J. Biol. Chem., Vol. 266, Issue 6, 3422-3426, 02, 1991
NMR relaxation properties of 77Se-labeled proteins
P Gettins and SA Wardlaw
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
A 77Se-containing moiety has been attached to cysteine residues in bovine
hemoglobin, reduced ribonuclease A, and glutathione by reaction with
[77Se]6,6'-diselenobis(3-nitrobenzoic acid). The resultant species contain
Se-S linkages that have 77Se NMR absorptions in the range range of 568-580
ppm. Spectra have been recorded at 4.7 and 9.7 tesla (T). For labeled
hemoglobin a line width of 250 Hz is seen at 4.7 T and 1000 Hz at 9.4 T.
This quadrupling of line width with doubling of observational field
strength is consistent with exclusive relaxation by the chemical shift
anisotropy (CSA) mechanism. These line widths are greater than expected for
a molecule the size of hemoglobin and indicate some aggregation at the high
concentrations used. Upon dissociation and partial unfolding of the
hemoglobin subunits, the line widths of the selenium resonance decrease to
35 and 120 Hz at 4.7 and 9.4 T, respectively. The spin-lattice relaxation
time (T1) for the dissociated hemoglobin at 9.4 T was found to be 220 ms.
Together with a value of 377 ms for the spin-spin relaxation time (T2),
determined from the line width, an estimate of the CSA was made. This gave
a value of 890 ppm, which is in accord with other values for Se(II) linked
only by single bonds. When this value for the CSA is used, together with
the CSA contribution to the line width, in estimating a correlation time
for seleno(3-nitrobenzoic acid) (SeNB)-labeled glutathione, a value of 4 x
10(-11) s is obtained. For SeNB-labeled denatured ribonuclease, four
distinct resonances are resolvable at 4.7 T and five resonances at 9.4 T.
From T1 values for these resonances and the value of 890 ppm for the CSA,
an appropriate correlation time of 0.1 ns was determined, which should
result in 77Se resonances of 0.2-1.0 Hz at 4.7 and 9.4 T, respectively.
Much greater apparent line widths are observed, which are attributed to
microheterogeneity resulting from formation of inter- and intramolecular
disulfide linkages. It is concluded that when there are no complications
from protein aggregation or chemical exchange, the CSA values anticipated
to exist in glutathione peroxidase or other selenoproteins should result in
resonances with line widths in the range from 27 to 170 Hz, depending on
field strength. These resonances should therefore be observable in the
intact protein, if 77Se-enriched material is available.
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