8/13/2019 lantanidos

1/4

Phorochemisrry and Pitotobiology Vol. 46 N o . 6 , pp 1067-1070, 1987Printed in Great Britain. All rights reserved

003 1-8655187 03.00 O.OOCopyright 987 Pcrgamon Journals Ltd

YEARLY REVIEW

LANTHANIDE IONS AS LUMINESCENT PROBES OFBIOMOLECULAR STRUCTURE

Introduction

Lanthanide luminescence provides a unique probeinto questions of biological interest. This reviewarticle summarizes contributions to the literature inthe last year in which light absorption or emissionspectroscopy of the trivalent lanthanides [Ln(III)]

was used as such a probe. Several optical propertiesmake the Ln(II1)s useful and convenient: their red-shifted visible emission, their long luminescent life-times, the sensitivity of their absorption and emis-sion to environment, the overlap of their excitedstate manifolds with the excited states of biologicalligands and finally, the variety of their emissionproperties which make intra-Ln(II1) transfer andluminescence quenching possible. The long historyof Ln(II1) substitution into biochemical environ-ments and spectroscopic analysis of the product isbest summarized in the review by Horrocks (1982).By far, the greatest amount of biological infor-mation has been obtained from Ln(II1) substitutionsinto Ca(I1) binding sites in proteins. The greatestsimilarity between Ca(I1) and the Ln(1II)s is theionic radius [Ca(II):1.06 A Ln(1II)s range from1.06 A to 0.85 A]. The obvious difference, thetrivalent charge, is accompanied by an increaseddifferential binding entropy. Ln(II1)s are muchmore highly solvated than Ca(II), and so must losemore of their solvation sphere to bind to the biologi-cal site. Thus there is a much greater increase in

the entropy of the system when the Ln(II1) is boundwhich contributes to the generally larger bindingconstants. It must be remembered that the Ln(II1)sare by no means an isomorphous replacement forCa(I1).

Ln I I I ) protein crystals

With this in mind, it is appropriate to reviewrecent crystallographic work in which Ln(1II)s havebeen substituted into biological calcium sites. Intheir extensive analysis of the structure of vitamin

D-dependent intestinal calcium binding protein,ICaBP, Szebenyi and Moffat (1986)- summarizework done using Ln(II1) substitution. Ln(II1)s havebeen shown to selectively displace Ca(I1) from cer-tain binding sites in crystals of parvalbumin (Pa)and ICaBP. In Pa, the E-F site but not the C-D sitebinds Tb(II1). In ICaBP, the C-terminal site butnot the N-terminal site binds Nd(1II). The crystalstructures of these Ln(II1)-protein complexes show

only slight displacements of the metal and littledistortion of the protein itself. These sites are char-acterized by having one bound water, multiplemobile amino acid side chains as ligands, and anoverall negative charge. Sites incapable of metalexchange presumably have limited ability to adjusttheir size and shape to accommodate the new metal.The authors advise caution in generalizing from dataderived from such substitutions.

Herzberg and James (1986) analyzed the bindingof Eu(III) , Tm(III), and Lu(II1) to half-saturatedCa(I1) crystals of troponin C (TnC). TnC binds fourCa(I1) and has two high-affinity structural sites inthe C-terminal domain and two low-affinity regu-latory sites in the N-terminal domain. Both highaffinity sites in the crystal exchanged their Ca(I1)for Ln(III)s, though to different degrees dependenton metal and the site. Ln(II1) binding at these

structural sites appears to be much the same asCa(I1) binding, with the Ln(II1) displaced from theCa(I1) only 0.3 8 at site I11 and 0.8 A at site IV.Ln(II1)s bound with much more varied affinities tothe empty low affinity site I and not at all to theempty low affinity site I of the crystal. With twowaters at this site, the Ln(II1) binding at site I isquite different than that expected for Ca(I1) bind-ing. The authors caution that metal binding to lowaffinity sites described in this work is not physiolog-ically relevant.

Calmodulin

Calmodulin (CaM), a major regulatory protein ineucaryotic cells, contains four similar binding sitesfor Ca(I1). General, though not universal, agree-ment is that Ca(I1) binds somewhat more stronglyto sites 111 and IV in the C-terminal domain. Bucci-gross and Nelson (1986a,b,c) have contributed sev-eral papers in the last year which help to clarifysome of the inconsistensies reported for Ln(II1)binding to CaM. By using EPR of a Tyr-99 spin

label they determined that the Ln(II1)s show differ-ential binding affinities to the four sites of CaM.Three types of binding behavior were observed:Ca(I1) like, or metal binds most strongly to sites 111and IV Lu(II1) and Er(II1); opposite of Ca(II), ormetal binds most strongly to sites I and 11, Eu(II1)and Tb(II1); unique behavior, La(II1) and Nd(II1).Exchange of lS3Gd, 45Ca, and I3Cd from labeledCaM by flow dialysis with competing ions was used

1067

8/13/2019 lantanidos

2/4

1068 PATRICIA . OHARA

to measure binding and dissociative rate constants.Binding constants were 25@500 fold higher forLn(II1)s than for Ca(I1) binding to CaM. Gd(II1)dissociation followed first order kinetics, whereasboth Cd(I1) and Ca(I1) dissociation was biphasic.Together with other data, this suggests that Cd(I1)is a better analogue for Ca(I1) than Tb(II1) in CaM.Martin and co-workers (1986) reported the kineticsof Tb(II1) and Cd(I1) dissociation from CaM andits tryptic fragments. Fluorescence changes in thecompeting metal chelator, Quin 2, were monitoredwith time after rapid mixing with metal saturatedprotein or fragments. The two fragments, one con-taining the C-terminal I and IV sites and the othercontaining the N-terminal I and I sites, showeddifferent dissociation constants for the differentmetals. For Cd(II), dissociation was most slow fromthe C-terminal fragment, suggesting that the metalis bound more tightly to sites 111 and IV. ForTb(III), dissociation was most slow from the N-terminal fragment, suggesting that this metal isbound more tightly to sites I and 11 These differ-ences are preserved in the intact protein. These datagive irrefutable evidence that different classes ofbinding sites exist in CaM. Whether these classesare physiologically relevant remains to be seen.

Wang (1986) has measured a 10.5 8 distancebetween both pairs of metal-binding sites on CaM

by intra-Ln(II1) energy transfer. The N-terminal,site I-site I distance was measured by quenching ofthe Eu(II1) emission (using direct excitation) byNd(III) , assuming that a t half saturat ion, all of theLn(II1)s are bound to sites I and 11. Though this isnot strictly true for Nd(III), the results of the studywould be valid regardless, and are, in fact, in goodagreement with crystallographic data. The C-ter-minal domain, site 111-site IV distance was deter-mined by quenching of Tb(II1) emission (selectedby using indirect excitation through tyrosines whichexist only at sites I and IV) and quenching thiswith Nd(II1). The distances from the N- and C-terminal domains to a labeled cysteine on troponinI in the binary complex was measured to be 27 and25 A, respectively.

Ca l1)-M g II) ATP ase fro m sarcoplasmicreticulum

By using laser excited Eu(II1) luminescence,Shamoo and coworkers have investigated the majorCa(I1) translocater from skeletal and cardiac musclesarcoplasmic reticulum, Ca(I1)-Mg(I1) ATPase.Eu(I1I) bound to the Ca(I1) translocating sites ofthe skeletal muscle ATPase inhibited Ca(I1) bindingand uptake, phosphoenzyme formation, and ATPhydrolysis activity (Gangola and Shamoo, 1987).Nonequivalence of the two Ca(I1) sites was evidentfrom both the complexity of the excitation profileand the double exponential fit of the luminescencedecay. ATP binding was seen to reduce the numberof water molecules bound at the two sites, from

four to zero at the short-lived site and from 1.5 to0.6 at the long-lived site, indicating major changesin the protein environment of the metal. Cardiacmuscle Ca(I1)-Mg(I1) ATPase was also character-ized by similar techniques and though different indetail, the general observations were the same asfor skeletal muscle, two Eu(II1) lifetimes wereobserved, and the number of water molecules ateach site is reduced upon ATP binding (Joshi andShamoo, 1987). Energy transfer experiments fromluminescent Ln(II1) donors at one Ca(I1) site toLn(II1) quenchers at the second Ca(I1) site yieldedintersite distances of S 9 8 (Herrmann et al. 1986).Other energy transfer experiments from Eu(II1) atone site to Cr-ATP at the Mg-ATP binding siteshowed that the distance between these sites is lessthan 10

A.A biologically relevant peptide, thought

to be a good candidate for the Ca(I1) transport sitewas synthesized and characterized by a variety oftechniques including Eu(II1) spectroscopy (Gangolaand Shamoo, 1986). The very unique octapeptide con-tains acidic amino acids (likely metal ligands) altemat-ing with proline residues which could potentiallyconstrain the peptide into a torus. The peptide bindsEu(II1) with a 1 1 stoichiometry and contains onemetal-bound water.

Ln I1I) substituted into other systems

Canada has recently examined the quenching ofTb(II1) luminescence by the anticancer drugs,cisplatin (1986) and adriamycin (1987) in tumorig-enic cells. Cisplatin quenches the Tb(II1) lumi-nescence in a static fashion suggesting either closecontact of the drug and the metal or conformationalchanges induced by cisplatin which affect the exci-tation of the metal. Adriamycin also quenches theemission from Tb II1) bound to the surface of thesecells, but by a Forster mechanism where thedonor-acceptor distance is 40 A . The data suggestthat both drugs or perhaps both drug receptors areintimately associated with the same Ca(I1) bindingprotein in the membrane.

The binding of Ln(I1I)s to oncomodulin, a cal-cium binding protein expressed nearly exclusivelyin tumor cells, was studied by both direct excitationof Eu(II1) and indirect excitation of Tb(II1) (Henzlet al . 1986). Like parvalbumin (Pa) the Eu(II1)excitation was very pH dependent, but unlike Pathe CD site was found to have similar affinities forTb(II1) and Ca(I1).

Lactalbumin is a low molecular weight proteinfrom mammalian milk that plays a crucial role inlactose biosynthesis and contains a high affinityCa(I1) site Kd = 0.2-3 nM). Musci and Berliner(1986) report measuring a Ca(I1)-Zn(I1) intersitedistance of 11.5 8 in lactalbumin by energy transferfrom Eu(II1) or Tb(II1) at the Ca(I1) site to Co(I1)at the Zn(I1) site.

The normal collagen aggregation to form fibrilsand filaments is fundamental to the formation of

8/13/2019 lantanidos

3/4

Yearly Review 1069

connective tissue. A breakdown in the fibrillo- Fluoroimmunoassaygenetic machinery can lead to disease. Drouven andEvans (1986) studied the activation of fibrillogenesisby Ln(II1)s with intact collagen and collagen pepsinfragments. Light scattering due to the polymeric

nature of the sample made luminescence measure-ments difficult, but Tb(II1) emission at 490 and 545could be observed on a broad background when thesample was excited at 290 nm.

The binding of Ln(II1)s to acidic and neutralphospholipids was investigated by direct laser exci-tation of bound Eu(II1) (Herrmann et al . , 1986).As expected, neutral phospholipids bound metalweakly, stripping off one or two waters of hydrationwhen added to aqueous Eu solutions. In contrast,acidic phospholipids stripped off all but one or twowater molecules from Eu. This work underlines thesignificant interaction of Ln(1II)s with membraneswhich should not be ignored when interpreting datafrom protein-membrane systems.

The phenomenology of blood clotting requires acomplex concert of multiple protein factors bindingto membranes stimulated by Ca(I1). Previousattempts to use Ln(II1)s to probe this heterogenoussystem have been complicated by the Eu(II1)induced precipitation and Tb(II1) induced aggre-gation of prothrombin (Pro). The successful use ofTb(II1) to mimic the Ca(I1) induced equilibrium

binding of Pro to phospholipid vessicles (PLV) hasrecently been reported (Sommerville et a l . , 1986).Eight binding sites were identified in the binding ofTb(II1) to FI, the N-terminal portion of Pro,whereas 11 Tb(II1) binding sites were identifiedwhen Pro was mixed with PLV. These results wereconsistent with the number of Ca(I1) and Mn(I1)ions needed to saturate Pro in the absence(8) andpresence(l1) of PLV. Three types of metal bindingsites were inferred. Decreases in the lifetime ofTb(II1) bound to Pro and Pro-PLV mixtures wastaken as evidence that all sites were at least partiallyexposed to the collisional quencher cobalt-EDTAand that n o buried sites exist though i t is not clearwhether intra-Ln(II1) energy transfer from a poten-tially buried Tb(II1) to an exposed Tb(II1) was con-sidered.

Newton and Huestis (1986) examined the aniontransporter in human erythrocytes by using the sen-sitization of Tb(II1) luminescence by the aniontransporter substrate, dipicolinic acid. The appear-ance of anion-bound Tb(II1) luminescence with 278nm excitation was correlated with anion efflux fromred blood cells, red cell membrane fragments, andband-3-vessicle complexes.

In another report, (Loscalzo and Rabkin, 1986)Ca(I1) binding sites on both the resting and acti-vated human platelet were probed by Tb(II1) lumi-nescence. Upon activation, the number of Ca(I1)binding sites increases by 35% and interestingly,the Tb(II1) luminescence increases 78%. The exactnature of the binding protein or proteins is, at thispoint, unknown.

Recently, Ln(II1) luminescence has come underclose clinical scrutiny as a replacement for the radio-active emission currently used to lend sensitivity to

immunological assays (Jackson and Ekins, 1986).Coupling of a radio-isotope (generally *I) to eitheran antigen of interest (radioimmunoassay-RIA) oran antibody of interest (immunoradiometric assay-IRMA), yields antigenic sensitivities as low asM ensitivities approaching and in some cases sur-passing this value have been reported using lumi-nescence from Ln(II1)s chelated to either anantigen (fluoroimmunoassay-FIA) or an antibody(immunofluorometric assay-IFMA). This is poss-ible due to the ability to recycle the probe duringthe course of the assay and to greater collection

capabilities and quantum yields. The practicaladvantages of the luminescent assays are that nodangerous radioactivity is used and the assays arecomplete in seconds rather than minutes. Severalreports document the use of Ln(1II)s and FIA andIFMA for the detection of the hormones; thyrotro-pin (Lawson et al. , 1986), follitropin (Bador et al .1987), and prolactin (Dechaud et al . 1986).Increases in sensitivity have been made by the useof laser-excitation and time resolution. These devel-opments open up the possibility for accurate androutine clinical assays using commercial luminome-ters which would reduce exposure of laboratorypersonnel to radiation, be more cost efficient, andpermit new diagnostic strategies.

New applications

Two groups (Austin et a l . 1987 and OHara eta l . , 1986) have recently initiated investigations ofprotein dynamics using Ln(II1) luminescence emis-sion as a probe. By examining the temperaturedependence of the shape of the decay of Tb(II1)emission in the protein calmodulin from 293 to 140K Austin and coworkers have noted the markeddeviation from simple exponential behavior below200 K . They have interpreted this in terms of arubber to glass phase transition of the polymerwhere there is a reduction in the number of confor-mational substates and thus a less efficient averagingbelow 200 K . OHara and coworkers examined thetemperature dependence of energy transfer in vari-ous Ln(III)/protein samples in the range from 290to 340 K. Changes in the normalized energy transferefficiency were interpreted in terms of generalizedprotein motions over 2, 10, and 45 A in differentprotein matrices.

Meares and coworkers (Wensel et a l . , 1986) haveused Ln(II1) spectroscopy to probe the electrostaticforce surrounding DNA, which is thought to regu-late and control protein binding to DNA and thusall aspects of DNA activity. By analyzing the ratesof bimolecular energy transfer from luminescentLn(II1) donors to transition-metal acceptors, the

8/13/2019 lantanidos

4/4

1070 PATRICIA . OHARA

distribution of ions around a polyelectrolyte suchas DNA can be numerically related to the spatialdistributions of the ions. Energy donors wereTb(II1) complexes which range in overall chargefrom -1 to 1 and energy acceptors were cobalt

complexes which range in overall charge from -1to +2. As expected, the results show that at 2 mMsalt, collisional frequency increases six-fold betweenmonovalent cations and 29-fold between mono anddivalent cations in the presence of 1 mM DNA.Catiodanion collisional frequencies are predictablyreduced. These direct experimental results werecompared with four theoretical calculations. Theclever strategy of probing the electrostatic field bycollisional quenching of Tb(II1) chelates can beextended to other structures of biological interestsuch as synthetic DNA oligomers, Z-form DNA,DNAldrug and DNA/protein complexes, as well asother types of macromolecules.

New developments in the use of circularly pola-rized luminescence of chiral Ln(II1) complexes tosystematically characterize Ln(II1) coordinationchemistry have been summarized by H. Brittainelsewhere in this issue and so will not be discussedhere except to say that such characterization isessential before a full understanding of the bioinor-ganic chemistry of the Ln(II1)s is possible.

Department of ChemistryAmherst CollegeAmherst, MA 01002 U S A

PATRICIA . OHARA

REFERENCES

Austin, R. B., D. L. Stein and J. Wang (1987) Terbiumluminescence lifetime heterogeneity and protein equilib-rium conformational dynamics. Proc. Natl. Acad. Sci.

Bador, R . , H. Dechaud, F. Claustrat and C. Desuzinges(1987) Europium and samarium as labels in time-resolved immunofluorometric assay of follitropin. Clin.Chem. 33 4 51.

Buccigross, J. M. and D . J. Nelson (1986) EPR studiesshow that all lanthanides do not have the same orderof binding to calmodulin. Biochem. Biophys. Res. Com-mun. 138 1243-1249.

Buccigross, J. M. and D. J . Nelson (1986) Comparisonof lanthanide and cadmium as calcium models: metal-ion induced conformational changes in spin-labeled cal-modulin. J . Less-Common Metals 126 343-350.

Buccigross, J . M., C. L. ODonnell and D. J . Nelson(1986) A flow-dialysis method for obtaining relativemeasures of association constants in calmodulin-metal-ion systems. Biochem. J . 235 677-684.

Canada, R. G . (1986) Terbium fluorescence studiesof cisplatin binding to GH3/B6 pituitary tumor cells.Biochim. Biophys. Acta 887 29-34.

Canada, R. G. (1987) Terbium luminescence studies ofadriamycin binding to GH3/B6 pituitary tumor cells.Biophysical J . 51 521a.

Dechaud, H., R. Bador, F. Claustrat and C. Desuzinges(1986) Laser-excited immunofluorometric assay of pro-lactin. Clin. Chem. 32 1323-1327.

Drouven, B. J . and C. H. Evans (1986) Collagen fibrillo-genesis in the presence of lanthanides. J Biol. Chem .261

USA 84 1541-1545.

11792-11797.

Gangola, P. and A. E . Shamoo (1986) Synthesis andcharacterization of a peptide segment of (Ca(I1)Mg(I1))-ATPase. J . Biol. Chem. 261 8601-8603.

Gangola, P. and A. E. Shamoo (1987) Characterizationof [Ca(II) Mg(II)]-ATPase of sarcoplasmic reticulumby laser-excited Eu II1) luminescence. Eur. J . Biochem.

Henzl, M. T., R. C. Hapak and E. R. Birnbaum (1986)Lanthanide-binding properties of rat oncomodulin.Biochim. Biophys. Acta 872 16-23.

Herrmann, T. R. , P. Gangola and A. E . Shamoo (1986)Estimation of inter-binding-site distances in sarco-plasmic reticulum (Ca(I1) Mg(I1)-ATPase usingEu(II1) luminescence energy transfer. Eur. J Biochem.

Herrmann, T. R . , A. R. Jayaweera and A. E. Shamoo(1986) Interaction of Eu(II1) with phospholipid vesiclesas monitored by laser-excited europium(II1). Lurnin.Biochem. 25 5834-5838.

Herzberg, 0 and M. N. G. James (1986) Crystallo-

graphic determination of lanthanide ion binding to tro-ponin C . FEBS 359 199, 279-282.Horrocks, W. Dew . (1982) Lanthanide ion probes of

biomolecular structure. In Advances in Inorganic Bio-chemistry (Edited by G . L. Eichorn and L. G. Marizilli),Vol. 4, pp. 201-261. Elsevier, Amsterdam.

Jackson, T. M. and R. P. Ekins (1986) Theoreticallimitations on immunoassay sensitivity. J . ZmmunolMeth. 87 13-20.

Joshi, N. B. and A. E. Shamoo (1987) Binding of Eu(II1)to cardiac sarcoplasmic reticulum (Ca(I1) and Mg(I1)-ATPase-laser excited Eu(II1) spectroscopic studies.Biophys. J 51 185-191.

Loscalzo, J. and D . Rabkin (1986) The interaction ofTb(II1) with the human platelet surface. Arch. Biochem.Biophys. 249 237-242.

Lawson, N., N . Mike, R. Wilson and H. Pandov (1986)Assessment of a time-resolved fluoroimrnunoassay forthyrotropin in routine clinical practice. C h Chem. 32684-686.

Martin, S . R., S. Linse, P. M. Bayley and S . Forsen(1986) Kinetics of Cd(I1) Tb II1) dissociation fromcalmodulin and its tryptic fragments. Eur. J Biochem.

Musci, G. and L. J. Berliner (1986) Intramoleculardistance measurements in alpha-lactalbumin. Biochem-istry 25 48874891.

Newton, A. C. and W. H. Huestis (1986) Efflux ofdipicolinic acid from human erythrocytes, sealed mem-brane fragments, and band 3-liposome complexes: afluorescence probe for the erythrocyte anion trans-porter. 156 56-60.

OHara, P. B., K. Gorski and M. Rosen (1986) Macro-molecular flexibility in the proteins calmodulin and trans-ferrin studied by thermal profiles of Forster energytransfer using bound lanthanide ions. Fed. Proc. 45160.

Sommerville, L. E., R. M. Resnick, D. D. Thomas andG. L. Nelsestuen (1986) Terbium probe of calcium-binding sites on prothrombin-membrane complex. J .Biol. Chem. 261 6222-6229.

Szebenyi, D. M. E . and K. Moffat (1986) The refinedstructure of vitamin D-dependent calcium-binding pro-

tein. J Biol. Chem. 261 8761-8777.Wang, C-L. A. (1986) Distance measurements betweenmetal-binding sites of calmodulin and from these sitesto Cys-133 of troponin I in the binary complex. J . Biol.Chem. 261 11106-11109.

Wensel, T. G., C. F. Meares, V . Vlachy and J . B. Mat-thew (1986) Distribution of ions around DNA, probedby energy transfer. Proc. Natl . Acad. Sci. USA, 833267-3271.

162 357-363.

158 555-560.

161 595-601.