"HE JOURNAL OF BIOLOGICAL CHEMISTRY 0 I991 by The American Society for Biochemistry and Molecular Biology, Inc.

Voi. 266, No. 6. Issue of February 25. pp. 3422-3426.1991 Printed in U. S A .

NMR Relaxation Properties of "Se-Labeled Proteins*

(Received for publication, July 26, 1990)

Peter Gettins$ and Sarah A. Wardlaw 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- teine residues in bovine hemoglobin, reduced ribonu- clease A, and glutathione by reaction with ['%e16,6'- diselenobis(3-nitrobenzoic acid). The resultant species contain Se-S linkages that have "Se NMR absorptions in the range range of 568-580 ppm. Spectra have been recorded at 4.7 and 9.7 tesla (T). For labeled hemoglo- bin 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 dou- bling 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 (TI) 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 (Tz), 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(I1) 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 glutathi- one, a value of 4 X lo-" s is obtained. For SeNB- labeled denatured ribonuclease, four distinct reso- nances are resolvable at 4.7 T and five resonances at 9.4 T. From TI values for these resonances and the value of 890 ppm for the CSA, an appropriate corre- lation time of 0.1 ns was determined, which should result in '%e 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 o r chemical exchange, the CSA values anticipated to exist in glu- tathione peroxidase or other selenoproteins should re- sult in resonances with line widths in the range from 27 to 170 Hz, depending on field strength. These res- onances should therefore be observable in the intact protein, if 77Se-enriched material is available.

* This work was supported by National Institutes of Health Grant GM39509. Funding for the 200- and 400-MHz spectrometers is sup- plied in part by the Center in MolecuIar Toxicology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduer- hement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

t: To whom correspondence should be addressed.

Selenium is an essential nutrient in mammalian systems (Burk, 1983). It has been found that selenium is a component of two different types of protein. One is glutathione peroxi- dase, which can reduce Hz02 and organic peroxides and thus seems to be necessary to protect cell membranes against oxidative damage (Floh4, 1971). This enzyme occurs as dis- tinct plasma (Maddipati and Marnett, 1987; Avissar et al., 1989) and red blood cell (Awasthi, et al., 1975) forms. The second protein is plasma selenoprotein-P (Burk and Gregory, 1982; Yang et al., 1987), which is of less well defined function but may serve as a selenium transport protein (Motsenbocker and Tappel, 1982). The expression of both types of protein is regulated by levels of dietary selenium (Toyoda et al., 1989; Yang et al., 1989). To analyze the functional roles of selenium in these and other proteins, it would be highly desirable to have a spectroscopic means of investigation.

77Se NMR spectroscopy is potentially an ideal means of examining the nature and interactions of selenium in macro- molecules, since it has spin = 1/2, and there is unlikely to be any background contribution from sites other than the one of interest. To date, however, very few studies have been re- ported (Luthra et al., 1982; Mullen et al., 1986; Dowd and Gettins, 1988). The 77Se nucleus has a chemical shift range of over 3000 ppm (McFarlane and McFarlane, 1983) (cf. approx- imately 200 ppm for "C) and therefore might be expected to be very sensitive to changes in bonding and conformation. However, this sensitivity leads to a potential problem in observing selenium resonances in macromolecules, since large anisotropies in chemical shift occur, which, in macromolecular systems, might result in very efficient spin-spin relaxation and consequently very broad resonances. Furthermore, the efficiency of the chemical shift anisotropy (CSA)' relaxation mechanism is strongly dependent on the magnetic field strength used. This may result in the apparently paradoxical situation of higher field spectrometers, with higher sensitivity giving poorer signal-to-noise ratios than lower field instru- ments, as a result of broadening of the resonance more than offsetting the increased signal strength.

To evaluate the significance of these problems we have examined the relaxation properties of selenium in a constant R-Se-S-R' linkage, as a function of both the size of the molecule and the magnetic field strength. The thiol-reactive reagent 6,6'-diselenobis(3-nitrobenzoic acid) has been used to label cysteine SH groups in glutathione, reduced ribonuclease, and bovine hemoglobin. 77Se NMR spectra have been obtained for each of these species at 4.7 and 9.4 T field strengths. It was found that, even for the smallest species, CSA is the dominant relaxation mechanism at 9.4 T. For the larger species, CSA is the only efficient relaxation mechanism at

The abbreviations used are: CSA, chemical shift anisotropy; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); DSeNB, 6,6'-disele- nobis(3-nitrobenzoic acid); T, tesla; SeNB, seleno(3-nitrobenzoic acid).

3422

77Se NMR of Proteins 3423

both field strengths. From a comparison of TI and Tz values for the labeled hemoglobin sample, a value for the CSA of 890 ppm was determined. These findings enable predictions to be made concerning observation of "7Se resonances in intact glutathione peroxidase or other biological macromolecules of interest.

MATERIALS AND METHODS

Synthesis of r7Se/6,6'-Diselenobis(3-nitrobenzoic Acid)-The pro- cedure of Luthra et al. (1981) was followed, with small modifications. [77Se]KSeCN was prepared by dissolution of 100 mg of elemental selenium (94% Y3e-enriched, Oak Ridge National Laboratory, Oak Ridge, TN) in a 10% molar excess of 3 M KCN (Campbell and McCullough, 1945). This was left overnight at room temperature and gave a colorless solution of KSeCN, which was used without isolation of the selenocyanate. 1.57 mmol of 5-nitroanthranilic acid (10% excess over KSeCN plus residual KCN) were added to a tube con- taining 0.175 g of KOH in 2.1 ml of water. This was warmed to dissolve the nitroanthranilic acid. A solution of 112 mg of NaNOz in 200 p1 of water was added, and the solution was cooled on ice. 583 p1 of concentrated HCl was added dropwise and the suspension stirred for 20 min. The diazotization reaction was terminated by the addition of 760 mg of solid sodium acetate, which raised the pH. The solution of KSeCN was then added dropwise, and the mixture was stirred for 45 min at 0 "C and a further 150 min at room temperature. The suspension was centrifuged and the product selenocyanate dried overnight in a desiccator. A 'H NMR spectrum of the product in de dimethyl sulfoxide confirmed the identity and purity of the product. Three resonances were obtained, from H(2) at 8.56 ppm, H(4) at 8.30 ppm and H(5) at 8.07 ppm. Coupling to selenium was seen at H(2) and H(5). The selenocyanate was dissolved in 2 ml of dimethylform- amide. To this was slowly added 2 ml of 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 selenide anion (Rheinboldt and Giesbrecht, 1955). The reaction was allowed to proceed at room temperature for 15 min before pouring the solution onto 20 ml of ice to which had been added 1 ml of concentrated HCl. This gave a yellow precipitate, which slowly con- gealed as a sticky orange solid. The supernatant was decanted, and the product was rinsed and then dissolved in a small volume of ethanol to which water was added until a fine milky yellow precipitate formed. This was then freeze-dried. 274 mg of diselenide was obtained (87% based on selenium). The UV spectrum in 0.2 M Tris gave the expected maximum at 348 nm and an extinction coefficient in agree- ment with the literature value (Luthra et al., 1981).

Preparation of SeNB-labeled Derivatives-10 pmol of glutathione in 200 p1 of 0.1 M Tris, 1 mM EDTA, pH 8.0, was added to 20 pmol of DSeNB in 1.8 ml of the same buffer. This immediately gave a dark orange solution as a result of formation of SeNB-. Over time, the free SeNB- oxidized to reform DSeNB resulting in a change in color to yellow and the absence of any 77Se signal from SeNB-. A NMR spectrum gave two resonances, one at 569 ppm from the glutathione-SeNB adduct and one at 474 ppm from DSeNB.

SeNB-labeled reduced ribonuclease was prepared by the same procedure shown in Luthra et al. (1982). In a typical preparation, ribonuclease A was dissolved in 8 M urea, 0.1 M Tris, 1 mM EDTA, pH 8, and heated at 50 "C for 30 min to effect denaturation. A 50- fold molar excess of dithiothreitol over disulfides was added, and the solution was left at 50 "C for 3 h. Free dithiothreitol was removed by three cycles of dilution/reconcentration in an Amicon ultrafiltration unit and the total free SH content of the solution checked by assay with DTNB. A value of 8.5 free SH groups (8 expected) per ribonu- clease was determined. A 2-fold excess of [7"Se]DSeNB in the same buffer was added and the reaction mixture left 30 min before a 5-fold concentration in an ultrafiltration cell. After correction of the A355,,,,,

for absorption by free DSeNB (estimated from the absorbance at this wavelength of the ultrafiltration cell flowthrough) and of the A280 nm for absorption by total SeNB groups, a value of 4.6 SeNB labels/ ribonuclease was determined.

SeNB-labeled hemoglobin was prepared by a procedure analogous to that used by Amiconi et al. (1971) to label human hemoglobin with DTNB, except that DTNB was replaced by [77Se]DSeNB and bovine hemoglobin was used in place of human hemoglobin. Bovine hemo- globin, 25 mg/ml in 0.1 M K2HP04, pH 8.0, was reacted with 2 mg/ ml [77Se]DSeNB in the same buffer. The reaction was allowed to proceed in the dark at room temperature for 2 h before dialysis for 6

days against six changes of 2 liters of 0.1 M KHZPO4 buffer, pH 6.3, to remove nonspecifically bound DSeNB. The degree of covalent labeling was determined by spectrophotometric assay, using dithio- threitol to displace SeNB from cysteine residues. Any residual non- specifically bound DSeNB gives rise to a rapid burst of SeNB- release followed by a much slower pseudo-first order change representing the removal of covalently attached SeNB. The latter absorbance change was used to quantitate covalent labeling (Amiconi et al., 1971). Typical stoichiometries of 3.6-3.9 labels/hemoglobin tetramer were found. These values were somewhat unexpected, since Amiconi et al. (1971) reported 2 thionitrobenzoate labels/tetramer using DTNB as the labeling reagent. In addition to the use of DSeNB rather than DTNB, we employed bovine rather than human hemoglobin, since the labeled product appeared to have higher solubility, which was important for the 77Se NMR studies.

77Se NMR Studies-NMR studies were carried out, either on a Bruker AC200 wide-bore spectrometer operating at 38.2 MHz for or on a Bruker AM400 operating at 76.4 MHz. Pulsing conditions were optimized for each sample and varied depending on the 2'1 value. TI measurements were made using an inversion recovery pulse se- quence with phase cycling to reduce artifacts and with delay cycling to reduce time-dependent effects during the course of long experi- ments. Low power WALTZ proton decoupling was employed. Samples were 2 ml contained in IO-mm NMR tubes equipped with vortex plugs. Chemical shifts are referenced relative to external (CH&Se at 0 PPm.

RESULTS AND DISCUSSION

r7Se]SeNB-labeled Bovine Hemoglobin-The 77Se NMR spectrum of SeNB-labeled bovine hemoglobin at 9.4 T is shown in Fig 1. The major resonance at 574 ppm is at the 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. It should be noted that, because of the nature of the assay for incorporated SeNB groups (see "Materials and Methods") the non-cova- lently bound DSeNB does not interfere with quantitation of covalently bound SeNB groups. The resonance at 574 ppm, representing SeNB attached to approximately 4 cysteines/ hemoglobin tetramer, is extremely broad, with a half-height line width of approximately 1000 Hz (Table I). When the spectrum of the same sample was recorded at 4.7 T, the half- height line width of the 574-ppm resonance was reduced to 220 Hz, i.e. a 4-fold reduction. This is exactly the effect expected if the only efficient relaxation mechanism is chem- ical shift anisotropy, since there is a dependence of TZ-' (and

600 500 PPM

FIG. 1. 76.4-MHz (9.4 T) 77Se NMR spectrum of 17'Se] SeNB-labeled bovine hemoglobin, containing approximately four SeNB groups covalently bound to cysteine through Se-S linkages and giving rise to the major resonance at 574 ppm.

bound [77Se]DSeNB. The sample was 2 ml of 150 mg/ml hemoglobin The smaller resonance at 474 ppm arises from residual non-covalently

in 0.1 M potassium phosphate buffer at pH 6.3.

3424 77Se NMR of Proteins TABLE I

"Se NMR oarameters of SeNB n o u m attached to cvsteine Sample Field

T SeNB-hemoglobin 4.7

9.4 SeNB-hemoglobin/ 4.7

urea 9.4 SeNB-ribonuclease A 4.7

9.4 SeNB-glutathione 4.7

9.4

Av112~

Hz 220

1000 38

120

58-160 30-42

2 8

T I

Im

NDd ND ND 220 700 300

2580 900

TIKXA; 1;

ms

194 ns 171 ns 30 ns

220 23 ns 1600 100 ps 390 100 ps

4110 40ps 1030 40 ps

Apparent line width including any contributions from heteroge- neity.

* CSA contribution determined from field dependence of Tl. Correlation time determined from Tl/Tz(CSA) and Equation 3,

from T,(CSA) and Au (see "Results and Discussion"), or from T,(CSA) (based on half-height line width) and Au.

ND, not determined.

thus of the line width) on the second power of the field strength (Equation I), whereas other relaxation mechanisms do not show field dependence of the spin-spin relaxation time, except for small effects when w7= - 1.

Since it appears from the observed field dependence of the line width that CSA is the only relaxation mechanism oper- ative in this system, Equation 1 can be used to estimate Au, the chemical shift anisotropy, if a value for the correlation time, r,, appropriate to the selenium nucleus, is available. If it is assumed that the SeNB moiety is rigidly held by the hemoglobin, an estimate of the correlation time can be ob- tained by using the rotational correlation time of the protein as a whole, which in turn can be estimated from reported values of the diffusion coefficient of hemoglobin. Such an estimate would give an upper value to 7,. Using Dzo, = 6.9 X lo7 cm2 s" (Field and O'Brien, 1955) a value of 24.1 ns can be calculated for the rotational correlation time. However, the concentration of the hemoglobin solution used, 150 mg/ml, is far from a dilute solution. The viscosity of a 150-mg/ml sample of hemoglobin was determined and gave q/qw = 1.47. When correction for this increased viscosity is made, the expected correlation time is increased to 35.4 ns. Using this value in Equation 1, a value for Au of 2084 ppm is calculated. This is slightly more than twice that used by Luthra et al. (1982) for the same Se-S linkage in denatured SeNB-labeled ribonuclease, although in their calculation the contribution to the resonance line width from CSA relaxation had not been determined; the correlation time of 1 ns was arbitrarily taken, and the value for Au was estimated from a selenoether, dioctylselenide (Odom et al., 1979). Nevertheless, the present estimate seems high in light of the experimentally determined value for Au of 375 ppm for HzSe (Tossell and Lazzaretti, 1988), approximately 400 ppm for diphenyl diselenide (Wong et al., 1984) and 1270 ppm for elemental selenium (Koma and Tanaka, 1972). Since it was not possible to rule out some degree of multimer formation in the concentrated hemoglobin solution, which would increase the effective correlation time and thus lead to erroneously high estimates for Au when using the calculated correlation time, an alternative way of esti- mating Au was sought that did not require a priori knowledge of the correlation time.

The spin-lattice relaxation time (TI) due to chemical shift anisotropy is given by Equation 2. Combining this with Equa- tion l, it can easily be shown that T1/T2 is simply related by Equation 3. This shows that, for wrC > 1, the ratio of relaxation

times depends very strongly on the correlation time, whereas for wr, < 1, the ratio is constant and equal to 7/6. This is shown graphically in Fig. 2. Thus, for a macromolecular system such as hemoglobin, where wrC > 1 and where CSA relaxation dominates, determination of TJT, should permit calculation of 7= and, hence, from either Equation 1 or Equa- tion 2, calculation of Au. Unfortunately, the signal from labeled intact hemoglobin is so broad that a TI determination would require prohibitive amounts of spectrometer time. A compromise was to dissociate the labeled hemoglobin into subunits by transferring it into 8 M urea. This effects disso- ciation and partial subunit unfolding (Kirshner and Tanford, 1964), though complete retention of the heme ligand, judged by the absence of heme in the ultrafiltration flowthrough of the dissociated sample, indicates that substantial structure remains and that 07~ is likely to be very much greater than 1. The 77Se NMR spectrum of this dissociated sample gave a single resonance a t 575 ppm with a half-height line width of 120 Hz at 9.4 T and 38 Hz at 4.7 T (Table I). The roughly 4- fold increase in line width in going from an observation field of 4.7 to 9.4 T indicates that CSA relaxation is again the only effective mechanism, which then permits use of Equations 1 and 2 for calculation of the unknown parameters Au and rE. Equation 1 indicates that the line width is approximately proportional to the correlation time, so that the %fold reduc- tion in line width upon dissociation into subunits indicates that the undissociated sample probably contained aggregates of higher molecular weight than the tetramer.

The considerably narrower lines of the dissociated SeNB- labeled hemoglobin sample permitted a TI determination to be performed using the inversion recovery pulse sequence. A value of 220 ms was determined at 9.4 T. Using this value, and the value of Tz of 377 ms estimated from the half-height line width of the resonance, a value of 22.9 ns for the corre- lation time was calculated. This compares with a value of 17.4 ns calculated for myoglobin from the diffusion coefficient in water at infinite dilution (Perkoff et al., 1962). The effect of the higher viscosity of 8 M urea compared with water, ?/to =

"*.' 0 11 10 9 8 7

- ' 0qCJ5

FIG. 2. Dependence of the ratio Tl/T2 (CSA) on correlation time (Equation 3) for two different field strengths. Solid line, 4.7 T; dashed line, 9.4 T. It can be seen that at short correlation time (TJ, the ratio is insensitive to 7 , and cannot be used to estimate its value, while at long 7, it is extremely sensitive, depending approxi- mately on 72. Because of the presence of wz in Equation 3, the curve is field-dependent, reflecting the field dependence of T2 at constant Tl for > 1.

I7Se NMR of Proteins 3425

1.776 centipoise, would increase the correlation time in pro- portion, while any unfolding might tend to decrease it. Since there is evidence from the retention of the heme ligand by the protein that the subunits are not completely unfolded, the value estimated here for the correlation time is reasonable and is the value used subsequently in calculation on other SeNB-labeled species described here. Using this value, Aa can be calculated as being 890 ppm, which is in much better accord with expectations.

r7Se/SeNB-labeled Glutathione-The SeNB-labeled glu- tathione sample was prepared in the NMR tube and then examined and thus contained resonances from both this spe- cies and from free DSeNB. These species gave resonances at 570 and 474-ppm, respectively. Tl measurements were carried out on this sample a t both 9.4 and 4.7 T and gave values of 0.90 and 2.58 s, respectively, for SeNB-labeled glutathione and 0.88 and 3.0 s, respectively, for DSeNB. If w c < 1, contributions to TI from dipole-dipole relaxation and spin rotation are field-independent. The difference in TI at the different fields is thus due to the field-dependent contribution from chemical shift anisotropy relaxation, which may then be calculated. A Tl(CSA) a t 9.4 T of 1.03 s was determined. Using this value and the value for Aa of 890 ppm determined above from the labeled hemoglobin sample, a correlation time of 4 X lo-" s is obtained, which is in the range expected for a molecule of this size (Levy and Edlund, 1975).

77Se-Labeled Ribonuclease A-Luthra et al. (1982) reported the 77Se NMR spectrum of DSeNB-labeled denatured and reduced ribonuclease A at 2.35 T. Reflecting the sensitivity of the selenium nucleus to its environment, at least four resonances were resolvable, arising from the distinct cysteines labeled. Although these authors did not perform any relaxa- tion measurements, they did report the line widths of the resolved resonances as being from 34 to 53 Hz. These values seemed to be considerably higher than might be expected from an unfolded small protein with short correlation time. This species was therefore reexamined and Tl values determined 8s a function of field strength.

Fig. 3a shows a 4.7-T 77Se NMR spectrum of freshly pre- pared SeNB-labeled reduced ribonuclease A. Four well re- solved resonances, at chemical shifts between 567 and 580 ppm are seen, resulting from labeling of cysteine residues on the protein. These results are in agreement with those of

I

I 1 lbl

I sea 560 sin 530 abn 410 460

PPM

FIG. 3. "Se NMR spectra of ["SeISeNB-labeled, reduced, denatured ribonuclease A. The group of resonances between 566 nnd 580 ppm is from cysteine residues covalently labeled with ["Se] SeNB. The resonance at 474 ppm is from free DSeNB. a, spectrum 3f freshly prepared material at 36.2 MHz; b, spectrum at 76.4 MHz recorded 8 days later.

Luthra et al. (1982). The large resonance at 474 ppm is from free DSeNB. Fig. 3b shows the spectrum of the same sample recorded 8 days later at 9.4 T. The changes include not simply increases in apparent line width but also small shifts in resonance position and the appearance of additional compo- nents to low field of the 580-ppm resonance. TI values of 0.7 s were found for all of the protein resonances at 4.7 T, whereas shorter values of 0.3 s for all resonances were found at 9.4 T. Since TI decreases as the field strength increases, must be less than 1, and the change must again be due to the field- dependent contribution to relaxation from CSA. Using the field independence of all spin-lattice relaxation mechanisms except CSA, when W T ~ < 1, the contribution from CSA relax- ation can be calculated at each field strength. Tl(CSA) values of 1.6 and 0.39 s are calculated at 4.7 and 9.4 T, respectively. Substituting the Aa value of 890 ppm, determined above, a rotational correlation time of 1.0 X 10"' s is found, confirming that the ribonuclease must be unfolded.

At this point it is instructive to estimate the line width of the resonances using the determined correlation time of 0.1 ns and Aa value of 890 ppm. If CSA were the only relaxation mechanism at each field strength, line widths of 0.23 and 0.95 Hz would result (Le. 1-2 orders of magnitude smaller than apparently observed). While other relaxation mechanisms clearly contribute at the lower field strength, as evidenced by the less than 4-fold decrease in TI upon increasing the field by a factor of 2, it seems extremely unlikely that they would add more than a few tenths of a hertz at either of the field strengths. This raises the question of why the resonances, both reported here and by Luthra et al. (1982), are apparently much greater than these values.

A possible answer is microheterogeneity in the sample. Ribonuclease contains four disulfides, which yield eight free SH groups upon reduction with NaBH4. Although assay by either DSeNB or DTNB confirms the presence of eight free SH groups immediately upon reduction and just before addi- tion of the reagent for preparative labeling, the stoichiometry of covalently attached SeNB, estimated from the total ligand present in the sample after concentration and the relative intensities of Se-S uersus Se-Se (DSeNB) resonances, is in the range of 2-4 labels/molecule. Furthermore, dilution and reconcentration of the sample always diminishes the stoichi- ometry of covalently attached SeNB. This is a consequence of the labeling reaction involving an equilibrium that, while favoring S-Se over Se-Se plus S-S, has an equilibrium con- stant that is not orders of magnitude greater than 1. The consequence of reformation of S-S linkages is the possibility of generating an extremely large number of slightly different ribonuclease molecules, for a given stoichiometry of SeNB label, that differ in S-S cross-linking patterns. These cross- links may be both intra- and intermolecular. To test for the latter a sodium dodecyl sulfate-polyacrylamide gel was run on both reduced and nonreduced samples of the SeNB-labeled ribonuclease. While the former gave the expected single band with appropriate mobility, much of the latter was so exten- sively intermolecularly cross-linked that it failed to enter a 15% gel. It therefore seems likely that the 77Se NMR spectrum of SeNB-labeled ribonuclease A consists of many sets of similar spectra, with major differences in chemical shift aris- ing from the different sites of labeling and much smaller variations arising from heterogeneity due to differences in disulfide-bonding patterns. The latter is evidenced by an apparent broadening of the resonances.

Consequences for 77Se NMR Observation of Macromolecu- lady Bound Selenium--In proteins the selenium-containing species most likely to be examined are selenomethionine,

I7Se NMR of Proteins TABLE I1

Predicted properties of 77Se resonances in intact biological macromolecules

Sample T: Au Field T? A V I , ~ ~

P P T S Hz Glutathione per- 32 350-900 2.3 2.1-0.32 1.7-11

oxidase 4.7 2.0-0.30 6.4-42

Selenoprotein-P 20 350-900 2.3 1.44-0.22 1.1-7.3 9.4 2.0-0.30 25-166

4.7 1.28-0.20 4.1-27 9.4 1.24-0.19 16-106

Seleno-tRNA' 15 3000-6000 2.3 16.3-4.1 64-258 4.7 13.4-3.4 229-923 9.4 12.7-3.2 881-3553

a Estimated from molecular weight. * CSA relaxation contribution only. e TI values in milliseconds.

selenocysteine, or a selenide sulfide, either endogenous, such as has been proposed to be involved in the catalytic cycle of glutathione peroxidase, or introduced, such as the SeNB labels used in the present study. For all of these selenium species, the bonding involves two single bonds to selenium in oxida- tion state 11. The chemical shift anisotropies might therefore be expected to be similar to those found here for cysteine- SeNB or elsewhere for HzSe (i.e. in the range from approxi- mately 350 to 900 ppm). Since chemical shift relaxation has been shown to be the dominant relaxation mechanism for the protein species examined here and is almost certainly likely to be the mechanism for any intact protein, predictions can be made for the relaxation properties expected in other sys- tems. As an example, glutathione peroxidase will be consid- ered, since this is an important mammalian selenoenzyme. It is a tetramer with subunits of 20-24 kDa. In its enzymatic reaction with peroxide and glutathione, the selenium species SeH (or Se-), SeOH, and Se-S have been proposed to occur. Using a rotational correlation time of 32 ns, estimated from its size relative to hemoglobin, the expected TI and T2 values at different field strengths can be calculated from Equations 1 and 2 and are given in Table I1 for different values of Au. From this it can be seen that relatively narrow lines can be expected at the lower field strengths of 2.3 and 4.1 T and that, for the larger anisotropies, there is a short TI , so that rapid pulsing could be employed to maximize signal-to-noise in a given length of time. These line widths are similar to or less than 'I3Cd NMR line widths seen for 0.5-2 mM samples of enriched 'I3Cd (Gettins, 1988; Geidroc et al., 1989). It can thus be expected that unless there is additional broadening, such as that due to chemical exchange, it should be possible to observe 77Se resonances in 77Se-enriched glutathione peroxi- dase or other intact proteins. With the possibility of express- ing such proteins in culture, the incorporation of I7Se should be quite feasible.

The other class of macromolecules for which selenium NMR might be useful is selenonucleotide-containing tRNAs, such as those found in Escherichia coli, Clostridium sticklandii, and Methanococcus vanniellii (Wittwer et al., 1984). For these

molecules, however, selenium NMR is likely to be less suita- ble, since the selenium in selenouridine is involved in a C=Se double bond, for which the chemical shift anisotropy is ex- pected to be much larger than those found for the single bond species already considered. Thus, it has been found for two small molecules containing C=Se linkages that the values of Au were 3000-6000 ppm (Wong et al., 1984). Even at the lowest field strength of 2.3 T, line widths of 64-258 Hz are expected (using a correlation time of 15 ns).

Acknowledgments-We are grateful to Dr. Leon W. Cunningham for determination of the viscosity of the hemoglobin solution. We thank Brenda Crews for help in preparation of some of the labeled species.

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