Photochrmittry and Photobiology Vol. 42, No. 4. pp. 441 - 450. lYS5 Printed in Great Britain. All rights reserved

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RESEARCH NOTE

VARIATION OF STERN-VOLMER QUENCHING CONSTANTS IN MICELLAR SOLUTION AS A FUNCTION

OF AQUEOUS OR MICELLAR QUENCHER GAVRIELLA GABOR* and NICHOLAS J. TURRO

Chemistry Department, Columbia University, New York. NY 10027. USA

(Received 28 November 1984; accepted 2 May 1985)

Abstract-The critical micelle concentration (CMC) of a fluorescent detergent may be measured by determining Stern-Volmer quenching parameters as a function of detergent concentration. The CMC‘s of a cationic detergent, 1 1-(3-hexyl-l-indolyl)undecyltrimethylammonium bromide (6-In-1 l’), and an anionic detergent, sodium ll-(3-hexyl-l-indolyl) undecyl sulfate (6-In-1 1-) were determined b this

in the micellar phase (e.g. benzophenone) were employed. Aqueous phase quenchers are more effective below the CMC and cause a decrease in the long wavelength portion of the fluorescence band of the indole moiety. Quenchers located in the micellar phase are more effective above the CMC and decrease the short wavelength portion of the fluorescence band of the system.

quenching procedure. Quenchers which were predominantly located in the aqueous phase (e.g. Co I+ ) or

INTRODUCTION

Fluorescence probes have been employed to obtain information of the structure and dynamics of the microenvironments of micelles (Hautala et al., 1973; Hautala and Turro, 1972; Khuanga U. et al., 1976; Kalyanasundaram and Thomas, 1977), as well as many macromolecules of biological interest (Edel- man and McClure, 1968; Brand and Gohlke, 1972). Generally, variations in fluorescence quantum yield, fluorescence lifetime or spectral distribution of fluorescence spectra are monitored and then corre- lated with microenvironmental properties. The indole group of the 11-(2-hexyl-l-indolyl)-undecyl deter- gents 1 and 2 has been employed (Schore and Turro. 1975; Turro et al., 1980) as a “built-in” fluorescence probe, Le., the detergent itself is functionalized. The fluorophore of 1 and 2 is characterized by a strong solvent dependence of its fluorescence maximum and of its fluorescence lifetime. For example, both 1 and 2, upon micellization exhibit a strong “blue shift” of their fluorescence maxima and a reduction of their

1 2

*To whom correspondence should be addressed. Present address: Israel Institute for Biological Research, Dept. of Chemistry, Ness-Ziona 70450, Israel.

?The slight variations in the concentration of the deter- gent at the inflection point result from the presence of another component (the quencher) in the solution (Fendler and Fendler, 1975).

fluorescence lifetimes relative to that in dilute aqueous solution (i.e. below the CMC).

MATERIALS AND METHODS

The preparations of the detergents 1 and 2 have been described previously. Aqueous solutions of 2 X M detergent (above CMC) and a given concentration of quencher were diluted with aqueous solution of the quen- cher to generate solutions that were at constant concentra- tion of the quencher. The fluorescence intensity was measured for each concentration of detergent and corre- lated with a comparable detergent solution of known fluorescence intensity, but without quencher. The intensi- ties were normalized for changes in absorption at the exciting wavelength. The ratio of quantum yields in the absence and presence of quencher were plotted against detergent concentration to produce the curves shown in Figs. 1 and 2.

RESULTS

The use offuorescence quenching as a method for determining the CMC of I and 2

The indole moiety of 1 and 2 is expected to be “exposed” to aqueous quenchers below the CMC, but is “protected” from aqueous quenchers above the CMC. Thus for a given quencher, a Stern-Volmer analysis would reveal a different quenching constant below and above the CMC. Therefore, the CMC may be determined from a plot of the Stern-Volmer quenching constant as a function of the detergent concentration.

The Co2+ ion, and 3-aminophthalimide were em- ployed as aqueous phase quenchers of the fluoresc- ence of 1 (Fig. 1). An increase in quenching efficiency (&JI$~) is observed upon dilution of a stock solution of detergent from M to about 2 x M t . A

447

448

t: 3

2 -

I -

&

GAVRIELLA GABOR and NICHOLAS J . TURRO

I I -

c_ 4. +el 20

18

16

14

I2

lo

a 6

4

Figure 1 .

i w Quenching efficiency (cb,,/&J of Co2+ in 6-In-ll+, and 6-In-I 1- detergent solutions through

CMC. [ C O + ~ ] = 2.5 X M.

0

0

Figure 2. Ouenching efficiency (+,,/+J of +.CO in 6-In-1 1 + (0. 0 ) and 6-In-I I (A. 0) detergent solutions, b2C0 (=benzophenone) concentrations: 7: IS x 10 ' M (0). 10 ' M (0). 2.86 x 10 ' M (A.

a).

Research Note 449

Table 1. The Ksy values (Stern-Volmer quenching constants) for “aqueous” and “micellar” quenchers in 6-In-1 1 solutions of various concentrations

Quencher Concentration of ouencher DetKonc. K,, W+,, Location

Cp’ + 2.5 X lo-’ aq 6-In-11+/6.6 x 765 17.0 1 x 107 185 1.3

TCE

6-In-ll-/6.7 x 1060 4.5 2 x 10- ‘‘ 830 1.3

7.4 x 10-3 mic 6-In-11’12.7 x lo-‘ 90 2.0 1 x 1 0 F 440f50 10.0

Benzophenone 1.43 X lo-‘ mic 6-In-llf/6.4 X lo-‘‘ 4480 1.5 1.3 X lo-’ 10200 2.6

2.86 x lo-‘‘ 6-In-ll-/1.37 x lo-‘‘ 1060 1.5 2.2 x 10-3 5500 2.7

3-Aminophthalimide - aq 6-In-ll+/h.6 x 10-‘ 15000 -

3.3 x 1080 -

similar change. although to a smaller extent, is obtained when the fluorescence of 2 is quenched by Co2+ (Fig. 1).

Benzophenone, a molecule expected to reside dominantly in the micellar phase, was also employed as a quencher of the fluorescence of 1 and 2 (Fig. 2). In the case of benzophenone the decrease of quen- ching efficiency as a function of the detergent concentration, is apparent for both 1 and to a smaller extent for 2 (Fig. 2). c $ ~ , / + ~ , in the presence of trichloroethanol (TCE)-another non-polar quen- cher (Eftink et a l . , 1977), was also considerably reduced on dilution of 1 (Table 1). It should be emphasized that in these experiments the macrosco- pic quencher concentration is held fixed as the detergent concentration is increased. Thus, the Stern-Volmer constant increases as the ratio [ben- zophenone]/[detergent] decreases. The portion of the curves in Figs. 1 and 2, for which on dilution of the detergent a well pronounced change of c$(, /+~ occurs, are related to the “CMC region”.

Table 1 summarizes the variation of the Stern- Volmer constant (K+,) derived from the data in Figs. 1 and 2 for premicellar and postmicellar concentra- tions of detergent.

Variation inpuorescence spectra induced by aqueous and micellar quenchers

Micelle formation occurs in solutions of 1 and 2 as they reach a concentration of A4 (Figs. 1 and 2): in good agreement with the CMC of 1.5 X A4 published previously (Schore and Turro, 1975). Addition of Coz+ to a solution of 1 at a concentration near the CMC caused a “blue shift” in the fluoresc- ence maximum. This result is consistent with the specific quenching of the aqueous phase by Co2+. Since the aqueous detergent exhibits a fluorescence maximum at 373 nm, whereas the micellar detergent exhibits a fluorescence maximum at 356 nm, specific

quenching of the longer wavelength fluorescence by the aqueous quencher leads to a “blue shift” of about 10 nm in the observed fluorescence maximum. In contrast, addition of benzophenone to solutions of 1 in the same concentration range, i.e. close to the CMC. results in a “red shift” (10 nm) of fluorescence. This result is consistent with specific micellar quen- ching of the fluorescence of 1.

DISCUSSION

The data in Figs. 1 and 2 and in Table 1 demon- strate that the Stern-Volmer constants for quenching of the fluorescence of detergents 1 and 2 are a strong function of detergent concentration. Furthermore, the constant may increase or decrease as a function of concentration depending upon whether the quencher is dominantly located in the aqueous phase or in the micellar phase. The CMC’s of 1 and 2 have been determined to be 1.5 x M by the fluorescence maximum shift method (Schore and Turro. 1975). Since it is in this concentration range that the quenching efficiency changes most dramatically, we conclude that the variation in Stern-Volmer con- stants as a function of the detergent concentration is due to the formation of micelles. This conclusion is consistent with the observed “red shift” of the fluorescence maximum of 1 upon addition of ben- zophenone and the observed “blue shift” of the fluorescence maximum of 1 upon addition of Coz+.

The change of the Stern-Volmer constant is always bigger for 1 as compared to 2 for a given quencher, indicating a larger change of the polarity on micelliza- tion. A similar difference in the polarity of the interiors of a positively charged, -N+(CH& groups covered -HDTBr-, and the negatively charged -0SO3- covered -SDS-, micelles was suggested recently by us (Turro et af., 1980). The change of K,, for 3-aminophthalimide is considerably greater, 15 fold. compared to 4 fold decrease for Coz+, on

450 GAVRIELLA GABOR and NICHOLAS J. TURRO

micellization implying that the larger organic mole- cule has less access to the indole moiety in the tightly packed 6-In-ll+ micelle.

Acknowledgements-The authors thank the National Insti- tutes of Health for their generous support for this work. Dr. Bernhard Kreutler is thanked for his assistance in preparing 6-In-ll-.

REFERENCES Brand, L. and J. R. Gohlke (1972) Ann. Rev. Biochem.

Edelrnan, G. M. and W. 0. McClure (1968) Acc. Chem. 41, 843-868.

Res. 1, 65-70.

Eftink, M. R . , J . L. Zajicek and C. A. Ghiron (1977) Biochem. Biophys. Acta 491, 473-481.

Fendler, J . H. and E. J. Fendler (1975) Catalysis in Micellar and Macromolecular Systems 306 pp. A. P.

Hautala, R. R. and N. J . Turro (1972) Mol. Photochem. 4, 545-550.

Hautala, R. R., N. E. Schore and N. J. Turro (1973) J . Am. Chem. Soc. 95, 5508-5514. And other references quoted therein.

Kalyanasundaram, R. and J . K. Thomas (1977) J. Phys. Chem. 81,2176-2180.

Khuanga, U., B. K. Selinger and R. McDonald (1976) Awt. J. Chem. 29, 1-12.

Schore, N. E. and N. J. Turro (1975) J . Am. Chem. SOC. 97,2488-2496.

Turro, N. J . , Y . Tanirnoto and G . Gabor (1980) Photochem. Photobiol. 31, 527-532.