based on increase at 420 nm after 10 min; excitation, 325 nm.

94

LAWS, POSNER, AND BRAND

0

4

5 t

BSA

15 I

x

10'

24 M

FIG. 5. The increase in fluorescence intensity of the la&one (I) as a function of BSA concentration. Titration was done at 20°C in 10 mM phosphate buffer, pH 7.6, with the dye concentration of 2.5 x 10m5M and excitation at 335 nm.

be expected to provide enough of a hydrophobic environment to cause an increase in fluorescence intensity. When differing amounts of BSA were added to separate cuvettes containing a set concentration of la&one (I) and the time course for the increase in fluorescence enhancement was monitored, the increased intensity found on extrapolation to time zero (about a 5-s mixing time) was used to obtain Fig. 5. Thus it was assumed that hydrolysis and the covalent reaction were sufficiently slow such that the zero-time point represented fluorescence due only to the free and adsorbed la&one and that contributions from hydrolyzed lactone or covalent reaction products were negligible. An emission profile of the

complex was quickly taken and is similar to the emission of I in organic solvents (Fig. 2). An associated quenching of protein fluorescence can also be observed. A similar volume of dioxane has no effect on the protein fluorescence. This enhancement of fluorescence intensity was used to obtain the titration data for the Scatchard plot (14) shown in Fig. 6. Various amounts of la&one (I) were added to individual cuvettes containing a set concentration of BSA, and the enhancement was obtained by extrapolation (over a 5-s interval) to time zero. The data in Fig. 5 were used in a double-reciprocal plot to find the maximum increase possible for a given lactone concentration, FBound. The fluores-

FIG. 6. A Scatchard plot based on the increase of lactone (I) fluorescence in the presence of BSA. The binding parameters were determined by linear least squares.

EXCITED-STATE

PROTON

TRANSFER

cence of free lactone, Ffree, bound la&one, F Bnund,and the observed fluorescence, FOBS, in any mixture of lactone and BSA were used to calculate the fraction, X, of la&one bound, (FOBS - FfreeY(FBound - Ffree) = X. An association constant of 7500 M-’ with 0.93 binding sites was the average binding parameter found by linear least squares. Covalent conjugates were then prepared in 10 mM phosphate, pH 7.6, as described in the experimental section. Different molar excesses of la&one (I) to BSA were tried to see the effect on the number of moles covalently bound. As indicated in Table II, increased conjugation occurs with increase in probe concentration, but only to a small extent. Even with a lo-fold excess only a little more than 1 mol of dye attaches per mol of protein. Increased covalent binding can be achieved under more alkaline conditions. A lo-fold excess of dye at pH 9.5 (10 mM bicarbonate) causes three times as much label to bind. At the lower pH of 6.2 (10 mM phosphate), however, there is less dye bound than at pH 7.6. This indicates that the low amounts of labeling seen could be due to the unavailability of pH-dependent amino groups. Determination of the labeling ratio has always been difficult for spectroscopic probes since extinction coefficients must be assumed. Radioactive labeling is another sensitive method that can be used to determine moles bound. However, introduction of radioactive atoms into a fluorophore is often difficult. Fortunately HNAA was easily made radioactive as indicated in the experimental section. Consequently, the optical density method was shown to be TABLE COVALENT

PH 7.6 7.6 7.6 7.6 6.2 9.5

LABELING Molar excess added 10 r 2” 1 10 10

II RATIOS

OF I + BSA

Moles bound 1.5 1.2 0.97 0.45 0.4 3.5

” 2 + It -c t

0.3 0.3 0.25 0.2 0.2 0.3

FLUORESCENCE

PROBE

95

high in its estimation of moles bound. There will be no general rule as to how extinction coefficients change upon conjugation of a dye with a protein. For example, optical methods of determining moles of fluorescein isothiocyanate have been shown to be low when checked by radioactive labeling (15) while the extinction coefficient of dansyl has been assumed to be too large for several proteins (16). Stability of the BSA-la&one linkage was checked by preparing conjugates and comparing certain fluorescent characteristics that would change if the probe was released into solution (see below). Periodically after labeling, the fluorescence was found not to change. After 72 h, the preparation was then applied to a second column to see if the linkage had been broken and the probe had been adsorbed to the BSA. The column would then remove any free dye, changing not only the spectral data but also the labeling ratio. However, both the moles bound and fluorescence profiles remained the same, indicating that the conjugate appears to be stable at least for several days. The excitation and emission spectra of the BSA-I conjugate, with and without 0.2 M sodium acetate, are shown in Fig. 7B and can be contrasted to the spectra of the reference compound II under similar conditions (Fig. 7A). Although the emission bands for the naphthol species (360 nm) and the naphtholate species (430 nm) appear to be the same between II and the conjugate, there are several differences between the two. First, the conjugate has more relative emission due to the O- group. This could be caused by a change in the quantum yield of the naphthol group relative to that of the naphtholate, by a change in the excitedstate rate parameters, by excitation of ground-state naphtholate that has been ionized by groups on the protein, or by more efficient proton transfer to the solvent or to groups on the protein. A second difference is the small effect of acetate on the conjugate compared to the model compound (II). This behavior may indicate that the probe is somewhat “buried” and is unable to use the added proton acceptor efficiently. Therefore, water molecules might also be relatively inaccessible to the probe, and

96

LAWS, POSNER, AND BRAND

WAVELENGTH

(NM)

FIG. 7A. Corrected spectra of -2.5 x 10e5 M II in 10 mM phosphate buffer, pH 7.6 (-) and with 0.2 M sodium acetate added (- - -) at 20°C. Excitation was done at 325 nm, and emission was observed at 360 nm.

0

250

320 WAVELENGTH

380

440

500

(NM)

FIG. ‘7B. Corrected spectra of the BSA-I conjugate under the same conditions as Fig. 7A. BSA concentration -0.8 mg/ml with -0.75 dye/BSA.

proton transfer would be mainly to a side chain group of the protein. An alternative explanation of this effect could be that the overall excited-state rate parameters have been changed due to the conjugation and proton transfer is occurring as efficiently as possible. Added proton acceptors would then be expected not to have any effect, even though they might be involved in the reaction. A third difference can be seen in the excitation spectra, in that the conjugate is undergoing resonance energy transfer from tryptophan, as indicated by the differences in the 280-nm region. The decay kinetics of II were found to be

quite similar to that of 2-naphthol(4; Laws and Brand, J. Phys. Chem., in press) at neutral pH. Allowing for the proton-accepting capabilities of phosphate, the lifetimes given in Table III are quite close to those of 2-naphthol. The protonated species decays as a single exponential while the ionized species is a double exponential, with essentially equal and opposite amplitudes, and the lifetime at 360 nm is equal to the short decay time at 440 nm. There was no evidence of more complex kinetics as shown by HNAA (5). However, the decay profiles of the two species obtained with the BSA-I conjugate

EXCITED-STATE

PROTON TRANSFER TABLE

II II II II Conjugate Conjugate Conjugate Conjugate

97

PROBE

III

DECAY~ARAMETERSFOR

Compound

FLUORESCENCE

II AND BSA-I”

[Acetate](M) 0 0

0.12 0.12 0 0 0.12 0.12

360 440 360 440 360 440 360 440

3.15 2.9 1.4 1.15 0.55 0.60 0.60 0.65

n Conditions: 20°C; pH 7.6, 10 IIIM phosphate; subtracted from conjugate at 360 nm.

are quite complex, each being best described as triple exponential decays, as shown in Table III. Certain aspects of the decay behavior can be explained. Although there is a small amount of ground-state naphtholate, a portion of the emission at 440 nm is coming from an excited-state reaction, exemplified in the negative (Ye. The short 71 indicates that the reaction is extremely rapid. Also of note is that the two wavelengths, i.e., the two emitting species, are kinetically related by common 0.6- and 9-ns lifetimes. As seen in the steady state, acetate hardly affects the kinetic behavior of the conjugate (Table III). In contrast, ace-

1.0 -0.47 1.0 -0.47 0.40 -0.23 0.46 -0.25

10.2

0.53

10.2 3.35 9.2 3.35 9.4

0.53 0.40 0.50 0.37 0.49

[BSA] = -1 mg/ml; -1

8.9 16.4 8.8 16.5

0.20 0.27 0.17 0.26

dye/BSA; S 1Q, 1 = 1; blank

tate facilitates proton transfer with the model compound II (causing T, to be smaller) as expected kinetically and shown with Z-naphthol (Laws and Brand, J. P&s. Chem., in press). Steady-state intensities for the two emitting groups were also monitored as a function of pH, as shown in Fig. 8. An emission polarizer oriented at 55” was used to avoid errors due to time-dependent polarization changes. For comparison the model compound (II) was similarly done and has an identical titration pattern to that of 2naphthol(3). The conjugate, however, has a large change in the ionized intensity at a pH

FIG. 8. The change in relative intensity as a function of pH for -1 x lo+ M (II) (M) and the BSA-I conjugate (Cl) at two emission wavelengths. The pH was changed by additions of HCl. Initial conditions were 0.05 M phosphate buffer plus 0.01 M NaCl. Excitation at 330 nm, 20°C.

98

LAWS,

POSNER,

higher than that seen by steady-state anisotropy for the N @ F transition (17; Laws and Brand, to be submitted). This effect could be caused by one of two processes. First of all, the intensity change could be indicating the beginning of the isomerization earlier than that seen by polarization. Since it appears that the dye may be close to a proton-accepting group, perhaps the unfolding process is first indicated by a slight movement of the acceptor away from the probe causing decreased proton transfer. A second explanation would be that the decrease is caused by the titration of the proposed acceptor group; thus with decreasing pH the concentration of the acceptor decreases, leaving solvent as the only acceptor. Since the pH region for this effect is between pH 3.5 and 5, the titrated group would appear to be a carboxyl side chain. Evidence that a protein carboxyl group is being titrated is given in Table IV which indicates the change of the fluorescence decay parameters with pH. Notice that as the pH is lowered to 2.7, r1 increases indicating a slowing down of the excited-state proton transfer reaction. Also note that the contribution of r3 decreases greatly with the amplitude associated with it (CYJ becoming very small; consequently 73 is increased to compensate due to analysis problems encountered with small amplitudes (5). This change with pH is fully reversible, as indiTABLE

PH 7.9 5.65 4.9 4.4 3.9 3.4 2.7

IV

DECAY PARAMETERS AS A FUNCTION

OF BSA-I OF pH”

a1

Tdns)

a2

73(ns)

a3

-24 -21.3 -23 -29 -19.5 -21 -27

8.9 9.3 9.45 10.6 10.6 10.5 10.2

48.5 49 51 60 74.5 76 71.5

16.0 16.0 15.9 18.6 18.7 19.5 21.4

27.5 29.7 26 11 6 3 1.5

TIW 0.6 0.8 1.0 0.6 0.9 1.45 2.4

n Conditions: 20°C; 10 mM phosphate; [BSA] = -4 mg/ml; -1.3 dyes/BSA; 336-nm excitation; 440nm emission; pH 3.9 and 5.65 points obtained by adding base after reaching pH 2.7.

AND

BRAND

cated in Table IV, by adding base to obtain the pH 3.9 and 5.65 results. DISCUSSION

Fluorescence probes, both covalent and noncovalent, have been used with great success in studying biochemical problems. Noncovalent dyes can best be used when they either have negligible quantum yields in aqueous solution, for example, the anilinonaphthalene sulfonate derivatives (18), or if they are insoluble in water such as diphenylhexatriene. The fluorescence of probes that undergo excited-state proton transfer is sensitive to proton donors and acceptors and also to the kinetics of proton transfer. Most proton transfer dyes, including naphthols, are not only relatively soluble in water but also have appreciable quantum yields. It was desirable to design a covalent probe capable of excited-state proton transfer. A logical choice as the “core” molecule is 2-naphthol since its photochemistry is well understood (3,4; Laws and Brand, J. Phys. Chew., in press). Finding 2-naphthol derivatives with groups reactive to protein side chains is difficult for two reasons. First, the hydroxyl group is capable of reacting with many of the linkage groups used with biomolecules; thus the activated dye can cross-react with itself. Therefore a hydroxyl blocking group is needed, complicating the labeling process with a deblocking step. Second, many functional groups when introduced into the naphthalene ring system tend to either alter the proton transfer capabilities of the dye, or enhance the rates of radiationless transitions. The la&one of HNAA overcomes these difficulties. The la&one (I) is unique in that the blocking of the OH is included in the activation step, while the covalent reaction is also the deblocking reaction. By being an active ester, it not only becomes reactive toward many nucleophiles, but also blocks the important hydroxyl group. And since the covalent attachment is at the carboxyl position, the hydroxyl is uncovered in the same step. The rates of hydrolysis of the la&one (I) are slow when compared to the rates of reaction with amines. Further-

EXCITED-STATE

PROTON TRANSFER

FLUORESCENCE

PROBE

99

more, since a la&one will be less reactive unique single site on BSA, i.e., affinity than acid chlorides (19), it might be ex- labeling (20). The complex decay kinetics pected to show more specificity in reactions found for the BSA conjugate may have their with functional groups on macromolecules. origin in multiconformational forms of the The experiments described here show protein or in excited-state reactions and are that increases in fluorescence intensity (es- thus not inconsistent with the notion of pecially that due to emission from naphthobinding to a single site. late) may be used to monitor the kinetics of Fluorescence probes that show dual emiseither hydrolysis or the reaction of the lac- sion such as naphthols should be of value tone (I) with amines. It was found that in the study of macromolecules. It has althe reaction of la&one (I) with bovine serum ready been found (Laws and Brand, to be albumin was more rapid than its reaction submitted) that the decay of the emission with corresponding concentrations of small anisotropy when the emission is observed organic amines. This has been rationalized at 360 nm is quite different from that obin terms of an “affinity labeling” (20) served at 440 nm. Studies of this type will mechanism. lead to a clearer understanding of the rotaIt was found that addition of I to BSA re- tional behavior of macromolecules. And as sulted in an instantaneous enhancement of shown here, it is advantageous to develop la&one fluorescence reminiscent of that ob- fluorescence probes based on well-underserved when the la&one is transferred from stood excited-state reactions. water to dioxane. Addition of I also quenches ACKNOWLEDGMENTS the tryptophan fluorescence of the protein. These findings are interpreted in terms of a We are grateful to Dr. W. J. Frazee for his help noncovalent adsorption of the lactone (I) to in the preparation of the radioactive probe. We also apthe protein. This is then followed by the preciate the advice of Dr. Y. C. Lee in regard to covalent reaction as reflected by the addi- synthetic chemistry. We thank Ms. G. Pavlovitz and tional time-dependent (in minutes) changes Mr. R. L. Modlin for initial help on this project, Ms. S. Thomas for help with computer programming, and in fluorescence intensity. Dr. S. Roseman for assistance in the use of the scintilEquilibrium studies, making use of the inlation counter. We thank Professor A. Nason for the stantaneous fluorescence changes indicated use of a Cary spectrophotometer. above, were consistent with a 1:l stoichiometry between the protein and the lactone. REFERENCES BSA is known to bind many types of com1. ANDERSON, S., AND WEBER, G. (1966)Arch. Biopounds (21), including small aromatic ions them. Biophys. 116, 207. like 1-anilino-CGnaphthalenesulfonate (22), 2. WAGGONER, A. S., AND STRYER, L. (1970) Proc. with varying stoichiometries. However, Nat. Acad. Sci. USA 67, 579. certain uncharged molecules like L-trypto3. WELLER, A. (1961) Progr. React. K&et. 1, 187. phan at neutral pH (23) have been shown 4. LOKEN, M. R., HAYES, J. W., GOHLKE, J. R., to bind with a 1:l relationship. Therefore AND BRAND, L. (1972) Biochemistry 11, 4779. the data suggesting that a 1:l equilibrium 5. GAFNI, A., MODLIN. R. L., AND BRAND, L. (1976) binding exists for I and BSA is not unJ. Pkys. Chem. 80, 898. reasonable. 6. STOEMER, R. (1900) Annals 313, 79. It has been shown here that the covalent 7. OGATA, Y., OKANO, M., AND KITUMURA. Y. (1951) J. Org. Ckem. 16, 1588. reaction between the lactone (I) and BSA 8. JULIA, M. (1953) Bull. Sot. Ckim. Fr., 640. leads to a 1:l labeling ratio at neutral pH. Even when a small excess of I is used it is 9. FURMAN, F. M., THELIN, J. H., HEIN, D. W., AND HARDY, W. B. (1960) J. Amer. C’kem. difficult to attach more than one dye per sot. 82, 1450. protein. However, under more drastic con- 10. TERENT’EV, A. P., KOST, A. N.. DZBANOVditions of alkaline pH, with a small (lo-fold) SKIY, N. A., AND MAROCHKO, S. V. (1953) Sb. excess of I, higher labeling ratios can be obStatei Obsckck. Khim,.; Akad. Nauk. SSSR 1, tained. The above results suggest that the 610. la&one (I) may, under mild conditions, first 11. EASTER, J. H., DETOMA, R. P., AND BRAND, L., adsorb to and then covalently react at a (1976) Biopkys. J. 16, 571.

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