Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Electron Magnetic Resonance Studies 201
Fig. 4. Q-band EPR spectra of a 2-mM Gd(III)-α-LA complex, pH 8.5 (50 mM
Tris-HCl buffer, T = 273 K). Experimental conditions were as follows: frequency,
34.56; GHz; microwave attenuation, –5 dB; modulation amplitude, 8 G; time constant,
1 s; scan time, 4 min; field set, 12,700 G; scan range, 2500 G. Reproduced with permission
from ref. 11.
sensitivity of the EPR spectrum to subtleties in electronic structure around manganese,
the EPR unfortunately fails here. This was also the problem at 35 GHz
(see Fig. 3B, 10°C), which was almost completely devoid of inhomogeneous
broadening contributions from second-order effects because of zero-field
splittings (12). On the other hand, the absence of spectral narrowing with increasing
temperature verified that the apparently homogenous line shape was not a
result of free, unbound Mn[II] (13).
3. Gadolinium [III]. Gd[III] is an almost perfect substitute for calcium. Here, the
EPR spectrum is interpretable to a greater level of detail than experienced with
Mn[II]. Figure 4 compares Gd[III] α-lactalbumin at Q-band (273 K). The differences
and deviations from highly symmetric environments are more discernible
particularly of lower temperatures (Fig. 5). The most prominent features were
clustered near g = 2 constitute a pattern expected for the central (M S = –1/2) fine
structure transition in a crystal field with intermediate rhombic symmetry (14).
The other two features at 11.74 and 13.70 kG (note 1.0T = 10 kG) apparently
belong to the outer (satellite) fine transitions. One can determine various energies,
quadratic zero-field splitting interactions, and its anisotropy.
4. Vanadyl [IV]. Chasteen showed many years ago that VO 2+ [IV] can substitute for
calcium, despite its unusually chemical structure and size (15). Spectrum 5 shows
the X-band spectrum of VO 2+ [IV] α-lactalbumin at 77 K. Analysis of the spectra,
by comparing model compounds, indicates that the VO 2+ [IV] was most closely
associated with four equatorial oxygen ligands (14). The linewidth of the m = 1/2
perpendicular line in deuterated water was reduced by 1.7 G, which correlates
with a single water ligand in the vanadyl protein complex. This is to be compared
with the X-ray crystal structure of human lactalbumin showing two water molecules
to a seven-oxygen coordinated calcium (16).