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Mumndl' .qufl‘ m " LIBRARY W Michigan State University This is to certify that the thesis entitled SYI~ITHE"IS AND FLUORESCENCE PROPERTIES OF , N-DAi-ISYL—MS —EIH<= moo emm _ =*_ecaox=o-_Am=eu-z 0mm mom - Nee .oe aueco—go._»mcuu owe :— cm on om cm as AllSNSlNI HONBOSSUOOTJ OHZITVNUON .v mg=m_m 43 N-dansyl-enkephalin, since dansylamide and N-dansyl-tyrosine both dis- played an emission peak at 516 nm in ethanol (exc 340 nm); and also, the presence of the gly-gly-phe-met side groups caused the emission maximum of dansyl to red shift further. The fluorescence emission Spectrum of met-enkephalin was found to closely resemble that of the free tyrosine residue with a maximum at 330 nm in 0.05 M NaHCO3 buffer pH 8.3 (exc 270 nm) due to the greater fluorescence quantum yield of the tyrosine amino acid (over that of phenylalanine) and due to FOrster type singlet-singlet energy transfer from the phenylalanine residue to the tyrosine residue. The emission peak of tyrosine has been shown to shift to longer wavelengths with increasing pH (Bishai et al., 1967); likewise, the fluorescence emission maximum of met5 -enkephalin was found to increase to 334 nm at pH 9.5 in the tris buffer which at pH 9.5 can permit a small percentage of the phenolic groups of met-enkephalin to become partially unprotonated, causing the very small (4 nm) shift in wavelength. The 330 nm value for the fluorescence emission maximum of met-enkephalin at pH 8.3 corresponds to a tyrosine with a protonated phenolic group. The attachment of the dansyl group to the N-terminal of mets- enkephalin was confirmed by its distinct ultraviolet absorption spectrum, as compared to that of the unbound dansyl group. The absorption Spectrum of N-dansyl-metS-enkephalin was found to have maxima at 260 and 342 nm, and a pronounced peak at 268 nm in ethanol. In comparison, the absorption spectrum of dansyl chloride in ethanol was established to have two maxima at 260 and 360 nm. When dansyl chloride reacts with amino acids or peptides, its absorption maximum at 360 nm is known to blue shift to shorter wavelengths (Gros and Labouesse, 1969; Hartley, 44 1970; Chen, 1967). The absorption maximum at 360 nm was determined to shift to 335 nm for dansyl glycine in ethanol. The ultraviolet absorp- 5-enkephalin was found to be very similar to tion spectrum of met tyrosine, having a maximum at 275 nm and a molar absorptivity of approxi- mately 1600, in 0.05 M NaHCO3 pH 8.3. It is suggested that the pro- nounced peak at 268 nm in the absorption spectrum of N-dansyl-mets- enkephalin corresponds to the tyrosine residue and the two maxima at 260 nm and 342 nm corresponds to the dansyl chromophore. The fluorescence and absorption changes Of the dansyl group were taken as verification of the synthesis Of N-dansyl-met5-enkephalin, and are consistent with the information in the literature on N-dansyl pep- tides (Hartley, 1970; Gros and Labouesse, 1969; Chen, 1967). Fournie- 5-enkepha1in, and Zaluski et a1. (1978) have synthesized N-dansyl-met employed NMR to confirm the position of the dansyl group, the presence of the phenolic proton at 9.3 ppm and the sulfamidic NH at 8.8 ppm. 4. Related compounds The fluorescence emission maximum of a dansyl derivative in ethanol was found to depend upon the nucleophilic strength of the substituent attached to the dimethylaminonaphthalene-sulfonyl group. in that, the emission maximum of the dansyl chromaphore Shifted towards longer wave- lengths as the basicity of the substituent increased, according to OH < Cl < NH2 5_NHR, see Table 1. The emission maximum of dansyl hydro- xide, i.e. dansyl sulfonic acid, was 42 nm less than that of dansylamide, which contains the strong base NH2 as dansyl substituent. Note: the 0H and Cl ions are very weak bases. The nature of the R group in dansyl derivatives with NHR substituents was determined to affect the extent of red shift in the dansyl fluorescence, but the size of the R group 45 Table 1. Fluorescence emission maximum of various dansyl derivatives in ethanol (exc A 340 nm). Compound A max 1. dansyl sulfonic acid 474 nm 2. dansyl chloride 480 nm 3. dansylamide 516 nm 4. dansyl glycine 519 nm 5. N-dansyl-l-tyrosine 516 nm 6. O-dansyl-l-tyrosine 548 nm 7. N,0-didansyl-l-tyrosine 522 nm 8. N-dansyl-metS-enkephalin 530 nm 9. N-dansyl-(D-a1a2)-met5-enkephalin 530 nm 46 was determined to have no effect on fluorescence, e.g. compare the emission maximum of N-dansyl-l-tyrosine at 516 nm to that of dansyl glycine at 519 nm and of N-dansyl-met5 5 -enkephalin at 530 nm in ethanol. The emission maximum of met -enkephalinamide-(CH2)2-dansyl has been reported at 520 nm in ethanol (F6urnie-Zaluski et al., 1978, a) compared to 519 nm for dansyl glycine. The dansylation procedures (in Section II, Experimental Details) were employed on free 1-tyrosine, in order to Obtain the fluorescence emission maximum of N-dansyl-l-tyrosine in ethanol and to confirm the synthesis of N-dansyl-met5 -enkepha1in. Under similar TLC conditions, the fluorescence and chromatographic properties of N-dansyl-l-tyrosine were identical to those reported by Felgner and Wilson (1977) for a commercially obtained sample of dansyl tyrosine, thus, verifying the formation of N—dansyl-l-tyrosine. N-dansyl-l-tyrosine was determined to be positive to the l-nitroso-Z-naphthol reaction, indicating that the phenolic hydroxyl of tersine was not dansylated. The fluorescence emission maximum of N-dansyl-l-tyrosine was found at 516 nm in ethanol. Since the unprotonated phenolic hydroxyl of tyrosine is considerably more reactive (basic) than the unprotonated amino group of tyrosine (Hartley, 1970), the fluorescence emission maximum of O-dansyl-l- tyrosine was found at 548 nm, red shifted by 32 nm, (see Table 1). Additionally, the electroegativity of oxygen is greater than that for nitrogen; one expects a greater solvent shift in a polar media. The fluorescence emission maximum of N,0-didansyl-l-tyrosine was found at 522 nm, close to the emission maximum of N-dansyl-l-tyrosine. 47 5. Energy transfer N-dansyl-metS-enkephalin has only two structures capable of fluores- cence, the phenol and dansyl moieties. Since there is considerable spectral overlap between the tyrosine emission spectrum and the dansyl groups absorption spectrum, efficient energy transfer from the tyrosine residue to the nearby ligand was established to occur in N-dansyl-mets- enkephalin via the FOrster's theory of singlet-singlet energy transfer (Fournie-Zaluski, 1978, a, b; Stryer, 1978, 1968), in that, the fluorescence emission of the tyrosine residue was completely quenched, and only the emission of the dansyl group had occurred upon excitation of the tyrosine residue at 270 nm. Energy transfer from tyrosine to dansyl was evidenced by the fluorescence excitation spectrum of N-dansyl- (D-a1a2)-met5 -enkephalin in ethanol (ems 530), which showed an additional excitation peak at 272 nm in the wavelength range of the tyrosine's absorption band, see Figure 3. The excitation spectrum of the free dansyl chloride in ethanol, displayed only one peak at 328 nm; which shifted to 356 nm when reacting with the enkephalins. The above results are in complete agreement with those reported by Fournie—Zaluski et al. (1978, a, b) on the energy transfer in metS-enkephalin dansylated at either the N- or C-terminals. The energy transfer from the tyrosine to the dansyl group has been demonstrated by a dramatic 94% decrease in the fluorescence emission of tyrosine at 305 nm as compared to non- labeled met5-enkephalin alone, at the same concentration in 0.05 M tris HCl buffer pH 7.4 (exc 275 nm) (Fournie-Zaluski et al., 1978, a). The FOrster theory of singlet-Singlet energy transfer has been employed to elucidate the conformational behavior of the enkephalins via evaluation of intramolecular distance between donor and acceptor 48 chromophores (Fournie-Zaluski et al., 1978, a, b; Schiller, 1977; Stryer, 1978, 1968). Fournie-Zaluski et a1. (1978, b) have determined that the intramolecular distance between tyrosine and dansyl in mets- 0 enkephalin-(CH2)2-dansyl is 13.7 A, which favors a folded conformation 5-enkephalin. Schiller (1977) has used the energy transfer from tyrosine to tryptophan in the biologically active (trp4)-met5- 5 (B) for met enkephalin to study the conformational behavior of met -enkephalin. In 5-enkephalin was replaced this analogue, the phenylalanine residue of met by tryptophan. He calculated that the Tyr-Trp separation is 9.3 A in aqueous solutions at pH 1.5 and 5.5, suggesting a folded conformation (8) for both the cationic and zwitterionic forms of the analogue (Schiller, 1977). The attachment of a dansyl group to the N- or C-terminal of (trp4)-met5-enkephalin may possibly be useful for confOrmational analysis of metS-enkephalin, since the spectral overlap of the tryptophan emission spectrum and the dansyl absorption spectrum is considerably better than that of tyrosine and dansyl (Chen, 1967). 6. Fluorescence intensity changes of N-dansyl-metS-enkephalin as a function of concentration The amount of N-dansyl-mets-enkephalin in solution should be reflected in the fluorescence intensity at 530 nm. In this experiment, the emission Spectra of N-dansyl-metS-enkephalin were recorded as a function of its concentration in ethanol. The fluorescence emission intensities were found to increase linearly as the N-dansyl-metS-enkephalin concentration increased from 6 x 10"7 5 M to 3 x 10'5 M, indicating that the emission of N-dansyl-met -enkepha1in was prOportional to its concentration in ethanol. The relative intensity for each concentration is graphically displayed in a semilogarithmic plot in Figure 5. The slope of the line was 49 1000 500 100 50 RELATIVE FLUORESCENCE INTENSITY 10 J, 1 I 1 1 l l l 1 1 J l 1 l x 10‘6 M 5 x 10'6 M . 1 x 10'5 M CONCENTRATION (molarity) Figure 5. Fluorescence intensity of N-dansyl-met5 -enkepha1in as a function of concentration. 50 calculated to be equal to l x 105 per molar. This calibration curve may be used to determine the amount of N-dansyl-metS-enkephalin in a solu- tion of unknown concentration. Also, the fluorescence emission maximum of N-dansyl-mets-enkephalin was established to decrease slightly at lower concentrations (data not shown), reflecting an aggregation effect caused by the hydrophobicity of the molecule at higher concentrations. 7. Effects of solvent dielectric constant on N-dansyl-metS-enkephalin fluorescence. The dansyl group has been identified as a fluorescent molecular probe of polarity (Stryer, 1968), and the fluorescence properties of N- 5-enkephalins have dansyl derivatives of amino acids, peptides and met demonstrated a sensitivity to the dielectric constant of the environment (Fournie—Zaluski et al., 1978, a; Hartley, 1970; Chen, 1967). The emission maximum of 3.6 x 10'6 M N-dansyl-met5 -enkephalin was established to shift 15 nm towards shorter wavelengths from water (dielectric con- stant, 78.5 D) to ethanol (dielectric constant, 25.8 D) (Figure 6), accompanied by approximately a four-fold increase in fluorescence in- 5-enkephalin tensity. Inversely, the excitation maxima of N-dansyl-met were shifted towards the longer wavelengths as a function of the ethanol concentration in water (Figure 7); similar to the shifts in wavelength 5-enkephalin, which was for the dansyl absorption maxima of N-dansyl-met due to H-bonding effects. The dependence of the emission maximum of N-dansyl-metS-enkephalin on the dielectric constant of the environment results from a reOrienta- tion of the solvent shell around the excited dansyl chromaphore; since the excited state of the dansyl group has a much higher dipole moment than the ground state (Stryer, 1968; Chen, 1967). Due to the solvation 51 545 540 (nm) 535 WAVELENGTH 530 1 I I ,I 44 LI 1, I 1 I one LI .4 I 1 I 41 I, 1 I 10 20 30 4O 50 60 70 80 90 100 ETHANOL CONCENTRATION (%) Figure 6. Fluorescence emission maximum of 3.6 x 10'6 M N-dansyl-mets- enkephalin as a function of ethanol concentration in water (ems A 530 nm). 52 :owpoczm a me :Ppmcaoxcm- See-_»meee-z z .AEc omm K meow Loam: cw comumgucmocoo pocmgum mo m e Lacy :eezmam>muumowvog we compmu?$wucmc~ .w ogzmwd m Ase. zo_euv .mvPEmcwszawxcwimumsiAmmFMIQV-Fzmcmc-z m>wuoeo_umg we mcvucwa one .m wgamwu umumaaucm muNEmcwpmzaoxcm- umeflmcpauovi_»mcmuizuz m m z: ¢_ 2: o.“ z: m.m III c.9— .II o.m— (Z-Ol x uiaaoud 6m/salomd) punoq apiweuileudaaua-Sqaw-(zele-g)-lxsupp-N-H€ ll o.o~ 0:98pm: 305 .5 ”U Neexo_ee z: oo_ --- II. o.m~ 59 non-dansylated enkephalin. The influences of cold N-dansyl-DALA on the binding of 3H-(D-ala2)-met5-enkephalinamide (3H-DALA) to brain slices 5-enkepha1inamide (DALA) . were found to parallel those of cold (D-ala2)-met N-dansyl-DALA was prepared by the standard procedures, as previously described. The identification of the dansyl derivatives of DALA was based on their fluorescence characteristics on silica gel 60 chromato- grams and in ethanol, see Figure 10. The number one derivative displayed a bright yellow-green color on the chromatogram, and had an emission maximum at 530 nm in ethanol, like N-dansyl-enkephalin. Even though the Rf value of this derivative was different from the Rf value for N-dansyl-enkephalin, the number 1 derivative was designated as N-dansyl- DALA. The amide group at the carboxyl terminal of N-dansyl-DALA may have caused it to travel similar to dansylamide. The number 2 derivative had a yellow-green center surrounded by a bright blue band as concentric circles on the chromatogram. The emission maximum was obtained at 474 nm in ethanol and, was attributed to the high energy emission of dansyl- sulfonic acid, since the position of the number 2 derivative on the chromatogram was similar to that of 3H-N-dansyl-DALA, see Figure 8. It is inferred that the number 2 derivative contains dansylsulfonic acid and unseparated N-dansyl-DALA. The number 3 and 4 derivatives were not identifiable but the fluorescence properties of the number 3 derivative do indicate a dansyl group without a sulfonamide bond. When exposed to UV irradiation, all of the dansyl derivatives of DALA showed some degree Of photolytic degradation on the chromatograms. The number one deriva- tive (N-dansyl-DALA) did not fade like the number 1 dansyl derivative of enkephalin (dansylamide). 60 deriv. der. # name color ‘ELA c::) l. N-dansyl- yellow-green 530 nm DALA 2. dansyl -0H yellow-green 474 nm and N-dansyl- surrounded DALA by blue (§::) 3. ? blue 465 nm 4. ? orange 465 nm - ' 561 nm (7“; initial spot Figure 10. Identification of dansyl derivatives of (D-a1a2)-met5- enkephalinamide by silica gel 60 thin-layer chromatography. 61 3 In the presence of N-dansyl-DALA, the binding behavior of H-DALA to brain slices was similar to its binding in the presence of the con- trol, DALA, see Figure 11. At 3 nM, the saturable binding of 3 nM 3H-DALA was the same for both N-dansyl-DALA and DALA, at approximately 3 0.113 pmoles/mg protein. The Opiate receptor binding of H-DALA was markedly reduced by both N-dansyl-DALA and DALA, at high incubating concentrations (300 nM); however, the reduction by DALA was 28% greater than N-dansyl-DALA. This suggests that the Opiate receptor affinity of N-dansyl-DALA is slightly less than the affinity of DALA. The number 2 derivative influenced the binding behavior of 3H-DALA in a manner similar to the control (DALA) except, at low concentrations, the number 3 2 derivative reduced the binding site concentration of H-DALA by 28% less than DALA, probably due to hydrophobic interactions between dansyl- sulfonic acid and the receptor site. The presence of N-dansyl-DALA or 3 DALA were found to enhance the binding Of H-DALA to the brain slices. The saturable binding of 3H-DALA alone (at 0.0 nM cold drug) was 0.062 3 pmoles/mg protein. However, naloxone (100 nM) inhibited the H-DALA binding by 76%, similar to the inhibition of 3 3 H-N-dansyl-DALA binding. The Opiate receptor binding of H-DALA in the presence of the number 3 or 4 derivative was analogous to the binding of N-dansyl-DALA or the number 2 derivative, respectively, indicating Opiate-like characteris- tics Of DALA. These results conclusively demonstrate that N-dansyl- DALA and DALA occupy the same binding sites. Fournie-Zaluski et a1. (1978, a) have reported that the opiate-like activity of N-dansyl-metS-enkephalin was considerably less than lens- 3 5 enkephalin. They found that the Ki value for the inhibition of H-leu - enkephalin binding to membranes by N-dansyl-metS-enkephalin was 50 nM, 62 amEIANmpmuav so oopsmcwPecamxcmumposiANm_muov1~>mcmunz mo mozmmmcq .mvwEmcvpmgaoxcmum Hosifimmpoiovuxm eo mcwvcpn souamuog mumFao .9, «came; on» :P mwuppm apnea o» ocwsmcp_mcamx:mim 3.29.85 28.232328 gas 2: can x: on a: o.m l punoq appueu;leudanua-S:IauI--(z!LE--c1)-HE I 9.2 (Z-Ol x uIazoud Bun/salon) 252.28 N. m «3:35ZfioxcormuoeANSTSI323-2 I 2:52:pagaoxcmrmugtfimfimmmw D I od— 63 compared to 5 nM for laps-enkephalin; and, in the inhibition of electric- 5 ally induced contractions of guinea pig ileum, N-dansyl-met -enkepha1in had a IC 50 value of 1306 nM compared to 67 nM for leus-enkephalin. The 5 low values for N-dansyl-met -enkephalin binding may be due to comparison to leus-enkephalin binding, instead of metS-enkephalin or naloxone. 5 However, the opiate receptor binding of (D-ala2)-met -enkephalin-(CH2)2- dansyl has been shown to be equal to or better than leu5 -enkephalin. It is agreed that N-substitution of enkephalin decreases its opiate receptor affinity (Pert et al., 1976). C. Interaction of Cultured Brain Cells with N-dansyl-mets-enkephalin 1. Microspectrofluorometric characterization of amygdaloid cells by N-dansyl-metS-enkephalin fluorescence The mapping of opiate receptor sites on amygdaloid cells was accomplished by N-dansyl-metS-enkephalin fluorescence; in that, enkephalin- dansyl-receptor complexes were visually located on single cells via fluor- escence microscopy. The enkephalin-dansyl-receptor complexes appear as a continuous turquoise covering on the surface membrane of the cell, with discrete blue patches, see Figures 12 and 13. In photomicrograph A of Figure 12 (phase-contrast), the precesses of the center cell pro- jected towards other nearby cells and appeared to come into close opposi- tion with their cell bodies. The left process of the center cell, having several bifurcations, extended over to a cell and appeared to wrap around approximately 30% of the cell body. A short process projected to a bottom large cell and wrapped around its cell body. In photomicrograph B of Figure 12, there was no fluorescence from the cell in the center of the picture, but the cells that were contacted by its neurites displayed a Figure 12. 64 Part A - This is a photomicrograph of cultured cells from the amygdalar region of a 15 days old postnatal rat brain. The cells were maintained 30 days in culture before fixation and staining for 74 hours with 2 x 10"6 M N-dansyl-metS-enkephalin in tris buffer. They are seen here under low intensity white light. The cell in the center of the picture is about 20 microns long and has five processes. The nucleus of the cell is situated to one-side of the cell body and is plainly visible. (0.5 cm = 10 microns). Part B - This is a photomicrograph of the same cells that were seen in the above picture, but using the fluorescence excitation system. 65 m. s n. .. N am I. to"? I 5 \ 66 Figure 13. Part A - This photomicrograph is of cells that are located in a different culture region. Part B - These are the same cells under fluorescence excitation. 67 68 considerable amount of dansyl fluorescence. The intense fluorescence from the cells contacted by the processes may indicate a large concentra— tion of enkephalin receptors located on the postsynaptic membrane of opiate susceptible neurons. The lack Of fluorescence from the center cell was understandable, because an enkephalin secreting neuron may not contain Opiate receptors. In Figure 13 (photomicrograph A), the process of the center cell, leading up to the group of cells, was of particular interest. The process bifurcated just before contacting a cell, traveled across the cell body, and appeared to terminate in a specific location. In photomicrograph B of Figure 13, note the intense blue patches at the termination Site of the bifurcated neurite. Also, the above cell had a discrete blue patch at a point of contact with the process. It was evidenced from these photomicrographs, that the blue clusters were varied in size and shape, and located at soma-soma or process-soma contacts between two cells. The Opiate receptor binding of N-dansyl-mets- enkephalin onto amygdaloid cells was not conclusively demonstrated by these results, because inhibition of the enkephalin-dansyl-receptor fluorescence by naloxone or enkephalin was not attempted with the amygdalar cultures here. However, the dansylated opioid was established to bind to Opiate receptor sites, in brain slices and homogenates, see page 5 The fluorescence emission spectrum of N-dansyl-met -enkephalin bound to the amygdaloid cells had a maximum at 445 nm with a shoulder 5-enkepha1in around 478 nm, see Figure 14. The binding of N-dansyl-met to the cell caused a blue shift in its fluorescence peak, indicating that the microenvironmental dielectric constant of the 1-dimethylamino- naphthalene ring was substantially decreased upon cellular engagement. 69 m beep. be nooseNLeoe we 5: mee ee Neon o5» Noe-_Nneoe-z z NI mom Nmm Asev zeozubm>