Eur. J. Bioohem. 34,535-538 (1973)

Spectral Studies of Interactions of Detergents with Tryptophyl Compounds

Jake BELLO and Helene R. BELLO Department of Biophysics, Roswell Park Memorial Institute, Buffalo, New York

(Received September 4/December 4, 1972)

The interactiom of sodium dodecylsulfate and hexadecyltrimethylammonium chloride with typtophyl model compounds were investigated by fluorescence, classical difference spectra and thermal perturbation spectra. Oppositely charged models and detergents interact t o produce marked spectral changes. Apolar interactions also contribute. The ease of interaction with sodium dodecylsulfate decreases in the order : tryptamine, Lys-Trp-Lys, Leu-Trp-Leu, Gly-Trp-Gly. Glu-Trp-Glu shows little or no interaction. Lys-Trp-Lys forms both soluble and insoluble com- plexes with sodium dodecylsulfate.

I n other papers on detergents [1,2] we presented evidence that the interaction of a charged detergent with tyrosyl compounds depends strongly on the presence of an opposite charge in the latter. I n this communication we demonstrate the same effect for tryptophyl compounds. This is of importance because it has been reported that tryptophyl models do not interact with detergent. For example, Rogers and Yusko reported that 1 mM sodium dodecylsulfate does not affect the fluorescence of N-acetyltrypto- phan amide [3].

MATERIALS AND METHODS

Tryptophyl peptides were obtained from Mann Research Labs., tryptamine hydrochloride from Calbiochem, sodium dodecylsulfate (Sequanal grade) from Pierce Chemical Co., and hexadecyltrimethyl- ammonium chloride from Eastman Organic Chemicals. Temperature perturbation spectra were done as described earlier [I, 41. I n this method, bringing identical solutions to Merent temperatures generates a difference spectrum. A Cary 15 spectrophotometer was used. The base line was obtained with both cuvettes a t 26 “C; then the upper, or “sample” cuvette was cooled, while the lower was kept a t room temperature. Classical difference spectra were made by the four-cell method [5] to compensate for the presence of detergent in only one of the trypto- phyl-containing cells. 1-cm cuvettes were used for all difference spectra, Fluorescence measurements were made with an Aminco-Bowman spectrophoto- fluorimeter.

RESULTS AND DISCUSSION I n Table 1 are displayed the results of fluorescence

measurements. It is apparent that 1 mM sodium dodecylsulfate significantly affects the emission spec- trum of tryptamine (in contrast t o the absence of effect with N-acetyltryptophan amide [3] and Gly- Trp-Gly), but that was 30 times that concentration of sodium dodecylsulfate has no effect on the emission of Glu-Trp-Glu. The effects on the wavelength of maximum emission for Glu-Trp-Glu, Lys-Trp-Lys and tryptamine in 0.03M sodium dodecylsulfate, and for indoleacetic acid in 0.03 M hexadecyltri-

Table 1. Fluorescence of {nndole CornpozLds in detergent solutions

The buffer used was 0.05M ammonium acetate pH 6.3. Excitation wavelength, 290 nm. Concentrations are 0.03 mg/ ml for the tripeptides and 0.01 mg/ml for tryptamine and indole-acetic acid. Emission was measured a t 90’ from the incident beam, using 2-mm slits in incident and emitted beams. Detergent I = sodium dodecylsulfate, detergent I1

= hexadecyltrimethylammonium chloride

Solute Emission wavelength

in 1 . Detergent in

buffer detergent

mM

Glu-Trp-Glu 30 I Gly-Trp-Gly 30 I

Leu-Trp-Leu 30 I Lys-Trp-Lys 30 I Trvotamine 30 I

Gly-Trp-Gly 1 1

~~

Tgptarnine 11 Indoleacetic acid 30 II

nm nm nm

361 361 0 363 358 - 5 363 363 0 363 358 - 5 361 351 -10 362 351 -11 362 358 - 4 365 358 - 7

536 Detergents with Tryptophyl Compounds Eur. J. Biochem.

270 290 310 330 350 Wavelength (nm)

Fig. 1. Classical difference spectra for tryptamine in sodium dodecylsulfate. The concentration of tryptamine was 0.1 mM and the buffer 0.05 M ammonium acetate pH 6.3. Each division on the vertical axis represents an absorbance differ- ence of 0.01 and A s of 100 M-l. cm-l. The baseline ( A E = 0) coincides with the horizontal line above 320nm. (---)

30 mM dodecylsulfate; (----) 1 mM dodecylsulfate

methylammonium clearly show that opposite charges in detergent and substrate facilitate the interaction between detergent and indole moiety.

Additional evidence for interaction was obtained from classical difference spectra as shown in Fig.1 for tryptamine in 30 and 10 mM sodium doclecyl- sulfate, molar ratios of detergent to tryptamine of 300 and 10, respectively. The difference spectrum for tryptamine in 5mM detergent (not shown) was nearly identical with that for 30 mM detergent.

Ananthanarayanan and Bigelow [6] showed that 5 0 / , sodium dodecylsulfate (approximately 0.15 M) produced a difference spectrum for indole with

= 950 M-l * cm-l (confirmed by us). However, in 30mM sodium dodecylsulfate we have found

for indole to be only 30 M-l - cm-l. Thus, a t low detergent concentration the interaction of this detergent with indole is much less complete than with the indole ring of the positively charged indole derivative tryptamine, which has only a small additional apolar region.

In Fig. 2 are shown temperature perturbation difference spectra, further demonstrating the impor-

Indoleacetic acid

250 270 290 310 330 35 Wavelength (nm)

Fig.2. Temperature-perturbation spectra of tryptophyl cinn- pounds in 30 m M detergent. Spectra were measured a t 8 "C, vs 20°C. Each division on the vertical axis represents an absorbance difference of 0.01. The concentration of trypto- phyl compounds was 0.075 mg/ml for the tripeptides, and 0.02 mg/ml for tryptamine hydrochloride and indoleacetic acid. The buffer was 0.05 M ammonium acetate pH 6.0. Baselines as for Fig.1. (----) Detergent; (-) buffer. The detergent for indoleacetic acid was hexadecyltrimethyl- ammonium chloride, for all other curves i t was sodium

dodecylsulfate

tance of the nature of the tryptophyl compound. It should be noted that the most useful feature of these spectra is the negative extremum at 290 to 300 nm, because this is least affected by the positive A e arising from the density difference between the cold and warm solutions. For aqueous solutions the density difference contribution a t 292 nm decrease lAel by about 7O/,, calculated from E~~~ of the direct absorption spectrum. (However, since modest con- centrations of detergent probably have only minor effects on the coefficient of thermal expansion of water, the observed difference between detergent and non-detergent experiments are significant a t any wavelength.) I n an earlier paper [7] we showed that spectral effects such as those of Fig.2 can be produced by changes in solvent. I n Fig.3 we show

J. BELLO and H. R. BELLO 537

270 290 310 330 35( Wavelength (nrn)

Fig. 3. Temperature-perturbation-dif ference spectra of indole in polar (----) and non-polar (-) solvents. The polar solvent was methanol-water (25: 75, vjv) and the non-polar solvent CCI,. The concentration of indole was 0.03 mg/ml (0.26 mM). Each division on the vertical axis represents an

absorbance difference of 0.01. Baseline as for Fig. 1

an extreme case of this for indole in CCI,, compared with indole in 25O/, methan0l-+75~/, water. The density difference for CCl, contributes about 3001, to the maximum a t 280nm and 25O/, to that a t 290nm. These effects do not alter the conclusion that the thermal perturbation spectra are highly sensitive to the environment of the chromophore.

The effect of detergent on the spectrum of trypt- amine does not appear to arise from neutralization of the positive ammonium groups of tryptamine by the detergent negative chrge, because in aqueous media in the absence of dodecylsulfate the thermal perturbation spectra of charged tryptamine and un- charged indole are similar, and the spectrum of tryptamine with sodium dodecylsulfate is quite differ- ent from that of indole without the detergent (Fig.2 and 3, 25O/, methanol spectrum). Further, since tryptamine is too small for burial of the indole ring within a folded conformation, the spectral changes must arise from a direct interaction of the detergent with chromophore.

of the thermal perturbation spectra (such as those of Fig. 2) toward sodium dodecylsulfate concentration from 0.1 to 100mM. It is interesting that Lys-Trp-Lys, with its greater net charge and more numerous possi- bilities for apolar interactions than tryptamine, requires a higher concentration of sodium dodecyl- sulfate for interaction. with the indole moiety, as

In Fig.4 is shown the response of

Z t I !

-0.01 c I,'. Leu-Trp-Leu

-0.02

0.1 03 1 3 10 30 100 [Dodecylsulfate] (rn M)

Fig. 4. Temperature-perturbation response of tryptophyl models relative to sodium-dodecylsulfute concentration. The absorb- ance differences were taken a t 290 nm from spectra such as those of Fig. 2. Concentrations of models: tryptamine hydro- chloride, 0.03 mg/ml ; tripeptides, 0.075 mg/ml, except 0.037 mg/ml for Gly-Trp-Gly. pH 6.0, 0.05 M ammonium acetate. Dashed portion of the Lys-Trp-Lys curve indicates

region of precipitation (see text)

indicated by fluorescence and thermal perturbation. However, we have no information on total binding to Lys-Trp-Lys, which may be greater than the binding to tryptamine. The mid-point for Lys-Trp- Lys is uncertain because of precipitation in the cold cuvette between 0.3 and 3 mM detergent. The result- ing concentration difference and turbidity generate a tryptophyl spectrum and a turbidity slope, super- imposed on the thermal perturbation spectrum. The effect is greatest a t about 1 mM detergent, decreasing in both directions and vanishing a t 0.3 and 3 mM detergent. The dashed portion of the curve was drawn through the region of precipitation, with mid-point a t 1 mM sodium dodecylsulfate. The preci- pitation and redissolution indicate that more than one kind of complex is formed between Lys-Trp-Lys and sodium dodecylsulfate, and is reminiscent of similar effects with some proteins. Some weak turbidity was shown by Gly-Trp-Gly in I mM sodium dodecylsulfate. The turbidity was not visible to the eye, but was manifested as the characteristic scattered-light slope in absorbance. This indication of an interaction at a concentration of detergent a t which there is little change in the thermal perturba- tion spectrum or in the fluorescence emission spectrum suggests an interaction between sodium dodecyl- sulfate and non-indole portions of Gly-Trp-Gly. The relative positions of Gly-Trp-Gly and Leu-Trp-Leu in Fig.4 suggest that the apolar groups of the latter contribute to binding of detergent to the indole.

538 J. BELLO and H. R. BELLO: Detergents with Tryptophyl Compounds Eur. J. Biochem.

At the ionic strength of 50 mM used in this work, the critical micelle concentration of sodium dodecyl- sulfate is approximately 2 mM [S]. Thus, the curves of Fig. 4 show that some chromophore-detergent interactions are complete below and some above the critical micelle concentration. Complexes formed a t these concentrations probably have different stmc- tures. The turbidity observed for Lys-Trp-Lys a t 0.3 mM detergent may arise from formation of an insolubIe complex of Lys-Trp-Lys with detergent monomer, and increasing solubilization above 1 mM detergent may arise from formation of a complex with detergent micelle. Reynolds and Tanford have presented evidence that only dodecylsulfate monomer, not micelle, binds to proteins [9].

The near absence of effect of sodium dodecylsul- fate on Glu-Trp-Glu shows that repulsive charges outweight both the potential apolar interactions of dodecylsulfate with the indole ring and the hydro- phobic portions of the glutamyl residues, as woll as the ion-dipole interactions between the anionic sulfate and the peptide groups.

For tryptamine the mid-point of 0.6mM detergent corresponds to a detergent to tryptamine ratio of 4, while the plateau is reached a t about a ratio of 6. The actual stoichiometry has not been measured for any of the substrates.

Fig.l and Table 1 indicate that the tryptarnine- dodecylsulfate interaction is not near completion a t 1 mM, while Fig.4 indicates that it is nearly complete. This may arise from several causes: pH difference, temperature effects, or different responses of the methods. (Indeed, the mid-point for trypt- amine can be shifted between 0.4 and 0.8 mM by using d e a t different wavelengths if the thermal perturbation spectra. Similar shifts occur for the other models, the relative positions being unchanged.) Additional detergent-tryptamine interactions may occur above 1 mM detergent, which are detectable by fluorescence, but not by some other methods of study. This emphasized the desirability of using several methods.

It may be that no one model adequately repre- sents all of the tryptophyls of a protein, nor any given tryptophyl a t all detergent concentrations. It is desirable to use several models to obtain a range of effects against which to compare the effect(s) on the protein. Polet and Steinhardt [lo] in studying differ- ence spectra of serum albumin in detergents did not use detergents in the model systems because the information then available indicated that “deter- gents are without effect on the optical properties of small peptides”. Nevertheless, they and others recognized that detergents may interact with the indole groups of proteins. We now see that deter- gent-reactive models are available. Refined inter- pretations of spectral data of proteins may require studies of the models used here, and of other models of more sophisticated design.

This work was supported by Grant GM 13485 from the Institute of General Medical Sciences, National Institutes of Health and Grant GB 20083 from the National Science Foundation.

REFERENCES 1. Pittz, E. P. & Bello, J. (1971) Arch. Biochem. Biophys.

2. Bello, J. & Bello, H. R. (1972) Biochim. Biophys. Actu,

3. Rogers, K. S. & Yusko, S. C. (1969) J . Biol. Chem. 244,

4. Bello, J. (1969) Biochemistry, 8, 4542. 5. Laskowski, M., Jr.. Leach, S. J. & Scheraga, H. A.

6. Ananthanarayanan, V. S. & Bigelow, C. C. (1969) Bio-

7. Bello, J. (1970) Biochemistry, 9, 3562. 8. Emerson, M. 3’. & Holtzer, A. (1972) J . Phys. Chem. 71,

9. Reynolds, J. A. & Tanford, C. (1970) Proc. Nutl. Acud.

10. Polet, H. & Steinhardt, J. (1968) Biochemistry, 7, 1348.

147, 284.

2r8,45.

6690.

(1960) J. Am. Chem. Soc. 82, 571.

chemistry, 8, 3717.

1898.

Sci. U. S. A . 66, 1002.

J. Bello and H. R. Bello Department of Biophysics, Roswell Park Memorial Institute 666 Elm Street, Buffalo, New York, U.S.A. 14203