Proc. Nat. ASad. Sci. USA Vol. 69, No. 3, pp. 769-772, March 1972

Circular Polarization of Fluorescence of Probes Bound to Chymotrypsin. Change in Asymmetric Environment upon Electronic Excitation* (circularly polarized luminescence/proteins/fluoresent probes) JOSEPH SCHLESSINGERt AND IZCHAK Z. STEINBERG Chemical Physics Department, Weizmann Institute of Science, Rehovot, Israel Communicated by Harold A. Scheraga, January 13, 197S

ABSTRACT The circular polarization of fluorescence excited. The site, strength, rigidity, and geometry of binding is related to the conformational asymmetry of the emitting of a fluorescent probe to a protein molecule may thus molecule in the first singlet excited state in the same way differ that circular dichroism is related to the conformational in the ground and excited states. The use of fluorescence asymmetry of the absorbing molecule in the electronic for the study of the binding and the environment of the ground state. By measurement of these optical pheno- chromophore in the ground state, therefore, involves the mena, the induced asymmetry of two chromophores bound basic assumption that in the case under investigation no to chymotrypsin (EC 3.4.4.5) when in the ground state Was compared with the induced asymmetry of the ligands major changes have occurred in the bound chromophore when in the excited state. The two chromophores studied upon electronic excitation. Detection and examination of such were 2-p-toluidinylnaphthalene-6.sulfonate (TNS), bound changes is therefore of much interest and importance. at a specific site which is not the active site of the protein, In this study, the changes in the interaction of a protein and an anthraniloyl group, bound at the active site of the molecule and an attached extrinsic chromophore caused by enzyme. Both chromophores showed a change in induced asymmetry upon electronic excitation, the effect being electronic excitation of the chromophore are followed by the particularly large in the case of the TNS chromophore. optical rotatory power of the chromophore in the ground It is thus concluded that the orientation of TNS in the state and the excited state. Asymmetric molecules or chromo- binding site, its freedom of rotation in its site, the strength phores situated in an asymmetric environment are, as a rule, of binding, or even the site of binding of the dye to the protein might have changed upon electronic excitation. circularly dichroic, i.e., they absorb left-handed and right- handed circularly polarized light to different extents. In Fluorescent small molecules bound to proteins are useful as other words, the probability of transition from the ground probes in the study of the structure of proteins. Thus, the to the excited state differs when such molecules or chromo- intensity and spectrum of emission of various fluorescent phores are exposed to right-handed or left-handed circularly dyes have yielded invaluable information concerning the polarized light. An absorption band is circularly dichroic degree of polarity of the sites at which the dyes are bound if the relevant excitation involves both an electric and a (1, 2). Similarly, the extent of linear polarization of the light magnetic transition dipole moment and if the two transition emitted by dyes bound to proteins has been used for the moments are not orthogonal in space (7). Analogously, when investigation of the rotational diffusion of the protein mole- the above conditions are fulfilled for a radiative transition cules, or of segments of the molecules, and has thus allowed from an excited state to the ground state, i.e., in luminescence, deductions concerning the size and rigidity of the macro- the light emitted by the luminescent molecules in solution molecules (2-4). will be partly circularly polarized (7, 8). If the absorption In the interpretation of fluorescence data in terms of and emission processes involve exactly the same pairs of molecular structure one should be aware of the fact that the quantum states, the extent of circular dichroism (CD) and the fluorescence emitted by a chromophore is related to, and degree of circular polarization of the luminescence are related yields information about, the electronically excited chro- to each other by a simple expression (see below). It may be mophore (5, 6). It is well known that molecules in the excited recalled that, with few exceptions, fluorescence involves a state may have both physical and chemical properties that transition between the same pair of electronic states (Si -- So) are markedly different from those of the molecules in the as the absorption process in the long-wavelength absorption ground- state. Though short (about 10-9-10-7 sec for fluores- band (So SO). The vibrational states involved and the cence), the lifetime of the molecules in the excited state is positions of the nuclei are, however, different in the two still long enough to allow many reactions and conformational processes. Changes in the conformation or environment of an changes to take place during the period the molecule is optically active chromophore are thus readily disclosed by a comparison of the CD of the chromophore and the circular Abbreviation: TNS, 27p-toluidinylnaphthalene-6-sulfonate. polarization of its fluorescence. Circular polarization of * Presented in part at the 41st Annual Meeting of the Israel fluorescence emitted by systems of biological interest has Chemical Society, October 1971. apparently not been reported before. t Part of Ph.D. thesis to be submitted by J. Schlessinger to the Haugland and Stryer have prepared an anthraniloyl Feinberg Graduate School, The Weizmann Institute of Science; derivative of a-chymotrypsin (EC 3.4.4.5) in which the Rehovot, Israel. attached chromophore is covalently bound at the active site 769 Downloaded by guest on September 29, 2021 770 Biochemistry: Schlessinger and Steinberg Proc. Nat. Acad. Sci. USA 69 (1972)

of the enzyme (9). This derivative was used to study the amplified and monitored. Full details of the instrument and rotational diffusion of a-chymotrypsin by the measurement the procedures for its calibration are described elsewhere of the linear polarization of the anthraniloyl fluorescence (9). (12). The spectral bandwidth for the excitation beam was McClure and Edelman have prepared another derivative of 30 nm, while the spectral resolution of the emitted light was a-chymotrypsin in which 2-p-toluidinylnaphthalene-6-sul- 15 nm. fonate (TNS) is bound noncovalently at a specific site, The protein solutions were deaerated by purified nitrogen which is not the active site (10). From the intensity and prior to the measurements of circular polarization of the spectrum of the fluorescence of the dye attached to the protein, luminescence and were kept under nitrogen in a hermetically it was deduced that the binding site is hydrophobic. The closed fluorescence cell during the measurements. The asymmetry induced in' these chromophores upon binding is exclusion of oxygen was necessary since the intense and reported herewith. Changes in the induced asymmetry of the continuous illumination of the solutions caused a decrease anthraniloyl and TNS chromophores were observed upon in fluorescence if oxygen was present, probably as a result electronic excitation of these chromophores, the change for of some photooxidation reactions. The deaeration was the TNS chromophore being particularly large. performed in the apparatus shown in Fig. 1. The solvent MATERIALS AND METHODS (and TNS in the studies of the chymotrypsin-TNS complex) was placed in the flask (A), and a weighed amount of protein a-Chymotrypsin was a product of Worthington Biochemical was placed in the test tube (B). Purified nitrogen gas was Corp., Lot no. CDI 8LK. p-Nitrophenyl anthranilate was a bubbled through the solvent for about 3 hr. The solvent was gift from Mr. A. Carmel, who prepared the material (Carmel, then transferred to the test tube (B). After the protein A., to be published). 2-p-Toluidinylnaphthalene-6-sulfonate completely dissolved in the solvent, the solution was trans- (TNS) was a gift from Dr. M. Shinitzky. All other reagents ferred to the fluorescence cell (C), and the Teflon stopcock were of analytical grade. (D) was closed. This procedure practically eliminated the Anthraniloyl chymotrypsin was prepared according to the detrimental effect of the illumination on the protein solutions. procedure developed by Haugland and Stryer (9). It was The exact concentration of the solutes was determined lyophylized and kept refrigerated after preparation. The spectrophotometrically. measurements were made within 1 week after preparation All measurements were made at room temperature (about of the protein derivative. 220). We prepared the chymotrypsin-TNS complex by mixing the protein with the dye, the total concentrations in the RESULTS AND DISCUSSION mixture being 4 X 10-4 M and 2 X 10-6 M, respectively. The absorption and CD spectra of the chymotrypsin-TNS Fresh preparations were used daily. complex in the range of 335-400 nm are presented in Fig. 2. Corrected fluorescence spectra were obtained with a The concentrations of the protein and dye were 4 X 10-4 M Turner 210 "spectro" fluorimeter. Measurements of CD and 2 X 10-5 M, respectively, in 0.1 M Tris buffer (pH were made with a Spectropolarimeter model 60, Cary (Palo 8.05). The CD is expressed as Ai = El- Er, i.e., the difference Alto, Calif.), with accessory 6001 for measurements of CD. between the molar extinction coefficients for left-handed and The circular polarization of the luminescence emitted by the right-handed circularly polarized light. The spectrum of the probes attached to a-chymotrypsin was measured with an instrument built in this laboratory (11). In this instrument the molecules studied in solutions are raised to an electronically excited state by monochromated unpolarized light. The Fluorescence a.-. 6 circularly polarized component of the emitted light at In -7 various wavelengths is selectively modulated by an electro- 4 50-V optic light modulator, and the ac and dc components of the electric signal generated by the modulated beam are 2 0~~~~~~~~~~~~~~~~ 25 C, I 10

-1 go / -l

X>-2t XeA i m -2 2 'C-3 ~0 0~

-5 IIfII.-5 340 380 420 460 500 540 Wavelength rnm) FiG. 2. Spectroscopic data for the chymotrypsin-TNS com- plex. Upper: absorption (e) and fluorescence spectra. Lower: = L.JC A circular dichroism (Ae), absorption anisotropy factor [g9 (Ae/ e)], and emission anisotropy factor [g. = (2Af/f)]. Protein FIG. 1. Experimental arrangement for deaeration of the sol- concentration: 4 X 10-4 M; TNS concentration: 2 X 10-6 M. vent by nitrogen gas and for subsequent preparation of the pro- Solvent: 0.1 M Tris buffer (pH 8.05). Temperature: 22°. The tein solution and filling of the fluorescence cell under nitrogen. signal to noise ratio in the g. measurements was about 10:1. Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Circular Polarization of Fluorescence 771 absorption anisotropy factor, g{7 = (Ae/e) (13), is also presented in Fig. 2. The absorption and CD spectra in the range 335-400 nm are caused by the TNS chromophore. The TNS moiety in the chymotrypsin-TNS complex is bound at a specific site on the enzyme which is not the active site. The induced CD at the absorption band of the TNS group is a reflection of the asymmetry of the binding site for TNS or of the asymmetric mode of binding of the dye at the binding site of the enzyme. The fluorescence spectrum and the degree of circular polarization of the light emitted from the chymotrypsin-TNS x-2- -6 complex in the wavelength range- 400-540 nm are presented on the right in Fig. 2. The protein and dye concentrations, 31 as well as the solvent, were the same as in the measurements 300 340 380 420 460 500 540 of absorption and CD. The circular polarization is expressed Wavelength (nm) by the emission anisotropy factor, gH = (2Af/f), f being the FIG. 3. Spectroscopic data for anthraniloyl-chymotrypsin fluorescence intensity, while Af is the intensity of the circu- Upper: absorption (e) and fluorescence spectra. Lower: circular larly polarized component in the emitted light under identical dichroism (Ae), absorption anisotropy factor [gm = (Ac/e)], and conditions. (Af is not presented since the instrument is emission anisotropy factor [g. = (2Af/f)]. Concentration of calibrated to yield the ratio of Af to f.) Left-handed and protein derivative: 4 X 10-6 M. Solvent: 0.2 M phosphate buffer right-handed circular polarizations are given plus and minus (pH 6.8). Temperature: 22°. The signal to noise ratio in the g. signs, respectively. It may be noted that the fluorescence measurements was about 20:1. spectrum shown in Fig. 2 agrees with that published previ- ously for this complex of chymotrypsin (1). The fluorescence spectrum and the spectrum of the emission The spectrum for the circular polarization of luminescence anisotropy factor, g6, of anthraniloyl-chymotrypsin in the shown in Fig. 2 is a reflection of the asymmetry induced in the wavelength range 380-530 nm are presented on the right in TNS chromophore by the protein environment when the Fig. 3. The concentration of the protein derivative was dye is in the excited state. If the conformation and environ- 8 X 10-4 M in 0.2 M phosphate buffer (pH 6.8). [The ment of the chromophore in the excited state are the same as fluorescence and absorption spectra of this derivative of those in the ground state, and if the absorption and emission chymotrypsin shown in Fig. 3 agree with those published processes involve exactly the same pairs of quantum states, (9). ] The spectrum of the circular polarization of the lumines- the emission anisotropy factor, ge, should be of the same cence is a reflection of the asymmetry induced in the anthra- sign and magnitude as the absorption anisotropy factor, niloyl chromophore when in the electronically excited state. ga (7, 8, 14). Obviously, the value of ge obtained for the It may be noted that, in the present case also, ga and ge chymotrypsin-TNS complex is markedly smaller, by about differ in magnitude from each other throughout their respec- a factor of 10, than the value of gp obtained for this complex. tive spectra. The discrepancy between ga and gB is, however, An explanation of the relatively low value observed for ge smaller for anthraniloyl-chymotrypsin (ge ga about 1:2.5) based on the assertion that most of the TNS chromophores than for the chymotrypsin-TNS complex (ge: ga about 1:10). dissociate from the protein upon electronic excitation, and This quantitative difference in behavior between the two thereby lose their asymmetric environment, is unacceptable, complexes of chymotrypsin is possibly due in part to the since unassociated TNS in aqueous solution has an extremely fact that the anthraniloyl group is covalently bound, while low quantum yield (15). Therefore, the fluorescence of the the TNS group is noncovalently bound, to the enzyme. free TNS is hardly detected by the measurements performed. The observations described above for the chymotrypsin- Furthermore, the emission of TNS in aqueous solution is at TNS complex may have far-reaching consequences. The wavelengths longer than those of the fluorescence emission mode of interaction of chymotrypsin and TNS apparently shown in Fig. 2. It must therefore be concluded that TNS differs when the latter is in the ground or excited state. still binds to chymotrypsin when in the excited state, but in a The binding constants for the association of TNS with fashion markedly different from TNS in the ground state. chymotrypsin as obtained by fluorimetric measurements Upon electronic excitation the orientation of the chromophore may, therefore, not apply to the association of the protein in the binding site, its freedom of rotation in the site, the with the dye when the latter is in the ground electronic strength of binding, or even the site of binding might have state. Similarly, the hydrophobicity of the environment of changed. TNS in the protein complex as deduced from the fluorescence The experiments described above for the chymotrypsin- spectrum and intensity strictly applies to the environment TNS complex were repeated with anthraniloyl-chymotrypsin. of the electronically excited dye. Some caution must therefore The absorption and CD spectra-of anthraniloyl-chymotrypsin be exercised in drawing conclusions concerning the detailed in the range of 310-390 nm are shown in Fig. 3. The concen- characteristics of the binding site of the dye in the ground tration of the protein derivative was 4 X 10-5 M, in 0.2 M state. phosphate buffer (pH 6.8). The CD observed at the measured This study illustrates the potentialities of measurements spectral range is due to an electronic transition in the anthra- of circular polarization of fluorescence as a tool in revealing niloyl moiety in the chymotrypsin derivative and is a reflec- changes in interaction between a protein and a bound small tion of the asymmetric environment that the active site of molecule upon electronic excitation. It is hoped that this new chymotrypsin offers to the extrinsic chromophore. tool will find further application in probing the environment Downloaded by guest on September 29, 2021 772 Biochemistry: Schlessinger and Steinberg Proc. Nat. Acad. Sci. USA 68 (1972) of chromophores, both intrinsic and extrinsic, in a variety 8. Emies, C. A. & Oosterhoff, L. J. (1971) J. Chem. Phys. 54, of systems of biological interest. 4809-4819. 9. Haugland, R. P. & Stryer, L. (1967) Conformation of Bio- polymers, ed. Ramachandran, G. N. (Academic Press, 1. Edelman, G. M. & McClure, W. 0. (1969) Accounts Chem. NewYork), Vol. 1, pp. 321-335. Res. 1, 65-70. 10. McClure, W. 0. & Edelman, G. M. (1967) Biochemistry 6, 2. Stryer, L. (1968) Science 162, 526-533. 559-566. 3. Weber, G. (1953) Advan. Protein Chem., 8, 415-459. 11. Steinberg, I. Z., Patent Application no. 36297, Israel. 4. Yguerabide, J., Epstein, H. F. & Stryer, L. (1970) J. Mol. 12. Steinberg, I. Z. & Gafni, A., Rev. Sci. Instrum., in press. Biol. 51, 573-590. 13. Kuhn, W. (1930) Trans. Faraday Soc. 26, 293-308. 5. Weller, A. (1961) Progr. React. Kinet. 1, 187-214. 14. Gafni, A. & Steinberg, I. Z. (1972) Photochem. Photobiol. 15, 6. van Duuren, B. L. (1963) Chem. Rev., 63, 325-354. 93-96. 7. Condon, E. U., Alter, W. & Eyring, H. (1937) J. Chem. Phys. 15. McClure, W. 0. & Edelman, G. M. (1966) Biochemistry 5, 5,753-775. 1908-1919.

Correction: In the article "Crystal and Molecular Struc- made. The last line of the legend to Table 4, p. 2347, ture of N,N'-Diethyl-N,N'-Diphenylurea", by Ganis, P., should read: "the nuclear fraction of 6.3 X 107 infected Avitabile, G., Benedetti, E., Pedone, C. & Goodman, M., cells was used for each hybridization." In Table 6, p. 2349, which appeared in the September 1970 issue of Proc. Nat. the right-center column head should read" "% Total cellu- Acad. Sci. USA 67, 426-433, the following correction lar radioactivity." should be made in Table 2, p. 428: The z coordinate of atom N1 is 0.3517, not 0.3317. Correction. In the article "Regulation of the Nucleolar Correction. In the article "DNA and Gene Therapy: Trans- DNA-Dependent RNA Polymerase by Amino Acids in fer of Mouse DNA to Human and Mouse Embryonic Cells Ehrlich Ascites Tumor Cells", by Franze-FernAndez, by Polyoma Pseudovirions," by Qasba, P. K. & Aposhian, M. T. & Pogo, A. O., which appeared in the December 1971 H. V., which appeared in the October 1971 issue of Proc. issue of Proc. Nat. Acad. Sci. USA 68, 3040-3044, the Nat. Acad. Sci. USA 68, 2345-2349, several corrections following corrections should be made. In the legend to should be made. On p. 2345, column 2, line 10 (from the Table 1, p. 3042, reference "(18)" should read "(13)". On top) should read: "10-40% sucrose gradient, contain- p. 3043, eleven lines from the bottom, right-hand column,

ing. . .," not "10-20%....." Due to editorial errors made in the sentence beginning "Assumptions b and c suggest" the PROCEEDINGS office, the following corrections should be should read "Assumptions a and b suggest". Downloaded by guest on September 29, 2021