‘H NMR Studies on Lanthanides Substituted Transferrins Luigi Messori and Mario Piccioli Department of Chemistry, University of Florence, Florence, Italy

ABSTRACT The binding of lanthanide(IlI) ions to human serum apotransferrin has been investigated through ‘H NMR spectroscopy. Several well resolved isotropically shifted signals have been observed between lOO/- 100 ppm for the Tm, Tb, Yb, and Dy derivatives. Significant spectroscopic inequivalence of the two metal binding sites has been revealed. Differences in the behavior of signals assigned to the C- and to the N-terminal site have been observed upon titration with sodium perchlorate.

The ability of transferrins to bind lanthanides has been extensively demonstrated by UV difference [l-3], CD [4], ESR [5, 61, and fluorescence spectroscopies [7- 111. After a long controversy it has been established that human serum apotransferrin can bind specifically two equivalents of Ln(III) ions at the same sites of iron(III) provided that bicarbonate concentration is relatively low and the pH is held between 7 and 9 [3, 5, 6, 121. Because paramagnetic lanthanides, with the only exception of Gd(III), are known to behave as good shift reagents [13, 141 and, in principle, can provide structural information on the metal environment, we have prepared a series of lanthanide derivatives of human serum transferrin and recorded their ‘H NMR isotropically shifted spectra. Lanthanide derivatives were prepared at pH 8, in the presence of 5 mM sodium bicarbonate by adding two Ln(II1) equivalents to a 1 mM solution of apotransferrin. The best ‘H NMR spectra in terms of the ratio between isotropic shifts and linewidths were obtained for the 2:l derivatives of thulium, dysprosium, terbium, and ytterbium. In addition, we observed that the linewidths of the isotropically shifted signals sizeably reduce if the spectra are recorded at relatively high temperatures. The strong dependence of the linewidths on temperature is probably to be ascribed to a relevant Curie type contribution to the T; ’ relaxation mechanisms [ 151. The ‘H NMR spectra of the four Ln,Tf derivatives at

Address reprint requests to: Dr. Luigi Messori, Department of Chemistry, University of Florence, Via Gino Capponi 7, I-50121 Florence, Italy. Journal of Inorganic Biochemistry, 0

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186 L. Messori and M. Piccioli

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(B) Tb,Tf. (C) Dy,Tf, and (D) Yb,Tf. All spectra have been recorded at pH 8.0 in TRWHCI 50 mM, in the presence of 5 mM sodium bicarbonate, 318 K. Each spectrum typically consisted of about 300,000 scans. recorded in block averaged mode, with a repetition rate of 90 ms.

3 18 K are shown in Figure 1. These spectra show a number of relatively narrow. paramagnetically shifted signals in the range lOO/- 100 ppm which can be tentatively assigned to protons located in the immediate proximity of the metals. It must be recalled that isotropic shifts induced by Ln(II1) ions are mainly dipolar in nature and can be roughly considered as a function of the three geometrical parameters r, 8, and w defining the orientation of the susceptibility tensor of the metal with respect to the x-ray coordinate system [ 161; in principle they can be used for the refinement of x..ray structure data and the determination ;,f the actual conformation in solution [IT]. Unfortunately, under the present conditions. despite many attempts to perform

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187

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signal correlation through the different LnTf derivatives, we do not have enough data -namely a sufficient number of specific signal assignments-as to determine unambiguously the orientation of the magnetic tensor. This prevents an exhaustive theoretical analysis of the observed shifts. On the other hand, ’ H NMR isotropic shifts can be advantageously used as an extremely sensitive probe of the conformational state of the metal centers to address two major problems of transferrin chemistry, namely the inequivalence of the sites and the effect of lyotropic agents [18, 191. The affinity constants of the two sites of transferrins for several metal ions have been determined; in most cases it has been found that they differ by at least one order of magnitude, the C-terminal site being the tighter one [20-241. A similar behavior has been found also for lanthanides [12]; as a consequence, stepwise titration of apotransferrin with lanthanide(III) ions should result in a roughly sequential filling of the sites. Provided that the two sites of transferrin give rise to distinct sets of ‘H NMR signals, a careful analysis of a series of spectra recorded at different Ln/Tf ratios should allow us to discriminate between signals arising from the C-terminal and from the N-terminal sites. Indeed, such a behavior was detected in the case of Yb(III) derivatives as shown in Figure 2. The final spectrum of the 2:l derivative (Fig. 2B) is characterized by five broad signals of similar intensity (a,b,c,d,e; signals c and d partially overlap) whereas for Ln/Tf ratios lower than 1 only signals b,c,e are detected (Fig. 2A). This suggests that the latter signals are specific to the tighter site whereas signal a and d belong to the weaker binding site. The present results indicate that the conformational properties of the two sites are sufficiently distinct; each of them can be easily discriminated under the resolution of the present technique. A clear confirmation of site inequivalence was obtained through the investigation of mixed transferrin derivatives containing an Al(W) ion in the C-terminal site and a Ln(III) ion in the N-terminal site. These derivatives were prepared by loading one equivalent of Al@) at pH 6, increasing pH up to 8, and adding one equivalent of Ln(III) [25]. In Figure 3 the spectra of AlcTb,Tf (Fig. 3A) and AlcYb,Tf (Fig. 3B) are compared with those of the respective Ln,Tf deriva-

188 L. Messori and M. Piccioli

FIGURE 3. 90 MHz ‘H NMR spectra of Al,Tb,Tf (A), Tb,Tf (BI, AI,Yb,Tf Yb,Tf (D). Experimental conditions are the same as those in Figure I.

(C), and

tives. Some of the signals of the Ln,Tf derivatives are missing in the spectra of the respective Al,Ln.Tf derivatives; the latter signals can therefore be assigned to protons of the C-terminal site. Again spectroscopic discrimination of the properties of the two sites has been achieved. Because the environment of the two metals in iron transferrins is very similar. even if not equivalent [26--281, it remains to be understood to what extent the marked inequivalence observed in LnTf derivatives depends on the greater resolution of the present technique; alternatively different metal ions could enhance the slight but intrinsic structural differences of the two sites. The sensitivity of Ln,Tf derivatives to conformational agents has also been tested. It is known that lyotropic agents, at high concentration, markedly modify both the structural and functional properties of the metal sites [29. 301; in particular, these conformational effecters make metal release much faster from the C-terminal site but slower from the N-terminal site [30, 311. The opposite k.inetic behavior of the two sites must meet a structural basis. For this purpose we have titrated Ln,Tf derivatives with sodium perchlorate in the range 0. I -0.X M. Addition of perchlorate results in conformational changes which are witnessed by small but significant variations in the ’ H NMR spectra as shown in Figure 4. Interestingly. signals arising from the N-terminal site are more affected than those arising from the C-terminal site (see, for example, the behavior of signals labeled a and b in Tb,Tf. respectively located at - 50 and -- 55 ppm). This suggests that the conformational change experienced by the N-terminal domain is larger than that experienced by the

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C-terminal domain; a different extent of the conformational searched structural basis for the different functional behavior.

change

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Authors acknowledge Professor Ivano Bertini for helpful suggestions.

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L. Messori,

M. Piccioli,

Received September 20, 1990; accepted October I, 1990

and A. Scozzafava.

99, 1101 Inorg.