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Interaction of a hydrophobic fluorescent probe with mouse embryo fibroblasts
Prinfed in Sweden Copyright Q 1973 by Academic Press, Inc. Ail rights of reproduction in any form reserved
Experimental
Cell Research 81 (1973) 139-142
INTERACTION FLUORESCENT
OF A HYDROPHOBIC
PROBE WITH MOUSE EMBRYO
FIBROBLASTS
GISELA WITZ, A. SIVAK and B. L. VAN DUUREN Laboratory
of Organic Chemistry and Carcinogenesis, Institute of Environmental Medicine, New York University Medical Center, New York, N. Y. 10016, USA.
SUMMARY Fluorescence photomicrographs show that the hydrophobic fluorescent probe l-anilinonaphthalene-8-sulfonate (ANS) binds to hydrophobic components of intact 3T3 ceils. Cells exposed to ANS exhibit fluorescence in the cytoplasm, intense nuclear membrane fluorescence, and well-defined fluorescent nucleoli. Fluorescence titrations of 3T3 cells with ANS show a decrease in fluorescence intensity, a blue shift of native cell emission with increasing ANS concentration and the appearance of a new peak due to ANS fluorescence. These fluorescence effects are ascribed to energy transfer processes involving bound ANS and the tryptophan and tyrosine residues of cellular proteins. ANS bound to 3T3 cells appears to quench the long wavelength component of the cellular tryptophan fluorescence, resulting in an unmasking of tryptophan and tyrosine emission at shorter wavelengths.
Hydrophobic fluorescence probes, such as l-anilinonaphthalene+sulfonate (ANS)have been used in conformational studies of proteins and cell membranes [l, 21. Compounds of this type do not fluoresce significantly in water; however, they fluoresce intensely in apolar environments, such as the hydrophobic regions of proteins or lipid micelles. The fluorescence characteristics observed yield information about the nature of their binding sites and hence also about the conformations of the proteins and/or lipids to which they are bound [3-51. This report describes the use of this method for the examination of such hydrophobic regions in 3T3 mouse embryo fibroblasts. The binding of ANS to the cells was examined by fluorescence microscopy and by fluorimetric titrations of 3T3 cells in suspension with ANS. The fluorescence microscopic exami-
nation yielded information about the intracellular localization of ANS and the fluorimetric titrations allowed the examination of the effect of ANS binding on native cell fluorescence.
MATERIALS
AND METHODS
Cell culture. The 3T3 cells were grown to confluence in 100 mm plastic dishes as described previously [6]. The 3T3 line of mouse fibroblasts was originally obtained in 1965 from Dr G. J. Todaro, Department of Pathology, New York University School of Medicine. The cultures used in these studies were obtained from frozen stocks derived from the original culture. After removal of the medium, confluent cultures were washed twice with phosphate buffered saline (PBS), pH 7.4, treated with 0.02% EDTA in PBS, and the cells dispersed by scraping with a Teflon policeman. The cell suspensions from six dishes were pooled and centrifuged. The cell pellet was washed with cold PBS, centrifuged and resuspended in 2 ml cold PBS. A uniform suspension of single cells was obtained by gentle pipetting. Exptl Cell Res 81 (1973)
Fig. 1. Fluorescence photomicrographs
of 3T3 cells treated with ANS. The photomicrographs (500 ): ) show diffuse fluorescence of the cytoplasm, heavy nuclear membrane fluorescence, and well-defined fluorescent nucleoli (a) 2.0 x 10” cells/ml, 2.0 x 1O-6 M ANS (as the magnesium salt); (6) 2.0 x lo6 cells/ml, 9.5 x 10m5M ANS (as the magnesium salt). The magnesium salt of ANS was prenared bv dissolving the free acid in hot water. filtering the solution through a layer of charcoal and addine: an excess of MzCl,.6H,O. The hot solution was saturated with MgCOi and the resulting needles were recrvstallized from saturated aaueous MgCO,. The magnesium salt had a melting-point of 301.5-303°C (d) and showed a single spot on a thin-layer chromatogram, silica gel, with ethanol as solvent; R,=0.83. The elemental analysis (C, H, N, Mg) of the salt agreed well with the calculated values for (C,,H,,O,S,), Mg. 5Hz0. ANS preparation.
RESULTS
After treatment with ANS, 3T3 cells exhibit a diffuse fluorescence of the cytoplasm (fig. 1). The plasma membrane does not fluoresce significantly. By contrast, the nuclear membrane fluoresces strongly and is sharply defined. The nucleus itself is non-fluorescent except for the nucleoli. At the highest concentration of ANS used (9.5 x 10e5 M per Fluorescence microscopy. The magnesium salt of ANS in PBS was added to aliquots of the cell 2 x lo6 cells/ml) there was no lytic effect on suspension. The concentration of the magnesium salt of ANS used ranged from 2.0 x 1O-5 M to 9.5 x 1O-5 3T3 cells; the higher concentration resulted in M per 2 x lo8 cells/ml. Samples of ANS-treated cells brighter fluorescence of bound ANS. were wet-mounted under coverslips on flat glass In the absence of ANS, 3T3 cells susslides. The cells were examined and photographed in a Zeiss photomicroscope equipped with an epipended in PBS show a single broad emission illuminator and a vertically mounted camera. The samples were excited at 400 nm by light from a peak with a maximum at 330 nm; this emis200 W Xenon lamp fitted with a grating monosion maximum is independent of the excitachromator. The emitted light was filtered through 500 nm barrier filters (Zeiss) in the eye-pieces and a tion wavelengths used, 295, 280 and 260 nm. 500 fl dichroic lens (Zeiss) in the epi-illuminator. Several authors have obtained evidence The cells were photographed at a magnification of that both tyrosine and tryptophan contrib500 x on 35 mm Kodak Plus X film with exposure times ranging from 1 to 2 min. ute to the fluorescence emission of certain proteins. This subject has been reviewed Fluorescence spectrophotometry. For the spectrophotometric studies, 3T3 cell pellets from 15 dishes recently [8]. of confluent cultures were obtained in a similar wav In the presenceof increasing concentrations as those for the microscopic studies, except that treatment with EDTA was omitted. The cell uellet of ANS, a blue shift of the native emission was suspended in 5 ml cold PBS, dispersed by gentle pinetting and stored in an ice-water bath until used maximum occurs and there is a progressive (about 2-3 h). Aliquots of 0.3 ml were withdrawn, decrease in fluorescence intensity, as the an auwopriate volume of lo-* M ANS added. and concentration of ANS is increased. The diluted with buffer to a final volume of 2.5 ml. Corrected emission spectra in quantum units were emission maximum at 485 nm which beobtained using a specially designed automatically comes apparent at higher concentrations corrected spectrofluorimeter [7]. Exptl Cell Res 81 (1973)
Fluorescent studies on mouse embryo ribroblasts
141
the blue shifts which result upon ANS binding. This binding probably causesalterations of the energy transfer processes involving tyrosine and tryptophan residues in proteins. The decrease in intensity of the native
emission
may
be
due
in
part
to
energy transfer from tryptophan [9] and tyrosine to ANS. Part of the quenching of the native emission is most likely due to ‘trivial’ quenching effects which are due to reabsorption of emitted native fluorescence by unbound ANS. The various causes of fluorescence quenching have been summarized in a recent review article [lo]. The blue shift of the native emission at a constant wavelength of excitation (280 nm) Fig. 2. Abscissa: wavelength (nm); ordinate: ref. fluorescence intensity. with increasing ANS binding suggests a Corrected fluorescence emission spectra of 3T3 cells in the presence of ANS. The cell density of each quenching of the long wavelength component sample was 1.3 x lo6 cells/ml; 5 nm slits were emof the tryptophan emission. This results in ployed both on the analyzer and exciter side and the an unmasking of the short wavelength comwavelength of excitation was kept constant at 280 nm. The final ANS concentrations (as the magnesium salt) ponents of the cellular tryptophan and also were: (1) 0.0 ANS; (2) 0.8 x 10d5M; (3) 2 x 1O-5M; particularly the tyrosine emission. This type (4) 4.5 x 10m5M; (5) 6 x 1O-5 M; (6) 8 x 1O-5 M; (7) 1.6 x 1O-4 M. of tyrosine emission has been observed as a shoulder at 303 nm in human erythrocyte membranes [ll]. The conclusion that the of ANS is due to the fluorescence of the probe bound in an apolar environment (fig. short wavelength components of cellular 2). This fluorescence increases with in- emission can be observed after ANS binding creasing ANS concentration. At any one is supported by the blue shifts in the presence ANS concentration, for example, 8 x 10e5M of a constant concentration of ANS with ANS, and a cell density of 1.34 x lo6 cell/ml, varying wavelengths of excitation. These studies indicate the feasibility of the native emission maximum varies with the wavelength of excitation, e.g. 322 nm at applying the hydrophobic fluorescent probe A,, 291; 318 nm at 1,, 280; and 312 nm at technique to whole cells in suspension. Since A,, 266, i.e. the blue shift in native fluorescence binding of ANS allows for observation of emission becomes larger with increasing emission components not accessible in the absence of ANS, refined studies of this shorter wavelengths of excitation. type should provide new information concerning cellular fluorescence and the effects DISCUSSION of various chemical agents on native emission. From the findings reported above, it appears We thank Dr S. Wolman, Department of Pathology, that the binding of ANS to 3T3 cells occurs for the cytological descriptions of the cells and for at least, in part, with cellular proteins. This the use of the Zeiss photomicroscope. We thank Miss Lydia McMorrow for the technical assistance conclusion is supported by the observed with the photomicroscopy. decreaseof the native emission together with The fluorescence Microscope Spectrum Analyzer Exptl Cell Res 81 (1973)
142 Gisela Witz et al. containing the grating monochromator was kindly loaned to us by the Farrand Optical Company, Valhalla, N.Y. The authors are indebted to Mr S. Cravitt of the Farrand Co. for arranging this loan. This work was supported by USPHS grants CA08580 and ES-00260.
REFERENCES 1. McClure, W 0 & Edelman, G M, Biochemistry 6 (1967) 559. 2. Azzi, A, Chance, B, Radda, G K & Lee C P, Proc natl acad sci US 62 (1969) 612. 3. Stryer, L, Science 162 (1968) 526. 4. McClure, W 0 & Edelman, G M, Biochemistry 5 (1966) 1908. 5. Dyckman J & Weltman, J K. J cell bio145 (1970) 192.
Exptl Cell Res 81 (1973)
6. Sivak, A & Van Duuren, B L, Exptl cell res 49 (1968) 572. 7. Cravitt S & Van Duuren, B L, Chem instr I (1968) 71. 8. Barenboim. G M. Domanskii. A N & Turoverov. K K, Luminescence of biopolymers and cells; p. 31. Plenum Press, New York-London (1969). 9. Wallach, D F H, Ferber, E, Selin, D, Weidekamm, E & Fischer, H, Biochim bioohvs _ - acta 203 (1970) 67. 10. Van Duuren, B L & Chan, T-L, Spectrochemical methods of analysis: quantitative analysis of atoms and molecules (ed J Winefordner) vol. 1, chapter VII pp. 388-450. Wiley, New York (1971). 11. Sonnenberg, M, Proc natl acad sci US 68 (1971) 1051. Received December 28, 1972
Experimental
Cell Research 81 (1973) 139-142
INTERACTION FLUORESCENT
OF A HYDROPHOBIC
PROBE WITH MOUSE EMBRYO
FIBROBLASTS
GISELA WITZ, A. SIVAK and B. L. VAN DUUREN Laboratory
of Organic Chemistry and Carcinogenesis, Institute of Environmental Medicine, New York University Medical Center, New York, N. Y. 10016, USA.
SUMMARY Fluorescence photomicrographs show that the hydrophobic fluorescent probe l-anilinonaphthalene-8-sulfonate (ANS) binds to hydrophobic components of intact 3T3 ceils. Cells exposed to ANS exhibit fluorescence in the cytoplasm, intense nuclear membrane fluorescence, and well-defined fluorescent nucleoli. Fluorescence titrations of 3T3 cells with ANS show a decrease in fluorescence intensity, a blue shift of native cell emission with increasing ANS concentration and the appearance of a new peak due to ANS fluorescence. These fluorescence effects are ascribed to energy transfer processes involving bound ANS and the tryptophan and tyrosine residues of cellular proteins. ANS bound to 3T3 cells appears to quench the long wavelength component of the cellular tryptophan fluorescence, resulting in an unmasking of tryptophan and tyrosine emission at shorter wavelengths.
Hydrophobic fluorescence probes, such as l-anilinonaphthalene+sulfonate (ANS)have been used in conformational studies of proteins and cell membranes [l, 21. Compounds of this type do not fluoresce significantly in water; however, they fluoresce intensely in apolar environments, such as the hydrophobic regions of proteins or lipid micelles. The fluorescence characteristics observed yield information about the nature of their binding sites and hence also about the conformations of the proteins and/or lipids to which they are bound [3-51. This report describes the use of this method for the examination of such hydrophobic regions in 3T3 mouse embryo fibroblasts. The binding of ANS to the cells was examined by fluorescence microscopy and by fluorimetric titrations of 3T3 cells in suspension with ANS. The fluorescence microscopic exami-
nation yielded information about the intracellular localization of ANS and the fluorimetric titrations allowed the examination of the effect of ANS binding on native cell fluorescence.
MATERIALS
AND METHODS
Cell culture. The 3T3 cells were grown to confluence in 100 mm plastic dishes as described previously [6]. The 3T3 line of mouse fibroblasts was originally obtained in 1965 from Dr G. J. Todaro, Department of Pathology, New York University School of Medicine. The cultures used in these studies were obtained from frozen stocks derived from the original culture. After removal of the medium, confluent cultures were washed twice with phosphate buffered saline (PBS), pH 7.4, treated with 0.02% EDTA in PBS, and the cells dispersed by scraping with a Teflon policeman. The cell suspensions from six dishes were pooled and centrifuged. The cell pellet was washed with cold PBS, centrifuged and resuspended in 2 ml cold PBS. A uniform suspension of single cells was obtained by gentle pipetting. Exptl Cell Res 81 (1973)
Fig. 1. Fluorescence photomicrographs
of 3T3 cells treated with ANS. The photomicrographs (500 ): ) show diffuse fluorescence of the cytoplasm, heavy nuclear membrane fluorescence, and well-defined fluorescent nucleoli (a) 2.0 x 10” cells/ml, 2.0 x 1O-6 M ANS (as the magnesium salt); (6) 2.0 x lo6 cells/ml, 9.5 x 10m5M ANS (as the magnesium salt). The magnesium salt of ANS was prenared bv dissolving the free acid in hot water. filtering the solution through a layer of charcoal and addine: an excess of MzCl,.6H,O. The hot solution was saturated with MgCOi and the resulting needles were recrvstallized from saturated aaueous MgCO,. The magnesium salt had a melting-point of 301.5-303°C (d) and showed a single spot on a thin-layer chromatogram, silica gel, with ethanol as solvent; R,=0.83. The elemental analysis (C, H, N, Mg) of the salt agreed well with the calculated values for (C,,H,,O,S,), Mg. 5Hz0. ANS preparation.
RESULTS
After treatment with ANS, 3T3 cells exhibit a diffuse fluorescence of the cytoplasm (fig. 1). The plasma membrane does not fluoresce significantly. By contrast, the nuclear membrane fluoresces strongly and is sharply defined. The nucleus itself is non-fluorescent except for the nucleoli. At the highest concentration of ANS used (9.5 x 10e5 M per Fluorescence microscopy. The magnesium salt of ANS in PBS was added to aliquots of the cell 2 x lo6 cells/ml) there was no lytic effect on suspension. The concentration of the magnesium salt of ANS used ranged from 2.0 x 1O-5 M to 9.5 x 1O-5 3T3 cells; the higher concentration resulted in M per 2 x lo8 cells/ml. Samples of ANS-treated cells brighter fluorescence of bound ANS. were wet-mounted under coverslips on flat glass In the absence of ANS, 3T3 cells susslides. The cells were examined and photographed in a Zeiss photomicroscope equipped with an epipended in PBS show a single broad emission illuminator and a vertically mounted camera. The samples were excited at 400 nm by light from a peak with a maximum at 330 nm; this emis200 W Xenon lamp fitted with a grating monosion maximum is independent of the excitachromator. The emitted light was filtered through 500 nm barrier filters (Zeiss) in the eye-pieces and a tion wavelengths used, 295, 280 and 260 nm. 500 fl dichroic lens (Zeiss) in the epi-illuminator. Several authors have obtained evidence The cells were photographed at a magnification of that both tyrosine and tryptophan contrib500 x on 35 mm Kodak Plus X film with exposure times ranging from 1 to 2 min. ute to the fluorescence emission of certain proteins. This subject has been reviewed Fluorescence spectrophotometry. For the spectrophotometric studies, 3T3 cell pellets from 15 dishes recently [8]. of confluent cultures were obtained in a similar wav In the presenceof increasing concentrations as those for the microscopic studies, except that treatment with EDTA was omitted. The cell uellet of ANS, a blue shift of the native emission was suspended in 5 ml cold PBS, dispersed by gentle pinetting and stored in an ice-water bath until used maximum occurs and there is a progressive (about 2-3 h). Aliquots of 0.3 ml were withdrawn, decrease in fluorescence intensity, as the an auwopriate volume of lo-* M ANS added. and concentration of ANS is increased. The diluted with buffer to a final volume of 2.5 ml. Corrected emission spectra in quantum units were emission maximum at 485 nm which beobtained using a specially designed automatically comes apparent at higher concentrations corrected spectrofluorimeter [7]. Exptl Cell Res 81 (1973)
Fluorescent studies on mouse embryo ribroblasts
141
the blue shifts which result upon ANS binding. This binding probably causesalterations of the energy transfer processes involving tyrosine and tryptophan residues in proteins. The decrease in intensity of the native
emission
may
be
due
in
part
to
energy transfer from tryptophan [9] and tyrosine to ANS. Part of the quenching of the native emission is most likely due to ‘trivial’ quenching effects which are due to reabsorption of emitted native fluorescence by unbound ANS. The various causes of fluorescence quenching have been summarized in a recent review article [lo]. The blue shift of the native emission at a constant wavelength of excitation (280 nm) Fig. 2. Abscissa: wavelength (nm); ordinate: ref. fluorescence intensity. with increasing ANS binding suggests a Corrected fluorescence emission spectra of 3T3 cells in the presence of ANS. The cell density of each quenching of the long wavelength component sample was 1.3 x lo6 cells/ml; 5 nm slits were emof the tryptophan emission. This results in ployed both on the analyzer and exciter side and the an unmasking of the short wavelength comwavelength of excitation was kept constant at 280 nm. The final ANS concentrations (as the magnesium salt) ponents of the cellular tryptophan and also were: (1) 0.0 ANS; (2) 0.8 x 10d5M; (3) 2 x 1O-5M; particularly the tyrosine emission. This type (4) 4.5 x 10m5M; (5) 6 x 1O-5 M; (6) 8 x 1O-5 M; (7) 1.6 x 1O-4 M. of tyrosine emission has been observed as a shoulder at 303 nm in human erythrocyte membranes [ll]. The conclusion that the of ANS is due to the fluorescence of the probe bound in an apolar environment (fig. short wavelength components of cellular 2). This fluorescence increases with in- emission can be observed after ANS binding creasing ANS concentration. At any one is supported by the blue shifts in the presence ANS concentration, for example, 8 x 10e5M of a constant concentration of ANS with ANS, and a cell density of 1.34 x lo6 cell/ml, varying wavelengths of excitation. These studies indicate the feasibility of the native emission maximum varies with the wavelength of excitation, e.g. 322 nm at applying the hydrophobic fluorescent probe A,, 291; 318 nm at 1,, 280; and 312 nm at technique to whole cells in suspension. Since A,, 266, i.e. the blue shift in native fluorescence binding of ANS allows for observation of emission becomes larger with increasing emission components not accessible in the absence of ANS, refined studies of this shorter wavelengths of excitation. type should provide new information concerning cellular fluorescence and the effects DISCUSSION of various chemical agents on native emission. From the findings reported above, it appears We thank Dr S. Wolman, Department of Pathology, that the binding of ANS to 3T3 cells occurs for the cytological descriptions of the cells and for at least, in part, with cellular proteins. This the use of the Zeiss photomicroscope. We thank Miss Lydia McMorrow for the technical assistance conclusion is supported by the observed with the photomicroscopy. decreaseof the native emission together with The fluorescence Microscope Spectrum Analyzer Exptl Cell Res 81 (1973)
142 Gisela Witz et al. containing the grating monochromator was kindly loaned to us by the Farrand Optical Company, Valhalla, N.Y. The authors are indebted to Mr S. Cravitt of the Farrand Co. for arranging this loan. This work was supported by USPHS grants CA08580 and ES-00260.
REFERENCES 1. McClure, W 0 & Edelman, G M, Biochemistry 6 (1967) 559. 2. Azzi, A, Chance, B, Radda, G K & Lee C P, Proc natl acad sci US 62 (1969) 612. 3. Stryer, L, Science 162 (1968) 526. 4. McClure, W 0 & Edelman, G M, Biochemistry 5 (1966) 1908. 5. Dyckman J & Weltman, J K. J cell bio145 (1970) 192.
Exptl Cell Res 81 (1973)
6. Sivak, A & Van Duuren, B L, Exptl cell res 49 (1968) 572. 7. Cravitt S & Van Duuren, B L, Chem instr I (1968) 71. 8. Barenboim. G M. Domanskii. A N & Turoverov. K K, Luminescence of biopolymers and cells; p. 31. Plenum Press, New York-London (1969). 9. Wallach, D F H, Ferber, E, Selin, D, Weidekamm, E & Fischer, H, Biochim bioohvs _ - acta 203 (1970) 67. 10. Van Duuren, B L & Chan, T-L, Spectrochemical methods of analysis: quantitative analysis of atoms and molecules (ed J Winefordner) vol. 1, chapter VII pp. 388-450. Wiley, New York (1971). 11. Sonnenberg, M, Proc natl acad sci US 68 (1971) 1051. Received December 28, 1972