Phorochemisrry and Pitotobiology Vol. 46, No. 6 , pp. 1067-1070, 1987 Printed in Great Britain.

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YEARLY REVIEW

LANTHANIDE IONS AS LUMINESCENT PROBES OF BIOMOLECULAR STRUCTURE

Introduction Lanthanide luminescence provides a unique probe into questions of biological interest. This review article summarizes contributions to the literature in the last year in which light absorption or emission spectroscopy of the trivalent lanthanides [Ln(III)] was used as such a probe. Several optical properties make the Ln(II1)s useful and convenient: their redshifted visible emission, their long luminescent lifetimes, the sensitivity of their absorption and emission to environment, the overlap of their excited state manifolds with the excited states of biological ligands and finally, the variety of their emission properties which make intra-Ln(II1) transfer and luminescence quenching possible. The long history of Ln(II1) substitution into biochemical environments and spectroscopic analysis of the product is best summarized in the review by Horrocks (1982). By far, the greatest amount of biological information has been obtained from Ln(II1) substitutions into Ca(I1) binding sites in proteins. The greatest similarity between Ca(I1) and the Ln(1II)s is the ionic radius [Ca(II):1.06 A, Ln(1II)s range from 1.06 A to 0.85 A]. The obvious difference, the trivalent charge, is accompanied by an increased differential binding entropy. Ln(II1)s are much more highly solvated than Ca(II), and so must lose more of their solvation sphere to bind to the biological site. Thus there is a much greater increase in the entropy of the system when the Ln(II1) is bound which contributes to the generally larger binding constants. It must be remembered that the Ln(II1)s are by no means an isomorphous replacement for Ca(I1). Ln(III) protein crystals With this in mind, it is appropriate to review recent crystallographic work in which Ln(1II)s have been substituted into biological calcium sites. In their extensive analysis of the structure of vitamin D-dependent intestinal calcium binding protein, ICaBP, Szebenyi and Moffat (1986)- summarize work done using Ln(II1) substitution. Ln(II1)s have been shown to selectively displace Ca(I1) from certain binding sites in crystals of parvalbumin (Pa) and ICaBP. In Pa, the E-F site but not the C-D site binds Tb(II1). In ICaBP, the C-terminal site but not the N-terminal site binds Nd(1II). The crystal structures of these Ln(II1)-protein complexes show only slight displacements of the metal and little distortion of the protein itself. These sites are characterized by having one bound water, multiple mobile amino acid side chains as ligands, and an overall negative charge. Sites incapable of metal exchange presumably have limited ability to adjust their size and shape to accommodate the new metal. The authors advise caution in generalizing from data derived from such substitutions. Herzberg and James (1986) analyzed the binding of Eu(III), Tm(III), and Lu(II1) to half-saturated Ca(I1) crystals of troponin C (TnC). TnC binds four Ca(I1) and has two high-affinity structural sites in the C-terminal domain and two low-affinity regulatory sites in the N-terminal domain. Both high affinity sites in the crystal exchanged their Ca(I1) for Ln(III)s, though to different degrees dependent on metal and the site. Ln(II1) binding at these structural sites appears to be much the same as Ca(I1) binding, with the Ln(II1) displaced from the Ca(I1) only 0.3 8, at site I11 and 0.8 A at site IV. Ln(II1)s bound with much more varied affinities to the empty low affinity site I and not at all to the empty low affinity site I1 of the crystal. With two waters at this site, the Ln(II1) binding at site I is quite different than that expected for Ca(I1) binding. The authors caution that metal binding to low affinity sites described in this work is not physiologically relevant. Calmodulin Calmodulin (CaM), a major regulatory protein in eucaryotic cells, contains four similar binding sites for Ca(I1). General, though not universal, agreement is that Ca(I1) binds somewhat more strongly to sites 111 and IV in the C-terminal domain. Buccigross and Nelson (1986a,b,c) have contributed several papers in the last year which help to clarify some 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 differential 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 111 and IV, Lu(II1) and Er(II1); opposite of Ca(II), or metal 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 labeled CaM by flow dialysis with competing ions was used
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PATRICIA B. OHARA four to zero at the short-lived site and from 1.5 to 0.6 at the long-lived site, indicating major changes in the protein environment of the metal. Cardiac muscle Ca(I1)-Mg(I1) ATPase was also characterized by similar techniques and though different in detail, the general observations were the same as for skeletal muscle, two Eu(II1) lifetimes were observed, and the number of water molecules at each site is reduced upon ATP binding (Joshi and Shamoo, 1987). Energy transfer experiments from luminescent Ln(II1) donors at one Ca(I1) site to Ln(II1) quenchers at the second Ca(I1) site yielded intersite distances of S-9 8, (Herrmann et al., 1986). Other energy transfer experiments from Eu(II1) at one site to Cr-ATP at the Mg-ATP binding site showed that the distance between these sites is less than 10 A. A biologically relevant peptide, thought to be a good candidate for the Ca(I1) transport site was synthesized and characterized by a variety of techniques including Eu(II1) spectroscopy (Gangola and Shamoo, 1986). The very unique octapeptide contains acidic amino acids (likely metal ligands) altemating with proline residues which could potentially constrain the peptide into a torus. The peptide binds Eu(II1) with a 1 : 1 stoichiometry and contains one metal-bound water. Ln(I1I) substituted into other systems Canada has recently examined the quenching of Tb(II1) luminescence by the anticancer drugs, cisplatin (1986) and adriamycin (1987) in tumorigenic cells. Cisplatin quenches the Tb(II1) luminescence in a static fashion suggesting either close contact of the drug and the metal or conformational changes induced by cisplatin which affect the excitation of the metal. Adriamycin also quenches the emission from Tb(II1) bound to the surface of these cells, but by a Forster mechanism where the donor-acceptor distance is 40 A. The data suggest that both drugs or perhaps both drug receptors are intimately associated with the same Ca(I1) binding protein in the membrane. The binding of Ln(I1I)s to oncomodulin, a calcium binding protein expressed nearly exclusively in tumor cells, was studied by both direct excitation of Eu(II1) and indirect excitation of Tb(II1) (Henzl et al., 1986). Like parvalbumin (Pa) the Eu(II1) excitation was very pH dependent, but unlike Pa the CD site was found to have similar affinities for Tb(II1) and Ca(I1). Lactalbumin is a low molecular weight protein from mammalian milk that plays a crucial role in lactose biosynthesis and contains a high affinity Ca(I1) site (Kd = 0.2-3 nM). Musci and Berliner (1986) report measuring a Ca(I1)-Zn(I1) intersite distance of 11.5 8, in lactalbumin by energy transfer from Eu(II1) or Tb(II1) at the Ca(I1) site to Co(I1) at the Zn(I1) site. The normal collagen aggregation to form fibrils and filaments is fundamental to the formation of

to measure binding and dissociative rate constants. Binding constants were 25@500 fold higher for Ln(II1)s than for Ca(I1) binding to CaM. Gd(II1) dissociation followed first order kinetics, whereas both 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 kinetics of Tb(II1) and Cd(I1) dissociation from CaM and its tryptic fragments. Fluorescence changes in the competing metal chelator, Quin 2, were monitored with time after rapid mixing with metal saturated protein or fragments. The two fragments, one containing the C-terminal I11 and IV sites and the other containing the N-terminal I and I1 sites, showed different dissociation constants for the different metals. For Cd(II), dissociation was most slow from the C-terminal fragment, suggesting that the metal is bound more tightly to sites 111 and IV. For Tb(III), dissociation was most slow from the Nterminal fragment, suggesting that this metal is bound more tightly to sites I and 11. These differences are preserved in the intact protein. These data give irrefutable evidence that different classes of binding sites exist in CaM. Whether these classes are physiologically relevant remains to be seen. Wang (1986) has measured a 10.5 8, distance between both pairs of metal-binding sites on CaM by intra-Ln(II1) energy transfer. The N-terminal, site I-site I1 distance was measured by quenching of the Eu(II1) emission (using direct excitation) by Nd(III), assuming that at half saturation, all of the Ln(II1)s are bound to sites I and 11. Though this is not strictly true for Nd(III), the results of the study would be valid regardless, and are, in fact, in good agreement with crystallographic data. The C-terminal domain, site 111-site IV distance was determined by quenching of Tb(II1) emission (selected by using indirect excitation through tyrosines which exist only at sites I11 and IV) and quenching this with Nd(II1). The distances from the N- and Cterminal domains to a labeled cysteine on troponin I in the binary complex was measured to be 27 and 25 A, respectively. Ca(l1)-Mg(II) ATPase from sarcoplasmic reticulum By using laser excited Eu(II1) luminescence, Shamoo and coworkers have investigated the major Ca(I1) translocater from skeletal and cardiac muscle sarcoplasmic reticulum, Ca(I1)-Mg(I1) ATPase. Eu(I1I) bound to the Ca(I1) translocating sites of the skeletal muscle ATPase inhibited Ca(I1) binding and uptake, phosphoenzyme formation, and ATP hydrolysis activity (Gangola and Shamoo, 1987). Nonequivalence of the two Ca(I1) sites was evident from both the complexity of the excitation profile and the double exponential fit of the luminescence decay. ATP binding was seen to reduce the number of water molecules bound at the two sites, from

Yearly Review connective tissue. A breakdown in the fibrillogenetic machinery can lead to disease. Drouven and Evans (1986) studied the activation of fibrillogenesis by Ln(II1)s with intact collagen and collagen pepsin fragments. Light scattering due to the polymeric nature of the sample made luminescence measurements difficult, but Tb(II1) emission at 490 and 545 could be observed on a broad background when the sample was excited at 290 nm. The binding of Ln(II1)s to acidic and neutral phospholipids was investigated by direct laser excitation of bound Eu(II1) (Herrmann et al., 1986). As expected, neutral phospholipids bound metal weakly, stripping off one or two waters of hydration when added to aqueous Eu solutions. In contrast, acidic phospholipids stripped off all but one or two water molecules from Eu. This work underlines the significant interaction of Ln(1II)s with membranes which should not be ignored when interpreting data from protein-membrane systems. The phenomenology of blood clotting requires a complex concert of multiple protein factors binding to membranes stimulated by Ca(I1). Previous attempts to use Ln(II1)s to probe this heterogenous system have been complicated by the Eu(II1) induced precipitation and Tb(II1) induced aggregation of prothrombin (Pro). The successful use of Tb(II1) to mimic the Ca(I1) induced equilibrium binding of Pro to phospholipid vessicles (PLV) has recently been reported (Sommerville et al., 1986). Eight binding sites were identified in the binding of Tb(II1) to FI, the N-terminal portion of Pro, whereas 11 Tb(II1) binding sites were identified when Pro was mixed with PLV. These results were consistent with the number of Ca(I1) and Mn(I1) ions needed to saturate Pro in the absence(8) and presence(l1) of PLV. Three types of metal binding sites were inferred. Decreases in the lifetime of Tb(II1) bound to Pro and Pro-PLV mixtures was taken as evidence that all sites were at least partially exposed to the collisional quencher cobalt-EDTA and that no buried sites exist though it is not clear whether intra-Ln(II1) energy transfer from a potentially buried Tb(II1) to an exposed Tb(II1) was considered. Newton and Huestis (1986) examined the anion transporter in human erythrocytes by using the sensitization of Tb(II1) luminescence by the anion transporter substrate, dipicolinic acid. The appearance of anion-bound Tb(II1) luminescence with 278 nm excitation was correlated with anion efflux from red blood cells, red cell membrane fragments, and band-3-vessicle complexes. In another report, (Loscalzo and Rabkin, 1986) Ca(I1) binding sites on both the resting and activated human platelet were probed by Tb(II1) luminescence. Upon activation, the number of Ca(I1) binding sites increases by 35% and interestingly, the Tb(II1) luminescence increases 78%. The exact nature of the binding protein or proteins is, at this point, unknown. Fluoroimmunoassay

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Recently, Ln(II1) luminescence has come under close clinical scrutiny as a replacement for the radioactive emission currently used to lend sensitivity to immunological assays (Jackson and Ekins, 1986). Coupling of a radio-isotope (generally *I) to either an antigen of interest (radioimmunoassay-RIA) or an antibody of interest (immunoradiometric assayIRMA), yields antigenic sensitivities as low as M . Sensitivities approaching and in some cases surpassing this value have been reported using luminescence from Ln(II1)s chelated to either an antigen (fluoroimmunoassay-FIA) or an antibody (immunofluorometric assay-IFMA). This is possible due to the ability to recycle the probe during the course of the assay and to greater collection capabilities and quantum yields. The practical advantages of the luminescent assays are that no dangerous radioactivity is used and the assays are complete in seconds rather than minutes. Several reports document the use of Ln(1II)s and FIA and IFMA for the detection of the hormones; thyrotropin (Lawson et al., 1986), follitropin (Bador et al., 1987), and prolactin (Dechaud et al., 1986). Increases in sensitivity have been made by the use of laser-excitation and time resolution. These developments open up the possibility for accurate and routine clinical assays using commercial luminometers which would reduce exposure of laboratory personnel to radiation, be more cost efficient, and permit new diagnostic strategies. New applications Two groups (Austin et al., 1987 and OHara et al., 1986) have recently initiated investigations of protein dynamics using Ln(II1) luminescence emission as a probe. By examining the temperature dependence of the shape of the decay of Tb(II1) emission in the protein calmodulin from 293 to 140 K, Austin and coworkers have noted the marked deviation from simple exponential behavior below 200 K. They have interpreted this in terms of a rubber to glass phase transition of the polymer where there is a reduction in the number of conformational substates and thus a less efficient averaging below 200 K . OHara and coworkers examined the temperature dependence of energy transfer in various Ln(III)/protein samples in the range from 290 to 340 K. Changes in the normalized energy transfer efficiency were interpreted in terms of generalized protein motions over 2, 10, and 45 A in different protein matrices. Meares and coworkers (Wensel et al., 1986) have used Ln(II1) spectroscopy to probe the electrostatic force surrounding DNA, which is thought to regulate and control protein binding to DNA and thus all aspects of DNA activity. By analyzing the rates of bimolecular energy transfer from luminescent Ln(II1) donors to transition-metal acceptors, the

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PATRICIA B. OHARA

distribution of ions around a polyelectrolyte such as DNA can be numerically related to the spatial distributions of the ions. Energy donors were Tb(II1) complexes which range in overall charge from -1 to +1 and energy acceptors were cobalt complexes which range in overall charge from -1 to +2. As expected, the results show that at 2 mM salt, collisional frequency increases six-fold between monovalent cations and 29-fold between mono and divalent cations in the presence of 1 mM DNA. Catiodanion collisional frequencies are predictably reduced. These direct experimental results were compared with four theoretical calculations. The clever strategy of probing the electrostatic field by collisional quenching of Tb(II1) chelates can be extended to other structures of biological interest such as synthetic DNA oligomers, Z-form DNA, DNAldrug and DNA/protein complexes, as well as other types of macromolecules. New developments in the use of circularly polarized luminescence of chiral Ln(II1) complexes to systematically characterize Ln(II1) coordination chemistry have been summarized by H. Brittain elsewhere in this issue and so will not be discussed here except to say that such characterization is essential before a full understanding of the bioinorganic chemistry of the Ln(II1)s is possible.

Gangola, P. and A. E. Shamoo (1986) Synthesis and characterization of a peptide segment of (Ca(I1) + Mg(I1))-ATPase. J . Biol. Chem. 261, 8601-8603. Gangola, P. and A. E. Shamoo (1987) Characterization of [Ca(II) + Mg(II)]-ATPase of sarcoplasmic reticulum by laser-excited Eu(II1) luminescence. Eur. J . Biochem. 162, 357-363. 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 sarcoplasmic reticulum (Ca(I1) + Mg(I1)-ATPase using Eu(II1) luminescence energy transfer. Eur. J . Biochem. 158, 555-560. Herrmann, T. R . , A. R. Jayaweera and A. E. Shamoo (1986) Interaction of Eu(II1) with phospholipid vesicles as monitored by laser-excited europium(II1). Lurnin. Biochem. 25, 5834-5838. Herzberg, 0. and M. N. G. James (1986) Crystallographic determination of lanthanide ion binding to troponin C . FEBS 359, 199, 279-282. Horrocks, W. D e w . (1982) Lanthanide ion probes of biomolecular structure. In Advances in Inorganic Biochemistry (Edited by G . L. Eichorn and L. G. Marizilli), Vol. 4, pp. 201-261. Elsevier, Amsterdam. Jackson, T. M. and R. P. Ekins (1986) Theoretical limitations on immunoassay sensitivity. J . Zmmunol. Meth. 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 of PATRICIA B. OHARA Tb(II1) with the human platelet surface. Arch. Biochem. Department of Chemistry Biophys. 249, 237-242. Amherst College Lawson, N., N. Mike, R. Wilson and H. Pandov (1986) Amherst, MA 01002, USA Assessment of a time-resolved fluoroimrnunoassay for thyrotropin in routine clinical practice. C h . Chem. 32, REFERENCES 684-686. Austin, R. B., D. L. Stein and J. Wang (1987) Terbium Martin, S. R., S. Linse, P. M. Bayley and S. Forsen luminescence lifetime heterogeneity and protein equilib(1986) Kinetics of Cd(I1) Tb(II1) dissociation from rium conformational dynamics. Proc. Natl. Acad. Sci. calmodulin and its tryptic fragments. Eur. J . Biochem. 161, 595-601. USA 84, 1541-1545. Bador, R . , H. Dechaud, F. Claustrat and C. Desuzinges Musci, G. and L. J. Berliner (1986) Intramolecular distance measurements in alpha-lactalbumin. Biochem(1987) Europium and samarium as labels in timeresolved immunofluorometric assay of follitropin. Clin. istry 25, 48874891. Newton, A . C. and W. H. Huestis (1986) Efflux of Chem. 33, 4%51. dipicolinic acid from human erythrocytes, sealed memBuccigross, J. M. and D. J. Nelson (1986) EPR studies brane fragments, and band 3-liposome complexes: a show that all lanthanides do not have the same order fluorescence probe for the erythrocyte anion transof binding to calmodulin. Biochem. Biophys. Res. Comporter. 156, 56-60. mun. 138, 1243-1249. Buccigross, J. M. and D. J. Nelson (1986) Comparison OHara, P. B., K. Gorski and M. Rosen (1986) Macroof lanthanide and cadmium as calcium models: metalmolecular flexibility in the proteins calmodulin and transferrin studied by thermal profiles of Forster energy ion induced conformational changes in spin-labeled calmodulin. J . Less-Common Metals 126, 343-350. transfer using bound lanthanide ions. Fed. Proc. 45, 160. Buccigross, J. M., C. L. ODonnell and D . J . Nelson (1986) A flow-dialysis method for obtaining relative Sommerville, L. E., R. M. Resnick, D. D. Thomas and G . L. Nelsestuen (1986) Terbium probe of calciummeasures of association constants in calmodulin-metalbinding sites on prothrombin-membrane complex. J . ion systems. Biochem. J . 235, 677-684. Biol. Chem. 261, 6222-6229. Canada, R. G. (1986) Terbium fluorescence studies of cisplatin binding to GH3/B6 pituitary tumor cells. Szebenyi, D. M. E. and K. Moffat (1986) The refined structure of vitamin D-dependent calcium-binding proBiochim. Biophys. Acta 887, 29-34. tein. J . Biol. Chem. 261, 8761-8777. Canada, R. G. (1987) Terbium luminescence studies of adriamycin binding to GH3/B6 pituitary tumor cells. Wang, C-L. A. (1986) Distance measurements between metal-binding sites of calmodulin and from these sites Biophysical J . 51, 521a. to Cys-133 of troponin I in the binary complex. J . Biol. Dechaud, H., R. Bador, F. Claustrat and C. Desuzinges Chem. 261, 11106-11109. (1986) Laser-excited immunofluorometric assay of proWensel, T. G., C. F. Meares, V. Vlachy and J. B. Matlactin. Clin. Chem. 32, 1323-1327. thew (1986) Distribution of ions around DNA, probed Drouven, B. J. and C. H. Evans (1986) Collagen fibrillogenesis in the presence of lanthanides. J . Biol. Chem. 261, by energy transfer. Proc. Natl. Acad. Sci. USA, 83, 3267-3271. 11792-11797.