Abstract
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.
Highlights
From the DeDartment of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 '
A 77Se-containing moiety has been attached to cys- Selenium is an essential nutrient in mammalian systems teine residues in bovine hemoglobin, reduced ribonu- (Burk, 1983).It has been foundthat selenium is a component clease A, and glutathione by reaction with ['%e16,6'- of two different types of protein
The spin-lattice relaxation time(TIf)or 77Se NMR spectroscopy is potentially an ideal means of the dissociated hemoglobin at 9.4 T was found to be examiningthe natureand interactions of selenium in macro
Summary
Typical stoichiometries of 3.6-3.9 labels/hemoglobin tetramer were found. These values were somewhat unexpected, since Amiconi et al. To this wasslowly added 2 molf a solution of sodium methoxide in methanol, prepared by the reaction of 0.175 g of sodium with 5 ml of anhydrous methanol This gives a deep violet solution of the selenideanion (Rheinboldt and Giesbrecht, 1955).The reaction position expected for SeNB attached to a cysteine sulfur (Luthra et al, 1982), whereas the minor peak at 474 ppm is from residual non-covalently associated DSeNB. Preparation of SeNB-labeledDerivatives-10 pmol of glutathione in 200 p1 of0.1 M Tris, 1 mM EDTA, pH 8.0, was added to 20 pmol of DSeNB in 1.8ml of the same buffer This immediately gave a dark expected if the only efficient relaxation mechanism is chemical shift anisotropy, since there is a dependence of TZ-'(and orange solution as a result of formation of SeNB-. The reaction wasallowed to bound [77Se]DSeNBT. he sample was 2 ml of 150 mg/ml hemoglobin proceed in the dark at room temperature for 2 h before dialysis for 6 in 0.1 M potassium phosphate buffer at pH 6.3
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