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PROBING THE METAL BINDING SITES OF α-LACTALBUMIN WITH LASER-EXCITED Eu(III)
IONS
J.-C.G. Bünzli, P. Morand, J.-M. Pfefferlé
To cite this version:
J.-C.G. Bünzli, P. Morand, J.-M. Pfefferlé. PROBING THE METAL BINDING SITES OF
α-LACTALBUMIN WITH LASER-EXCITED Eu(III) IONS. Journal de Physique Colloques, 1987, 48
(C7), pp.C7-625-C7-627. �10.1051/jphyscol:19877151�. �jpa-00226973�
JOURNAL DE PHYSIQUE
Colloque C7, suppl6ment au n012, Tome 48, dhcembre 1987
PROBING THE METAL BINDING SITES OF a-LACTALBUMIN WITH LASER-EXCITED Eu(II1) IONS
J.-C.G. BUNZLI, P.D. MORAND and J.-M. PFEFFERLE
Institut de Chimie Minerale et Analytique, Universite de Lausanne, Place du Chdteau 3 , CH-1005 Lausanne, Switzerland
cc-lactalbumin, also known as the B-protein of the lactose synthetase enzymatic complex, is a soluble milk protein which is secreted throughout lactation and functions as an "on-off' switch in the synthesis of lactose. The protein has a molecular weight of 14,200 and contains 123 amino acid residues arranged in a sequence similar to that of egg-white lysozyme [I]. The protein in its native form contains one or two Ca(I1) ions per mole but despite extensive investigation, mainly by Kronman and coworkers [2,3,4,8], the number and location of the sites for attachment of calcium remain ill-defined. We have initiated a study to determine further the number and nature of the protein metallic sites using Eu(II1) as a luminescent binding probe.
Experimental Details
Proteix calcium-free bovine a-lactalbumin (apo-BLA) was prepared from native BLA (Sigma Chemical Co., type I, lot 14F-8030; 2.2 mol ca2+ per mol BLA) by the method of Kronman and Bratcher [3]. The cal ium content, as determined by plasma emission, was found to vary between 0.02 and 0.05 mol Ca" per mol protein. The protein concentration was determined by measuring the absorba ce at 280 nm ( 6 = 29,400 ~ - l . c m - ' [4]).
Laser-induced
2'
luminescence: high resolution (0.2A)
5 ~ 0-
7 ~ 0 excitation spectra were scanned between 577 and 581 nm using an argon laser-pumped tunable dye laser (Coherent CR8-CR599/Rhodamine 6G). The emission was monitored at 615 nm. The spectra were fitted by means of a curve-resolution program, using Gaussian firnctions. All measurements were performed on protein solutions in D20 (Ciba-Geigy, 99.95%) containing 0.02 M Tris (Fluka, puriss. biochim.) and 0.01 M KCl.Luminescence lifetime measurements were obtained using a pulsed XeCl laser-pumped tunable dye laser (Lambda Physik EMG 101 MSC
-
FL 3001/Rhodamine 6G). The laser pulse rate, width and energy were 2 Hz, 20 ns and 10 mJ, respectively.pH tifrafions: monotonic pH titrations were carried out at 2S°C on M solutions of protein in 0.01 M KC1 using a Metrohm Model E636 Titroprocessor equipped with a combination glass microelectrode and a 1 rnl automatic buret. The titrating solutions (CaC1 and LnC13. in 0.01 M KCl) were prepared from the corresponding hydrated salts and standarbsed by titrat~on with EDTA. pH readings were taken after 1 min stabilisation following addition of titrant.
Calibrations were performed with p:I 4 and 7 Metrohm buffers. All protein and salt solutions were stored in polypropylene flasks.
Results and Discussion
5 owing to the non-degenerate nature
04
the F(II1) ground ( 7 ~ ) and excited emissive ( Do) states, the number of components of the D + Fo excitation ban! reflects the number of chemically different environments around the E~(I!!I) ion. In order to gain insight into the nature of the Eu(II1)-binding sites on BLA, laser excitation spectra were recorded under varying condi- tions of pD and metal-to-protein ratio.Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19877151
C7-626 J O U R N A L D E PHYSIQUE
The effect of changing the Eu:BLA ratio at constant pD is shown in Figure 1. The curve resolution reveals that the number of distinct chemical environments around Eu(II1) increases with increasing metal loading, to a maximum of 4. By comparison with spectra taken in the absence of BLA under otherwise identical conditions, the fourth band in Figure lc, labelled IV, can be readily attributed to the solvated Eu-TRIS 1:l complex. The other three bands, labelled I- 111, are associated with metal binding sites on BLA.
Following the variation of band intensity as a function of increasing Eu:BLA ratio (Figure 2), we note that the sites associated with bands I and I1 are occupied simultaneously at low metakprotein ratio. Interestingly, around 1 equiv. of added Eu(III), the growth of band 111 is accompanied by marked changes in the intensities of band I and 11. These results point to there being a cooperative interaction in which binding to site 111 causes a modification of the respective affinities of sites I and I1
.
The charge surrounding the Eu(II1) ion can be estimated [5] from the value of the energy of the 5 ~ 0 level. In the case of BLA, one calculates a charge of -4, -3 and 0 (uncertainty: +I) for sites I, I1 and I11 respectively. Measurement of emission lifetimes of Eu(II1) bound to a protein in H O / D 2 0 mixtures provides information on the number of H 2 0 molecules bound to the metal ion
[g].
T h ~ s number has been found to be 2.1 t0.3 for site 1 (pD 3.9) and 4.0 k0.4 for site 111 (pD 6.48) [7]. Finally, the delayed growth of band I11 (Figure 2), its absence at p D < 3.9 and results from studies involving bovine serum albumin (BSA)-
on which Eu(1J.I) binds non-specifically-
suggest that site I11 is a low affinity, low specificity site.
Finally, simpler experiments such as pH titrations can provide valuable information on metal binding to BLA, particularly with respect to calcium. Indeed, addition of metals such as Ca(II), Zn(I1) and Ln(II1) to protein solutions causes a small pH depression. This may be due to protons being released from His groups (pKa- 6) or, to a lesser extent, from groups being exposed to the solvent upon metal uptake by the protein. Interestingly, Ca(I1)- and Eu(II1)- binding to BLA differ significantly (Figure 4). We can interpret the Eu(II1) titration curves (C,D) in the light of what we know from excitation spectra. Thus, the first region (0-1 equiv.) corresponds to filling sites I and I1 while the pronounced pH depression after 1 equiv. of Eu(II1) is due to binding to site 111. From the reduced pH variation in curve C compared to D in the 0-1 equiv. range we deduce that Eu(II1) replaces Ca(I1) at a calcium binding site. It is also important to note that the pH drop is greatest after 1 equiv., which strongly suggests the presence of His groups at site 111.
Turning to Ca(II), titration of Ca-BLA (curve A) from an initial pH of 6.10 leaves the pH unchanged while titration of apo-BLA causes a pH variation up to 0.5 equiv. of added Ca(I1) (curve B). Note that the magnitude of the pH drop in the 0-0.5 equiv. range is identical in both Ca(I1) and Eu(II1) titrations of apo-BLA (curves B and D), which lends support to the assertion that initial Eu(II1) binding occurs at a calcium site. In this regard, although previous data [8]
indicate that there is a single calcium binding site on BLA, our results from laser excitation spectra constitute clear evidence for simultaneous Eu(II1) binding at sites I and 11. Therefore, one cannot rule out the possibilily that calcium binding follows the same scheme. However, results from our p H measurements clearly demonstrate that Ca(I1) does not bind to site 111.
Conclusions
Our results have shown that there are three binding sites for Eu(II1) on a-lactalbumin, at least one of which is a calcium binding site. Binding of Eu(II1) to site I11 results in an alteration of the affinities of sites I (decrease) and I1 (increase). A rearrangement of the protein structure also occurs, but whether this triggers or results from binding to site I11 cannot be established unequivocally.
Acknowledgements
Support of this work through grants from the Swiss National Science Foundation and the Fondation Herbette (Lausanne) is gratefully acknowledged.
References
1. Hill, R.L.; Brew, K.; Adv. Enzymol., 1975,43,411.
2. Kronman, M.J.; et aL; Biochemktiy, 1964,3, 1152. ibid., 1965,4,518,526.
ibid., 1966, 5, 1970. Idem, J. BioL Chem., 1981, 256, 8582.
3. Kronman, M.J.; Bratcher, S.C.; J. BioL Chem., 1983,258,5707.
4. Kronman, M.J.; Andreotti, R.E.; Biochemistry, 1964,3, 1145.
5. Albin, M.; Horrocks, W.De W. Jr.; Znorg. Chem., 1985,24,895.
6. Horrocks, W. Dew. Jr.; Schmidt, G.F.; Sudnick, D.R.; Kittrell, C.; Bernheim, R.A.;
J. Am. Chem. Soc.. 1977.99.2378. Horrocks. W. Dew. Jr.: Sudnick. D.R.:
J. Am. Chem. SOC.; 1979; 10i, 334.
7. Biinzli, J.-C.G.; Proceedings, LASER M2P Conference, Lyon, July 7-9, 1987.
8. Bratcher, S.C.; Kronman, M.J.; J. Biol. Chem., 1984,259, 10875.
Figure 1. Curve-resolved E U ~ + excitation spectra BLA in D20/Tris buffer.
Protein concentration 6.47.10~~ M in 0.02 M Tris and 0.01 M KC1 (pD 6.96).
[Eu3+]:[BLA] = (a) 0.53, (b) 1.06 and (c) 1.76. (Aem= 615 nm).
Figure 2. Plot of the intensity of bands
-
I-IV as a function of increasing Eu3+
:BLA>, . C .
-0 ratio (BLA qded). [Eu3+] = 2.10-~ M (0-1
C - *.--- equiv), 9.10- M (1-4 equiv) in 0.02 M
- -
I Tris and 0.01 M KC1 (pD 6.96)./ ,,P' i ..-'
-
, , , , , , ,0 1 2 3
[ E U ~ + I I [BLAI
Figure 3. pH titration of (A) Ca-BLA and (B) apo-BLA with Ca(I1); (C) Ca- BLA and (D) apo-BLA with Eu(II1) at 2S°C and 0.01 M KCI. [Ca-BLA: Ca- containing BLA (1 or 2 Ca2+ /mole)].
