J . Phys. Chem. 1991, 95, 1585-1589 From empirical correlations, the large upfield shift of the axial methylene hydrogens was found to be associated with the number of carbon atoms situated gauche to it.293 The results presented here prove that the gauche methyl carbons are directly responsible. Part of this shift is due to the increase in the charge density at the hydrogens resulting from methyl substitution. The second significant factor is the difference in the local anisotropic contributions and in particular the change in the chemical shift tensor at the C p position from methylene to that of methine. We note that the local anisotropic contributions appear to be transferable for similar local structures; for example, compare axial hydrogens C and F and equatorial hydrogens D and G in Table V. These values may be useful in the interpretation of the spectra of similar molecules.
Conclusions The gas-phase CH stretching overtone spectrum of TMCH-cis can be interpreted within the local mode description, in which the chemically distinct C H bonds each give rise to a single absorption peak. The peaks have been assigned on the basis of the bond
1585
lengths obtained from ab initio molecular orbital calculation with full geometry optimization. Local-mode parameters indicate that increasing anharmonicity is associated with increasing bond length. There are no anomalous structural differences found for the axial C H methylene bonds. The large upfield shift of the latter hydrogens observed in the 'H NMR spectrum is found to be attributable, in part, to the increased shielding from the &methine carbon atoms. Both axial and equatorial methylene hydrogens are shifted upfield relative to those in cyclohexane, because of the increased charge density on the hydrogens due to methyl substitution. The parameters calculated for the TMCH molecules may be transferable to other molecules with similar structures.
Acknowledgment. We are grateful to the Natural Sciences and Engineering Research Council of Canada for financial support. We are also grateful to Professor N. Muller for correspondence that kindled our interest in this project. We thank Silicon Graphics Inc. for the provision of time on a 4D/240 computer and Mr. Len Zaifman for facilitating the calculations.
Fluorescence Decay Kinetics of Tyrosinate and Tyrosine Hydrogen-Bonded Complexest K. J. Willid and A. G.Szabo* Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario Kl A OR6,Canada (Received: September 4, 1990)
We have examined the time-resolved fluorescence of aqueous tyrosine in the presence of high buffer base concentrations, to model the interactions between tyrosyl residues and potential proton-accepting side chains in proteins. The decay kinetics were not consistent with the formation of excited-state tyrosinate by proton transfer. Rather they indicated the presence of ground-state hydrogen-bonded complexes that persisted in the excited-state. Fluorescence properties of the tyrosine hydrogen-bonded complexes were correlated with the pKa of the proton acceptor and compared with those of tyrosinate. We conclude that the results have significance in the interpretation of fluorescence data from proteins.
Introduction Ionization of the phenolic hydroxyl group of tyrosine in alkaline media to form tyrosinate is accompanied by red shifts of the 275-nm absorption peak to 295 nm and the 305-nm fluorescence maximum to 340 nm.' In the ground state the pKa for this ionization is near 10; tyrosine, however is far more acidic in the first excited singlet state with an estimated pKa* of 4-5.2 Despite the acidic excited-state pKa, the quantum yield of tyrosine fluorescence is constant over the range pH 4-8. This implies that in dilute buffers the rate of deprotonation is slow relative to the rate of deactivation of the excited state.3 It has been reported that a second emitting species, with a tyrosinate-like emission, is observed in the fluorescence spectra of tyrosine solutions with a pH < IO if a high concentration of a proton-accepting buffer such as acetate2 or phosphate4" is present. Excited-state proton transfer from tyrosine to the proton acceptor, resulting in excited-state tyrosinate, was proposed to account for these observations.* The excited-state acid-base behaviour of tyrosine has significance in the interpretation of the fluorescence properties of proteins. In peptides and proteins at neutral pH, proton-accepting groups such as the side chains of aspartate or glutamate residues could act as proton acceptors and facilitate excited-state proton transfer from tyrosine residues leading to tyrosinate emission. Unfortunately, the spectroscopic properties of tyrosinate are similar to those of tryptophan, complicating the interpretation of protein spectral data. Two reports from this laboratory6,' of protein 'Issued as NRCC Publication No. 31926. *NRCC research associate. *Towhom correspondence should be addressed.
0022-3654/91/2095-1585$02.50/0
tyrosinate fluorescence have been retracted,8 since it was later shown that the 345-nm emission was dependent on the method of preparation. A recent review by Ross et a1.8 highlights the difficulty of unambiguous assignment of tyrosinate emission in proteins. It is also clear from their review that the fluorescence decay kinetics of tyrosinate and tyrosine in the presence of proton-accepting species have not been investigated in detail. In this study we report the results of picosecond time-resolved fluorescence measurements on tyrosine solutions containing high concentrations of proton acceptors. Proton acceptors, with base strengths typical of amino acid residue side chains, were chosen to model tyrosine interactions in proteins.
Experimental Section L-Tyrosine (Aldrich) was recrystallized three times from water. Tyrosine solutions were prepared to give an optical density of less (1) Cornog, J. L.; Adams, W. R. Biochim. Biophys. Acra 1963,66,356. (2) Rayner, D. M.; Krajcarski, D. T.; Szabo, A. G. Can. J . Chem. 1978, 56, 1238. ( 3 ) Feitleson, J. J . Phys. Chem. 1964, 68, 391. (4) Shimizu, 0.;Watanabe, J.; Imakubo, K. Phorochem. Phorobiol. 1979, 29, 915. ( 5 ) Alev-Behmoaras, T.; Toulmt, J.; HblEne, C. Phorochem. Photobiol.
. - .-30.- , 533.
1979. -.
(6) Szabo, A. G.; Lynn, K. R.; Krajcarski, D. T.; Rayner, D. M. FEBS Lett 1978, 94, 249. ( 7 ) MacManus, J. P.; Szabo,A. G.; Williams, R. E. Biochem. J. 1984,220, 261. (8) Ross, J. B. A.; Laws, W. R.; Rousslang, K. W.; Wyssbrcd, H. R. In Fluorescence Specrroscopy II. Biochemical Applications; Lakowicz, J. R., Ed.; Plenum: New York, in press.
Published 1991 by the American Chemical Society
1586 The Journal of Physical Chemistry, Vol. 95, No. 4, 1991
Willis and Szabo
TABLE I: Effect of Potential Acceptors on the Fluorescence Decay Parameters of Tyrosine in Aaueous Solutiona proton acceptor PK,b PH r I ,ns 72, PS :A,, nm F$ (340 nm) H2P04-8 2.15 4.5 1.863 f 0.003 HC02‘ 3.75 6.0 0.802 f 0.001 244 f 15 315 0.020 225 f 4 320 0.1 I6 CH,COC 4.17 6.0 0.667 f 0.001 Imh 6.99 7.5 0.551 f 0.001 71 f 1 335 0.120 HP0427.20 8.0 0.611 f 0.001 I36 f 1 330 0.421 (CH2OH)jCNH2 8.36 8.0 2.165 f 0.002 109 & 1 335 0.135
x2c 1.054 1.067 1.067 1.028 1.014 1.049
SVRJ 2.01 1.83 1.88 1.91 1.98 1.91
”The excitation wavelength was 287 nm. Parameters are given for a global fit to IO data sets collected at emission wavelengths 300-360 nm. The nominal concentration was 0.8 M; conjugate base (proton acceptor) concentration will depend on the solution pH. Lifetimes are shown with their standard errors as recovered from a given data set. *pK, values from ref 14. ‘Emission maximum of the spectrum associated with f 2 (from DAS). dThe fractional fluorescence of component r2at 340 nm. CThereduced x2 for the global fit. ’The serial variance ratio for the global fit. gSingleexponential in the range 300-360 nm. *Imidazole. than 0.1 at the excitation wavelength (7 ns. "Hydrogen-bonded tyrosine" has a red-shifted absorption spectrum (compared to H 2 0 , Figure 3) and may absorb at wavelengths frequently used to excite tryptophyl residues in proteins (295-300 nm). A survey of hydrogen bonding in globular proteins, based on X-ray data,27.shows some 60% of tyrosine residues are hydrogen bonded to protein groups. Since the emission from hydrogen-bonded tyrosine is in the range 3 10-340 nm, it can easily be mistaken as a minor tryptophyl component.
Acknowledgment. The expert technical assistance of Mr. D. T. Krajcarski is gratefully acknowledged. We also thank Mr. B. Lei for purifying the imidazole sample. We thank Drs. J. B. A. Ross and W. R. Laws for helpful discussions. (25) Hasselbacher, C. A.; Galati, L. T.; Contino, P. B.; Laws, W. R.; Ross, J. B. A. Biophys. J . 1990, 57, 55a. (26) Turner, R. J.; Roche, R. S.;Mani, R. S.;Kay, C. M. Biochem. Cell Biol. 1989, 67, 179. (21) Baker, E. N.; Hubbard, R. E. frog. Biophys. Molec. B i d . 1984,44, 91.
Solid-state 2H and 'H NMR of Guest and Host Motions in the Hofmann-Type and Related Benzene Clathratest Shin-ichi Nishikiori,' Christopher I. Ratcliffe,* and John A. Ripmeester Division of Chemistry. National Research Council of Canada, Ottawa, Ontario, Kl A OR9 Canada (Recriwd: June 29, 1990)
The motional behavior of the guest benzene molecule in the Hofmann-type benzene clathrate Cd(NH3)2Ni(CN)4.2C6D6 and related benzene clathrates Cd(en)Ni(CN),.2C6D6 (en = ethylenediamine) and Cd(dma)zNi(CN),.( 1 /2)C6D6 (dma = dimethylamine) were investigated by 2H NMR line shape and spin-lattice relaxation time studies. All the benzenes undergo reorientation about their 6-fold axes below room temperature with activation energy 15.0 ( I ) , 16.4 ( I ) , and 7.23 (4) kJ/mol, respectively, determined from the T I studies. In the case of the former two clathrates a second motion of the benzene was observed concomitant with the thermal decomposition process of their host lattices. The motional behavior of the bridging en in the host lattice of Cd(en)Ni(cN),-2C6H6was also revealed by 'H and 2H NMR, calorimetric, and powder X-ray diffraction experiments. There are four well-defined solid phases. In the low-temperature phase 1V the en is essentially static. For phases 1,H,and 111 a model was developed that involves reorientation of the en among four sites, 90° apart, about one of the crystal symmetry axes: In phases I1 and 111 the sites at 0 and 180 have lower energy than the sites at 90 and 270O. The energy difference AE decreases with increasing temperature, so that in phase 111 the motion involves mainly the two low-energy sites, but in phase IT AE approaches zero, at the transition to phase I, in a critical manner. In the room-temperature phase 1, the four sites are equivalent and the motion has an activation energy of 22.8 (1) kJ/mol. ZH NMR results for Cd( ND3),Ni(cN),.2C6H6 are compatible with ND, groups reorienting very rapidly about their 3-fold axes, coincident with crystal 4-fold axes, in a flat or almost flat potential.
Introduction
The Hofmann-type benzene clathrate Cd(NH3)2Ni(CN)4. 2C6H, is one of a number of clathrate compounds that are formed with a metal complex host and small organic guest molecules.' The host metal complex [Cd(NH&Ni(CN),] consists of two parts: a two-dimensional cyano metal complex [Ni(CN),Cd] formed with sauare-Dlanar nickel atoms: octahedrallv coordinated cadmium atoms with bridging cyano groups and ammine ligands coordinating to trans positions Of the Octahedra' cadmium atom in the [Ni(CN),Cd]. Layers of these host metal complexes build 'NRCC No. 32521. *On leave from the Department of Chemistry, College of Arts and Sciences, The University of Tokyo, Meguro, Tokyo 153, Japan.
0022-3654/91/2095-1589$02.50/0
up the host lattice of the Hofmann-type clathrate. Benzene molecules are enclathrated in the cavities between the layer^.^,^ Various structural modifications of the Hofmann-type clathrates can be obtained by replacing the ammine ligands in the host metal complex with other ligands. As a result, many related clathrates have been synthesized and their stuctures have been obtained by X-ray diffraction s t ~ d i e s . ' In ~ ~the ~ ~1960$, several wide line ' H ( I ) Iwamoto, T. The Hofmann-type and Related Inclusion Compounds. In /nc/usion Compounds; Atwood, J. L., Davies, J, E,D.,MacNic-1, D,D,, E&; Academic press: London, 1984; vel. 1, 29, (2) Sasaki, Y. Bull. Chem. Soc. Jpn. 1969, 42, 2412. (3) Nishikiori, S.; Kitazawa, T.; Kuroda, R.; Iwamoto, T. J . Inclusion Phenom. 1989, 7, 369-377. (4) Nishikiori, S.;Iwamoto, T. Chem. Leu. 1982, 1035.
Published 1991 by the American Chemical Society
