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PROBING BIOMOLECULAR STRUCTURES BY MEANS OF LASER-EXCITED LANTHANIDE
PROBES
J.-C.G. Bünzli
To cite this version:
J.-C.G. Bünzli. PROBING BIOMOLECULAR STRUCTURES BY MEANS OF LASER-EXCITED LANTHANIDE PROBES. Journal de Physique Colloques, 1987, 48 (C7), pp.C7-607-C7-610.
�10.1051/jphyscol:19877147�. �jpa-00226968�
PROBING BIOMOLECULAR STRUCTURES BY MEANS OF LASER-EXCITED LANTHANIDE PROBES
Universit6 de Lausanne, Institut de Chimie Minerale et
Analytique, Place du Chateau 3, CH-1005 Lausanne, Switzerland
1. INTRODUCTION
The trivalent 1 anthanide ions have very specific spectroscopic and magnetic properties which make them ideal probes in studies of biological systems
[I].This is particularly true in the investigation of metal-containing macromolecules in which the metal ion is spectroscopically silent, e.g. Ca(II), Mg(II), or Zn(I1).
Lanthanide probes are provided for solving analytical problems (trace analysis pro- cedures for biological material, with the help of macrocyclic ligands [ z ] ) or for gaining insight into the structure of metal-ion sites. We shall focus this lecture on the latter aspect, especially in the case of calcium-containing materi a1
[ 3 ] .The use of Ln(II1) as replacement probes for Ca(I1) is made possible by the analogy in the chemical and physico-chemical properties of these ions
: (i)they are spherical with approximately the same ionic radius, ca. 1.1
Afor a coordina- tion number of
8to 9,
( i i )they are hard cations with essentially non-directional and ionic bonding, and
(iii)their aquo-ions have similar coordination numbers (9-
10for Ca(I1) [4] and ca.
9for Ln(II1) [5]) and are kinetically highly labile.
2. THE Eu(II1) AND Tb(II1) IONS AS LUMINESCENT PROBES
The electronic configuration of the Eu(1II) ion is 4f6. The ground state is
7 ~ o
and there are long-lived luminescent excited states such as
'DOand (Fig.
1). Transitions between the sublevels are in principle forbidden by an electric di- pole mechanism. However, mixing of vibronic or charge-transfer states with the electronic states, as well as the J-mixing arising from the spin-orbit coupling, make the wavefunctions composite so that the selection rules do not apply strictly.
The transitions may be observed with a very weak intensity, comparable to that of the allowed magnetic dipole transitions. Since the 4f orbitals are shielded by the 5s2 5p6 sub-shell, all these transitions appear as narrow bands and their energy is not much affected by the chemical environment of the ion. The Tb(II1) ion has a 4f8 configuration; its fundamental level is 7 ~ s and the long-lived
'DI,level gives rise to intense luminescence. However, its inverted multiplet electronic structure makes the spectra more difficult to interpret than Eu(II1) spectra.
In view of the weakness of the transitions, it is best to use sensitive lumi-
Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19877147
C7-608 JOURNAL DE PHYSIQUE
nescent techniques, r a t h e r than absorption measurements. The E u ( I I 1 ) i o n may be se- l e c t i v e l y e x c i t e d by t u n i n g a dye l a s e r t o t h e energy o f t h e 5 ~ o + 7 ~ 0 t r a n s i t i o n . This t r a n s i t i o n occurs around 580 nm, t h a t i s i n a s p e c t r a l range i n which t h e b i o - l o g i c a l m a t e r i a l s are u s u a l l y transparent. The T b ( I I 1 ) i o n i s c o n v e n i e n t l y e x c i t e d by t h e 488 nm l i n e o f an argon l a s e r . The f o l l o w i n g i n f o r m a t i o n may be gathered by means o f luminescent experiments.
Number o f m e t a l - i o n s i t e s : E u ( I I I ) , T b ( I I 1 )
a) by 1 uminescent t i t r a t i o n : t h e t o t a l E u ( I I 1 ) luminescence i s monitored versus t h e amount o f metal i o n added t o t h e b i o l o g i c a l m a t e r i a l (Fig. 2). For t h i s type o f experiment, T b ( I I 1 ) i s o f t e n used and t h e e x c i t a t i o n o f t h e l a n t h a n i d e i o n may t a k e p l a c e through energy t r a n s f e r from chromophoric groups (e.g. tryptophane o r t y r o s i n e ) t h a t absorb i n t h e UV.
b) by m o n i t o r i n g t h e E u ( I I 1 ) + 7 ~ o t r a n s i t i o n under h i g h r e s o l u t i o n (< 0.1 A , e x c i t a t i o n spectroscopy). Indeed, n e i t h e r t h e i n i t i a l nor t h e f i n a l s t a t e s can be s p l i t by l i g a n d - f i e l d e f f e c t s since J = 0 f o r both states. One s p e c i f i c chemical environment t h e r e f o r e generates o n l y one 0-0 t r a n s i t i o n ( F i g . 3 ) . Some care has t o be excercized. When t h e l o c a l symmetry i s h i g h o r when an i n v e r s i o n center i s pre- sent, t h e 0-0 t r a n s i t i o n i s f o r b i d d e n by t h e symmetry-related s e l e c t i o n r u l e ; t h e t r a n s i t i o n i s nevertheless sometimes observed, b u t i s extremely f a i n t . Such a s i t u - a t i o n r a r e l y p r e v a i l s i n b i o l o g i c a l molecules, t h e l o c a l symmetry o f t h e metal s i - t e s being u s u a l l y q u i t e low. Another d i f f i c u l t y a r i s e s when two ( o r s e v e r a l ) metal i o n s i t e s generate 0-0 t r a n s i t i o n s t o o c l o s e i n energy t o be resolved i n t o d i s t i n c t components.
Sum o f t h e l i g a n d formal charges : E u ( I I 1 )
The nephelauxetic e f f e c t s h i f t s t h e energy o f t h e 0-0 t r a n s i t i o n according t o t h e f o l l o w i n g experimental equation [ 6 ] :
v[cm-l] = 17l273 + 2.29 q
-
0.76 q2,i n which q i s t h e sum o f t h e formal charges o f t h e l i g a n d s bonded t o t h e metal i o n . This r e l a t i o n s h i p i s accurate t o o n l y
+
1 u n i t o f charge and must b e used w i t h care. It provides however u s e f u l i n f o r m a t i o n f o r t h e assignment o f t h e 0-0 t r a n s i - t i o n t o a given chemical environment.Symnetry o f t h e m e t a l - i o n s i t e s : E u ( I I 1 )
The l o c a l symmetry o f each o f t h e metal-ion s i t e s i s determined by a n a l y s i s o f t h e 5 ~ o + 7 ~ J t r a n s i t i o n s according t o g r o u p - t h e o r e t i c a l p r i n c i p l e s , a f t e r selec- t i v e e x c i t a t i o n o f each s i t e . There may be complications a r i s i n g from v i b r o n i c t r a n s i t i o n s , from t h e i n t e r a c t i o n between t h e phonon d e n s i t y o f s t a t e s and e l e c t r o - n i c s u b l e v e l s [7], and from energy m i g r a t i o n from one s i t e t o another one. I n t h i s l a t t e r s i t u a t i o n , time-resolved spectroscopy might be needed.
The presence o f an i n v e r s i o n center i s p a r t i c u l a r l y easy t o recognize since i n t h i s case magnetic d i p o l e t r a n s i t i o n s o n l y are allowed and t h e emission spectrum i s dominated by t h e 5 ~ 0 + 7 ~ 1 t r a n s i t i o n .
High energy v i b r a t i o n s , e.g. 0-H o r N-H s t r e t c h e s , p r o v i d e an e f f i c i e n t path- way f o r t h e r a d i a t i o n l e s s d e e x c i t a t i o n o f L n ( I I 1 ) ions. Comparison between t h e l i f e t i m e 'C o f t h e e x c i t e d 5 ~ o ( o r 5 ~s t a t e i n t h e presence o f H20 and D20 allows ~ ) t h e d e t e r m i n a t i o n o f t h e number n o f water molecules d i r e c t l y bonded onto t h e L n ( I I 1 ) ion, a f t e r s u i t a b l e c a l i b r a t i o n [ 8 ] , cf. F i g . 4 :
n = 1.05 ( l / z H Z 0
-
1 1 ~,
~Ln = E u ( I I 1 ) ~ ~ ) n = 0.23 ( ~ / ' c H ~ ~-
l / ' C D 2 ~ ),
Ln = T~(III)S i m i l a r r e l a t i o n s h i p s may a l s o be e s t a b l i s h e d f o r T b ( I I 1 ) and f o r o t h e r quenchers, e.g. N03- [9].
Distance betdeen two m e t a l - i o n s i t e s : L n ( I I 1 )
I f two metal s i t e s a r e populated by two d i f f e r e n t and p r o p e r l y chosen L n ( I I 1 ) ions, p a r t o f t h e e x c i t a t i o n energy o f one i o n i s t r a n s f e r r e d t o t h e o t h e r ion, r e s u l t i n g i n a decrease o f t h e l i f e t i m e o f t h e e x c i t e d s t a t e . The y i e l d o f t h e energy t r a n s f e r q i s r e l a t e d t o both t h e l i f e t i m e s o f t h e donor i o n i n absence ( 7 0 ) and i n presence (T) o f t h e acceptor, and t h e d i s t a n c e r between t h e donor and t h e acceptor :
rl = ~ - T / ' c ~ = 1 / ( 1 + r 6 / ~ 0 6 ) ,
where Ro i s t h e c r i t i c a l d i s t a n c e f o r 50 % t r a n s f e r ; Ro depends upon an o r i e n t a t i o n f a c t o r , t h e quantum y i e l d o f t h e donor, t h e r e f r a c t i v e index, and t h e o v e r l a p i n t e - g r a l between t h e emission spectrum o f t h e donor and t h e absorption spectrum o f t h e acceptor. For an a p p l i c a t i o n t o a calcium-containing p r o t e i n , calmodul i n , see
[ l o ] .
ACKNOWLEDGMENTS
T h i s research i s supported by t h e Swiss N a t i o n a l Science Foundation and by t h e Fondation H e r b e t t e (Lausanne).
REFERENCES
1. F.S. Richardson, Chem. Rev. 82, 541-52 (1982).
2. 6. Mathis and J.-M. Lehn, French Patent 2 570 703 (1986).
3. J.-C. 6. Bunzli, P.D. Morand, and J.-M. P f e f f e r l 6 , poster communication, LASER M2P Conference, Lyon-Villeurbanne, 7-9 J u l y 1987.
4. M.M. Probst, T. Radnai, K. Heinzinger, P. Bopp, and B.M. Rode, J. Phys. Chem.
89, 753-59 (1985).
5. S.F. L i n c o l n , Adv. Inorg. B i o i n o r g . Mechan. 4, 217-86 (1986).
6. M. A l b i n and W. dew. Horrocks Jr., Inorg. Chem. 24, 895-900 (1985).
7. J.-C. G. B u n z l i , B. K l e i n , D. Wessner, and N.W. Alcock, I n o r g . Chim. Acta 59, 269-74 (1982); P. Caro, O.K. Moune, E. Antic-Fidancev, and M. Lema'itre-Blaise, J. Less-Common Met. 112, 153-73 (1985); J.-C. G. B u n z l i , G.A. Leonard, D.
Plancherel, and 6. Chapuis, Helv. Chim. Acta 69, 288-97 (1986).
8. W. dew. Horrocks and D.R. Sudnick, J. Am. Chem. Soc., 101, 334-40 (1979).
9. 3.-C. 6. B u n z l i and J.-R. Yersin, I n o r g . Chim. Acta 94, 301-308 (1984).
10. C.-L. A. Wang i n "New F r o n t i e r s i n Rare E a r t h Science and Applications1', Xu Guangxian and Xiao Jimei, eds., Science Press B e i j i n g , Vol I. 146-149 (1985).
C7-610 JOURNAL DE PHYSIQUE
CONFIGU- TERMS LEVELS SUBLEVELS RATION
Fig. 1. P a r t i a l energy l e v e l diagram f o r E u ( I I 1 ) .
+5 3 residual
Fig. 2. Luminescent t i t r a t i o n o f apo-BLA w i t h EuC13 : [BLA] = 4 - 1 0 - ~ M i n D20, pD = 6.7 ( b u f f e r : TRIS). Top curve : t i t r a t i o n , bottom curve : blank.
Fig. 3. E x c i t a t i o n spectrum o f Fig. 4. Luminescent decay constants o f t h e 0-0 t r a n s i t i o n . S o l u t i o n o f E u ( I I 1 ) bound t o BLA vs. t h e molar f r a c t i o n BLA 9 * 1 0 - ~ M i n D20 c o n t a i n i n g o f H20. Bottom curve : s i t e I, [BLA] = EuC13, TRIS (pD = 6.46) and KC1 2 . 1 6 - 1 0 - ~ M, TRIS 0.02 M, KC1 0.01 M, pD = 0.01 M. The spectrum i s r e s o l - 3.9; r e s u l t : 2.1
+
0.3 H20 per E u ( I I 1 ) i o n ved b y Gaussian a n a l y s i s . Botton curve : s i t e 111, [BLA] = 6 . 2 6 - 1 0 - ~M, TRIS 0.02 M, KC1 0.01 M, pD = 6.48; r e - s u l t : 4.0
