A Hydrophobic Quencher of Protein Fluorescence: 2,2,2-Trichloroethanol

Biochimica et Biophysica Acta, 491 (1977) 473-481

© Elsevier/North-Holland Biomedical Press BBA 37618 A HYDROPHOBIC QUENCHER TRICHLOROETHANOL

OF P R O T E I N F L U O R E S C E N C E :

2,2,2-

MAURICE R. EFTINK*, JAMES L. ZAJICEK and CAMILLO A. GHIRON Department of Biochemistry, University of Missouri, Columbia, Mo. 65201 (U.S.A.)

(Received September 28th, 1976)

SUMMARY Previously the neutral fluorescence quenching probe, acrylamide, was employed to determine the degree of exposure of tryptophan residues in proteins. A less polar neutral quencher 2,2,2-trichloroethanol (trichloroethanol) was used in the present work to investigate whether it would preferentially interact with apolar regions of proteins. For most proteins studied, the degree of quenching by trichloroethanol is found to be about the same as with acrylamide. However, for human and bovine serum albumin hydrophobic interactions between trichloroethanol and these proteins occur, leading to an exalted quenching. The fluorescence quencher thus senses the presence of a hydrophobic domain in the vicinity of the tryptophan residues in these proteins. Trichloroethanol is shown to induce conformational changes in certain proteins and to be a potentially useful quencher for proteins having predominantly tyrosine emission.

INTRODUCTION In earlier studies the neutral quenching probe, acrylamide, was employed to determine the degree of exposure of tryptophan residues in proteins [1]. Fluorescence quenchers that are able to interact with proteins can potentially provide additional information concerning the microenvironment in the vicinity of the tryptophan residues. Such is the case with ionic quenchers. Due to electrostatic interactions between these quenchers and the surface of proteins, the distribution of charged groups around a tryptophan residue can be revealed by their quenching action [2-4]. For example, by using ionic quenchers such as I - , NO a, and Cs ÷, Ivokova et al. [2] and Lehrer [3] have found that the single tryptophan residue in human serum albumin is guarded by nearby positively charged groups of the protein. This approach can be extended by the use of a hydrophobic quenching probe. If such a probe were able to interact with hydrophobic pockets in the microenvironment of a fluorescing residue, an enhanced degree of quenching would be expected. A * Present address: Department of Biochemistry, University of Virginia School of Medicine, Charlottesville, Va. 22903, U.S.A.

474 hydrophobic compound which appears to quench indole fluorescence with an efficiency near unity (apparently by an electron abstraction mechanism [5]) is trichloroethanol [6]. In a study employing an indole--micelle complex as a model for a protein, trichloroethanol was found to have an exalted quenching ability, as compared to the quenching by polar acrylamide molecules. This was attributed to the tendency of the less polar trichloroethanol molecules to become accumulated into the oily interior of the micelles, thus facilitating the quenching of the solubilized indole group. If similar oily domains exist in proteins in the neighborhood of tryptophan residues, they should be sensed in a similar fashion by this quencher. The quenching of several proteins by trichloroethanol was therefore studied, and a comparison was made to the quenching by acrylamide in order to detect any instances of enhanced quenching by the hydrophobic probe. EXPERIMENTAL

Chemicals. 2,2,2-Trichloroethanol was obtained from British Drug Houses Chemicals, Ltd., Poole, England, and was used without further purification. A mixture (50:50, v/v) of trichloroethanol with ethylene glycol (spectral quality, MathesonColeman-Bell) was prepared and used as a stock solution for quenching studies. It was found that the ethylene glycol aided in dissolving the trichloroethanol into aqueous solutions. Indole was twice recrystallized from hexane. L-Tyrosine (a product of Sigma Chemical Co.) was used in these studies due to the availability of a fluorescence lifetime at p H 7.0 for this phenolic derivative [7]. Proteins. RNAase T1 (Asperigillus oryzae), nuclease (Staphylococcus aureus), monellin (Dioscorephyllum eumminsii), pepsin A and trypsin were obtained from Worthington Biochemical Corp. Glucagon, RNAase A, human serum albumin, fllactoglobulin and a-chymotrypsinogen were obtained from Sigma Chemical Co. Insulin was from Schwarz/Mann. Corticotropin (procine, an Armour product) and bovine serum albumin (three times recrystallized) were provided by Dr. B. Campbell, University of Missouri. Trypsin and pepsin were further purified as previously described [1]. Human serum albumin was defatted by the charcoal filter procedure of Chen [8]. Proteins were dissolved in freshly prepared buffers before the fluorescence studies were performed. Commonly a small amount of protein (usually in a solid form) was dissolved into about 2.5 ml of the buffer in a fluorimetric cuvette. Human and bovine serum albumins, pepsin, chymotrypsinogen, and fl-trypsin were dissolved in 0.02 M sodium acetate buffer at pH 5.5. RNAase T1, RNAase A, monellin, and corticotropin were dissolved in 0.01 M Tris buffer at p H 7.0. fl-Trypsin was also studied in pH 3 HC1 solution. Glucagon was first dissolved at low pH (approx. 3) and the solution was subsequently adjusted to pH ~ 7 to avoid aggregation, fl-Lactoglobulin was dissolved in 0.1 M NaCl, p H 2.0, solution. Under such conditions, fl-lactoglobulin exists as a monomer [9]. Insulin was studied at pH 1.85 in 0.1 M NaCI at 0.06 mg/ml where it is predominantly a monomer and at 2.0 mg/ml where it is > 80 ~ dimer [10]. If it appeared necessary, the protein solutions were filtered before use. Fluorescence intensity measurements were made on a Farrand Mark I spectrofluorimeter equipped with a magnetic stabilizer for the xenon arc light source. Bandwidths of 5 to 2 nm were routinely used. An excitation wavelength 2 of 295 nm was

475 used to ensure that the light was absorbed almost entirely by tryptophan groups. Those proteins that did not contain tryptophan residues were usually excited at 280 nm. Protein solutions having an absorbance of 0.1 or less at the excitation wavelength were usually used. The fluorescence of a protein, monitored at its emission 2 . . . . was quenched by the progressive addition of small aliquots of 5.2 M trichloroethanol in ethylene glycol. (See refs. 1 and 6 for a description of experimental procedures.) The solution was then mixed by gentle inversion of the cuvette. Care was taken to add the trichloroethanol/ethylene glycol solution against the inside wall of the cuvette. Blowing of the quencher solution into the aqueous solution was avoided. These precautions were taken in order to minimize the formation of trichloroethanol droplets in the solution, which might act to denature the protein. All quenching experiments were performed at room temperature. Care was taken to avoid prolonged illumination of the samples with ultraviolet light since it was found that trichloroethanol greatly promotes photolysis of indole, indole side chains of proteins, and tyrosine. Data Analysis. Quenching data was analyzed according to the Stern-Volmer equation:

F0 F

-- 1 + K~, [Q]

(1)

where Fo and F are the fluorescence intensities in the absence and presence of quencher, respectively, and Ksv is the dynamic quenching constant. Ksv is equal to kqzo, where zo is the fluorescence lifetime of the excited state (in the absence of quencher) and kq is the bimolecular rate constant for the quenching reaction. In previous studies the magnitude of kq has been used as an index of the exposure of fluorescing residues in proteins [1]. This relationship is based on the fact that for an efficient quencher, the magnitude of kq is equal to the frequency at which the probing molecule encounters the residue. In certain cases a more detailed treatment of quenching data can be made by means of the following modified Stern-Volmer equation:

Fo

F -- (1%- K~v [Q]) exp (V[Q])

(2)

The added factor in this equation describes the quenching of the fluorescence that occurs by a static process. V, the static quenching constant, is related to the probability of finding a quencher molecule in the vicinity of the fluorescing group. (See ref. 6 for discussion.) If the two molecules are adjacent to each other at the moment that photon absorption occurs, the newly formed excited state will be quenched instantaneously (or statically). In a randomly distributed aqueous system there will be a small probability of finding such neighboring pairs, even if there is no significant attraction between the molecules. If there is an interaction between the quencher and the chromophore (or between the quencher and the microenvironment of the chromophore) the static quenching process will be more significant. As discussed previously, when dealing with proteins containing more than one tryptophan residue, the analysis of quenching data is complicated and only an effective dynamic quenching constant, Ks,(eff) can be obtained [1].

476 RESULTS The quenching of aqueous indole solutions by trichloroethanol results in a Stern-Volmer plot that is curved upward as shown in Fig. 1. Trichloroethanol quenching of tyrosine gave a similar result. The curvature indicates that a static component must be included to describe the kinetics of the reaction. When analyzed according to Eqn. 2, a dynamic quenching constant of 21 M-1 and a static quenching constant of 2.0 M -1 are found for indole. For tyrosine, values of ll.8 M -1 for Ksv and 1.1 M -1 for V are found. From the Ks~ for indole and the fluorescence lifetime of 4.3 ns, a quenching rate constant, kq, of 5.0 x l09 M - l . s -1 is calculated. A kq of 4.5 × 10 9 M - l . s -1 is calculated for tyrosine from the lifetime of 2.6 ns [7]. These values approach the diffusion limited rate constant of about 7 x l 0 9 M -1" s -1 that is theoretically obtained from the Smoluchowski equation for molecules the size of indole and tyrosine [6]. This argues that trichloroethanol quenches the fluorescence of both indole and tyrosine with an efficiency of near unity (i.e. almost every collisional encounter results in quenching). The V value of 1-2 M-~ indicates that instantaneous quenching occurs when the quencher happens to be within 8-10/~ of the center of the aromatic rings at the moment that the latter become excited [6]. Alternatively, V can be described as being a weak association constant between trichloroethanol and the ground state of either indole or tyrosine.

5C

F

,o

~o

f 3O

F'[:I

'®

20 10

I 005

I 010 [Trlcn~oroet hanoi] M

o'is

Fig, 1. Trichloroethanol quenching of indole in water.

Addition of trichloroethanol to protein solutions results in the expected loss of fluorescence both for proteins showing tryptophan and tyrosine emission. In many cases, however, trichloroethanol appears to also cause aggregation and precipitation of proteins, detectable by an increase in the turbidity of the solution. This complicates the analysis of the quenching experiments. Typically, plots ofEqn. 1 for proteins show a gradual upward curvature. Ideally, such a curvature would be attributed to static quenching. However, the deviation could also be due to aggregation in some instances. It is difficult to judge the effect that internal screening and internal reflection of scattered light will have on the intensity of the emitted light. Despite these complications, the initial slopes of the standard Stern-Volmer plots can be obtained. Aggregagation is usually minimal below 0.2 M trichloroethanol. The initial slope can be considered to be an effective dynamic quenching constant, Ksv(eff). According to this

477 treatment, static q u e n c h i n g must be completely disregarded in most cases due to the experimental difficulties associated with using the organic probe. I n Table I the Ks~(eff) are listed for trichloroethanol q u e n c h i n g of a series of proteins. The Ks~ a n d K~v(eff) f o u n d for acrylamide q u e n c h i n g of these same proteins are also tabulated for c o m p a r i s o n [1]. Acrylamide q u e n c h i n g of proteins having tyrosine fluorescence has n o t been studied due to the large m o l a r a b s o r b a n c e of this reagent at wavelengths < 295 nm. I n Figs. 2 a n d 3 the Stern-Volmer plots for some proteins of particular interest are shown. The plots for h u m a n a n d bovine serum a l b u m i n in Fig. 2 show a very large degree of q u e n c h i n g by trichloroethanol. The h u m a n serum a l b u m i n plot shows a gradual u p w a r d curvature which is n o t accompanied by a large increase in the turbidity of the solution. F o r fl-lactoglobulin a n d fl-trypsin the Stern-Volmer plots, as shown in Fig. 3, reveal a very dramatic u p w a r d TABLEI COMPARISON OF TRICHLOROETHANOL AND ACRYLAMIDE QUENCHING CONSTANTS a

Indole Corticotropinb Glucagon c Pepsin a Monellinb Nuclease c fl-Trypsin a Chymotrypsinogena RNAase TI b fl-Lactoglobulin f Human serum albumin~ Human serum albumina Bovine serum albumina Tyrosineb RNAase A b Insulin (monomer)h Insulin (dimer) ~

Trichloroethanol (M- 1)

Acrylamide (M- ~)

K~ (eft)

K,v

V

30.5 13.0 10.5 9.5 J 5.2 5.2 2.4 j 1.0j 1.1

2.5 0.1 1.0

21.0 11.0 10.0 7.5 4.1 8.0 4.0 2.0 1.0 0.6 18.5 20 230 11.8 8.3 6.0 3.7

v 2.0 1.0

0.3 0.25

1.6 J

5.0 25

3.3 3.1 3.7 J

0.6 0.8

1.1

Quenching studies at room temperature excited at 295 nm, and 280nm for proteins that contain no tryptophan; fluorescence monitored at the emission ;t~x. For trichloroethanol the quenching constants are the initial slopes of Stern-Volmer plots ( = K~v (eft)). Only in the case of glucagon and human serum albumin was aggregation of the protein by trichloroethanol not apparent and analysis according to Eqn. 2 attempted. b pH 7.0, 0.01 M Tris buffer. c pH 6, 0.1 M NaCI. d pH 5.5, 0.01 M acetate buffer. pH 7.5, 0.01 M Tris/0.05 M NaC1/0.01 M CaCI~. t pH 2.0 HCI/0.1 M NaC1. BpH 2.5, 0.01 M formate buffer. h pH 1.85, 0.1 M NaCI, 0.06 mg/ml. pH 1.85, 0.1 M NaCI, 2.1 mg/ml, because of this high protein concentration an excitation wavelength of 290 nm and an emission wavelength of 325 nm were used to avoid screening effects. J Values are K , (eft) since these proteins contain more than one tryptophan residue.

478

?0

_o F

oc

5C

Fo F '~ [.,s~ ' :~

c

3£

1C

001

002 003 [TrJchloroethanol] M

004

Fig. 2. Trichloroethanol quenching of bovine (0) and human (O, A) serum albumins. Both proteins were studied in a 0.01 M acetate buffer (pH 5.5).

i10

F F

0

0I

02

OS

CI4

05

LTrichloroet hanoi ] M

Fig. 3. Trichloroethanol quenching of fl-lactoglobulin (0) and fl-trypsin (pH 3.0, 0 ; pH 5.5, II). Conditions given in Table I.

479 curvature. Again, aggregation of the protein does not appear to be responsible for these breaks in the curves, since the turbidity of the protein solution does not increase significantly until much higher concentrations of trichloroethanol are added. For other proteins such as nuclease, RNAase T1, and monellin, sharp upward curvatures may also exist, but aggregation of the proteins occurs in the same concentration range and complicates the interpretation. Since trichloroethanol is transparent in the ultraviolet (ez4° = 0.29) a greater selection of excitation wavelengths is possible. This enables one to study the excitation wavelength dependence of the quenching constants. For example, the K~v for trichloroethanol quenching of N-acetyl-L-tryptophanamide have a constant value of 11.7 ± 0.7 M -~ (V --~ 2.0 M -1) from 240 to 295 nm. This argues that the lifetime of N-acetyl-L-tryptophanamide fluorescence is independent of the excitation wavelength [11]. The protein monellin was studied over this same excitation wavelength range and was found to also have a constant K~ of 4.0 ± 0.2 M -1. DISCUSSION As seen in Table I the effective trichloroethanol quenching constant for most proteins is similar to that found for acrylamide. Since Ksv ~ kq~ro, for the single tryptophan containing proteins, the quencing rate constant must be quite similar for both trichloroethanol and acrylamide in most cases. Small differences between the kq values might be accounted for by the fact that the probes are of slightly different molecular shape and size. A fluorophor could be located in a crevice of such dimensions that permits one of the quenchers to penetrate with greater ease than the other. However, this effect seems to be minor as most of the trichloroethanol quenching constants differ from the acrylamide values by less than 50 ~. The degree of quenching by trichloroethanol seems to be determined predominately by the same factors that govern the quenching by acrylamide, namely, the degree of exposure of the fluorescing residues. An absence of enhanced quenching indicates that trichloroethanol does not interact significantly with the protein matrix. Either the fluorophors are not located in large hydrophobic regions of the marcomolecule, or the oily regions surrounding the fluorophors are not capable of expanding to accomodate the probe. For those proteins containing more than one tryptophan residue, the Ksv(eff) (average value for all fluorophors) are also similar for the two quenchers. Although a direct interpretation of Ksv(eff) in terms of individual residue exposure cannot be made, the similarity of the values for both quenchers again argues that enhanced quenching by trichloroethanol is not significant. However, this is not the case for the human and bovine serum albumins. For this pair of proteins an unusually high degree of quenching by trichloroethanol is observed. Also, as seen in Fig. 2 there is a pronounced upward curvature in the SternVolmer plot for human serum albumin. The datum for human serum albumin is reminescent of that obtained for trichloroethanol quenching of the model indole-micelle complexes (see Fig. 7 in ref. 6). The smooth positive curvature is diagnostic of a static quenching component. Treatment according to Eqn. 2 yields a K~v = 20 M-1 and V = 25 M-~ for human serum albumin. The resultant static and dynamic constants are both extraordinarily large suggesting that there is a high local concentration of trichloroethanol in the immediate vicinity of the indole ring due to hydro-

480 phobic interactions between the probe and protein. The accumulation of trichloroethanol can best be described by interpreting V as being an association constant (or partition coefficient). The V value is about ten times larger than that found for the randomly distributed situation for indole in water. This suggests that the accumulation of quencher into human serum albumin can be expressed as an increase in the local concentration by a similar factor. The dynamic constant is also much higher than the value for acrylamide quenching. Again a high local concentration of trichloroethanol could account for this finding. Those trichloroethanol molecules that are bound very near the indole ring would quench its fluorescence statically; quencher bound at hydrophobic sites further away would have to diffuse towards the fluorophor in order to dynamically quench it. This implies that accumulated trichloroethanol molecules are relatively free to diffuse within the protein. The Stern-Volmer plot for bovine serum albumin appears linear within expreimental error and has a slope even larger than that for human serum albumin. The Ksv(eff) is so large that vicinal hydrophobic interactions between the quencher and protein must again be invoked. Quenching probably occurs by both the static and dynamic modes (fluorescence lifetime measurements would be required in order to dissect the two quenching components), but the heterogeneity (two fluorophors) of the fluorescence may cause a collapse of the data to a linear plot [l ]. The fact that the quenching patterns for trichloroethanol and the serum albumins are similar to that for trichloroethanol and the model micelle system previously studied is probably not just a coincidence. Wishnia and Pinder [12] have found bovine serum albumin to have an extraordinary capacity to bind a large number of small hydrocarbon molecules. In this respect, bovine serum albumin was found to behave much like a sodium dodecyl sulfate detergent micelle. The implication of this similarity is that the serum albumins possess a large hydrophobic region capable of accomodating several apolar molecules [13-16]. The question remains whether the binding sites preexist or are induced as part of the binding process. Besides binding to proteins, organic compounds such as trichloroethanol may also have a tendency to cause structural alternations in proteins [17, 18]. Inoue and Timasheff [9, 19] have studied the effect of a similar substance, 2-chloroethanol, on the structure of fl-lactoglobulin. At concentrations of 2-chloroethanol less than 10 ~ , little change in the state of the protein is observed. At concentrations between l0 and 2 0 ~ , a change in the conformation of a-lactoglobulin to one possessing more ahelix content occurs. Disruption of the native globular state is accompanied by a preferential interaction of 2-chloroethanol molecules with the a-helix rich state. (2-Chloroethanol was found to be a very inefficient quencher of both tryptophan and tyrosine fluorescence.) The Stern-Volmer plots for quenching of several proteins is marked by an abrupt upward curvature at high trichloroethanol concentrations, as shown in Fig. 3 for fl-lactoglobulin and fl-trypsin. These breaks are not due to aggregation of the proteins, as the turbidity of the protein solutions do not show a significant increase at these trichloroethanol concentrations. The abrupt onset of quenching must be the result of a conformational change induced by the organic perturbant. At concentrations of trichloroethanol above about 0.3 M, the fluorescence becomes drastically quenched. In fact, Fo/F reaches a value much larger than that

481 expected for a fully exposed indole ring. This agrues that the induced conformational change does not simply result in an increase in the exposure of the tryptophans. Preferential association of the organic perturbant with the sections of the protein containing the fluorophor must also occur. This is similar to the finding of Inoue and Timasheff [9, 19] that the native-+a-helix-rich conformational change was accompanied by binding of the perturbant. One might crudely describe this process as a "soaking up" of trichloroethanol by the protein. Since this process occurs very abruptly at [trichloroethanol] = 0.3 M, the conformational change would appear to be a cooperative disruption of the protein's structure. In these proteins there must be some resistance to the rearrangement associated with the "soaking up" process [20]. Note that the break for fl-trypsin shows a pH dependence. This is probably related to the increased structural stability of fl-trypsin at higher pH values [21]. The apparent accumulation of quencher into hydrophobic pockets of the serum albumins, may be analogous to the conformational change/"soaking up" process described for the other proteins. However, due to their "conformational adaptability" [16] the perturbation that is resisted by the other proteins appears to be readily tolerated by the serum albumins. ACKNOWLEDGEMEN TS We express our thanks to Dr. J. Franz, Department of Biochemistry, University of Missouri, for the use of his spectrofluorimeter. This research was supported by Research Grant URC-NSF-1241 from the University of Missouri, and by the Department of Biochemistry and School of Medicine, University of Missouri. This work is presented by Maurice R. Eftink in partial fulfillment of the requirements for the Doctor of Philosophy degree at the University of Missouri. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

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