Purification of a Thioredoxin System from Yeast*

SUMMARY In Escherichia coli the two proteins thioredoxin and thioredoxin reductase, together with NADPH, form a hydrogen transport system (“thioredoxin system”) which functions during the enzymatic reduction of ribonucleotides. We have now isolated thioredoxin and thioredoxin reductase from baker’s yeast. Yeast thioredoxin was obtained in two forms (thioredoxin I and II). Purified thioredoxin II appeared to be a homogeneous protein with a molecular weight of 12,600. In its oxidized form (thioredoxin-S), it contained a single disulfide bridge which was reduced with NADPH by thioredoxin reductase from yeast (but not from E. coli). The reduced form of thioredoxin (thioredoxin-(SH)z) served as hydrogen donor for E. coli ribonucleotide reductase. The oxidation-reduc-tion potential of the couple thioredoxin-(SH)z-thioredoxin-Sz was -0.24 volt at pH 7. The amino acid composition of yeast thioredoxin II showed considerable differences from that of E. coli thioredoxin. Yeast is a flavoprotein with FAD as the prosthetic group.

In Escherichia coli the two proteins thioredoxin and thioredoxin reductase, together with NADPH, form a hydrogen transport system ("thioredoxin system") which functions during the enzymatic reduction of ribonucleotides.
We have now isolated thioredoxin and thioredoxin reductase from baker's yeast.
Yeast thioredoxin was obtained in two forms (thioredoxin I and II).
Purified thioredoxin II appeared to be a homogeneous protein with a molecular weight of 12,600. In its oxidized form (thioredoxin-S), it contained a single disulfide bridge which was reduced with NADPH by thioredoxin reductase from yeast (but not from E. coli).
The reduced form of thioredoxin (thioredoxin-(SH)z) served as hydrogen donor for E. coli ribonucleotide reductase.
The oxidation-reduction potential of the couple thioredoxin-(SH)z-thioredoxin-Sz was -0.24 volt at pH 7. The amino acid composition of yeast thioredoxin II showed considerable differences from that of E. coli thioredoxin.
Yeast thioredoxin reductase is a flavoprotein with FAD as the prosthetic group.
Thioredoxin was identified as the hydrogen donor in the reduction of ribonucleotides to deosyribonucleotides in Escherichia coli B (1). It is a small protein and, as isolated, contains a single disulfide bridge made up from the 2 half-cystine residues of the molecule.
This disulfide bridge is reduced with NADPH by a specific flavoprotein, thioredoxin reductase (2) : thioredoxin Thioredoxin-Sz + NADPH + H+ T \ reductase (1) thioredoxin-(SH)z + NADP+ The dithiol form of thioredoxin is then used for the reduction of the hydroxyl group of the ribotide. This reaction is catalyzed by ribonucleotide reductase: * This work was supported by grants to P. Reichard Thioredoxin thus participates in a cyclic oxidoreduction reaction and, together with thioredoxin reductase and NADPH, forms a hydrogen carrier system (thiorcdoxin system) which makes available the reducing power of NADPH for the reduction of ribonucleotides (3). Both thioredoxin (4) and thioredoxin reductase (5) were obtained in pure form from E. coli B. Thioredoxin systems were also shown in other sources, such as Lactobacillus leichmannii (6), Novikoff hepatoma (7), regenerating rat liver,' and phageinfected E. coli (8). They were defined by their ability to act as hydrogen carrier systems during ribonucleotide reduction. The present investigation was started with a 2-fold purpose: (a) we wished to isolate pure thioredoxin from a source different from E. coli in order to learn something about the general principles involved in the mechanism of action of thioredoxin, and (b) we wanted to investigate the possibility that thioredoxin systems may function as hydrogen carriers in enzymatic reductions other than that of ribonucleot'ides.
Baker's yeast appeared to be a suitable starting material to obtain sizeable amounts of thioredoxin and its reductase. Furthermore, earlier work by others had shown that in yeast hydrogen carrier systems with properties reminiscent of the thioredoxin system might participate in the reduction of sulfate (9) and methionine sulfoxide (10). In this paper, we describe the preparation and characterization of a highly purified thioredoxin system from baker's yeast. In the accompanying paper (11) we show that this system can function as hydrogen carrier both in the reduction of sulfate and methionine sulfoxide.
The yeast was obtained as a 50% suspension in water which was centrifuged for 20 min at 5000 x g and the cell paste was washed with 0.9% NaCl. The cells were then collected by centrifugation and could be stored frozen at -20" without apparent loss of activity.
Before extraction the frozen cells were disintegrated three times by treatment with high pressure (12).
All other reagents were of analytical grade and were obtained from commercial sources.

lllethods Preparative Polyacrylamide Gel Electrophoresis
The runs were done in a Buchler polyacrylamide gel electrophoresis apparatus (Poly-Prep) equipped with a semipermeable rigid glass membrane as described by Thelander (5).

Acid Analyses
The amino acid composition of thioredoxin II was determined according to Spackman, Stein, and Moore (15) with a Spinco model 120B amino acid analyzer equipped with high sensitivity cuvettes (16). The samples were hydrolyzed under reduced pressure for 24, 60, and 96 hours. Extrapolations to zero time were made for serine. The sulfur-containing amino acids were analyzed after performic acid oxidation as described by Moore (17). Tryptophan was estimated either spectrophotometrically by the method of Bencze and Schmid (18) or by the method of Spies and Chambers (19).

Peptide Map
A peptide map of a tryptic digest of carboxymethylated (20) thioredoxin II (0.8 mg) was made as described by Holmgren (21) for thioredoxin from E. coli B essentially with the method of Harris and Perham (22).

Protein Determinations
During early stages of purification, protein was determined by the method of Biicher (23). After the Sephadex G-50 step the absorbance at 280 rnp of the protein solutions was used; a solution containing 1 mg of protein per ml was assumed to give an absorbance of 1 .O at 280 rnp (1 Asso unit).
With pure thioredoxin II, we found by refractometry in a Spinco model E analytical ultracentrifuge (24) that 1 Asgo unit corresponded to 1.08 mg of the protein. The initial protein concentration was determined in the ultracentrifuge with a capillary type synthetic boundary cell. The partial specific volume of thioredoxin II was calculated to be 0.736 ml per g from the amino acid com-position (26). A value of 0.724 ml per g was assumed for the partial specific volume of thioredoxin reductase (5).

Thioredoxin Assays
We used three different methods, similar to those used earlier for the assay of E. coli thioredoxin (1). Method l-This assay depended on the cross-reaction between reduced yeast thioredoxin and ribonucleotide reductase from E. coli. With an excess of the E. coli enzyme the formation of dCDP from CDP according to Equation 2 of the introduct,ory section depended on the concentration of thioredoxirr-(SH)z in the incubation mixture.
After incubation for 30 min at 25" the reaction was terminated by addition of 1 ml of M HClOd and the amount of dCDP formed was determined.
One unit of thioredoxin activity is defined as the amount of thioredoxin required to allow the formation of 1 mpmole of dCDP under the above conditions. Specific activity is defined as units per mg of protein.
Method Z-Here the reaction represented by Equation 1 of the introductory section was carried out in the presence of an excess of 5,5'-dithiobis(2nitrobenzoic acid) which reacted with the reduced thioredoxin to form a colored product and to regenerate thioredoxin-&.
Since thioredoxin is recycled, the rate of color formation was a relative measure of the amount of thioredoxin present.
Each of two cuvettes contained: 100 pmoles of potassium phosphate buffer (pH 7.0), 10 pmoles of EDTA, 0.1 pmole of NADPH, and about 30 pg of thioredoxin reductase from yeast (after DEAE-cellulose chromatography). Thioredoxin was added to one of the cuvettes and the volumes of both cuvettes were adjusted to 1 ml with water.
The absorbance at 412 mp of the thioredoxin-containing cuvette was read at I-min intervals with the other cuvette as a blank.
Usually three different determinations could be made simultaneously. One unit of thioredoxin is defined as the amount of protein which at room temperature under the above conditions gave an increase in the absorbance at 412 rnp of 1 per min.
Method S-Since Reaction 1 at neutral pH goes to completion the amount of thioredoxin-Sz present in a sample can be determined by measuring the total amount of NADPH which disappears during the reaction.
Each of two cuvettes contained: 0.1 pmole of NADPH, 120 pmoles of potassium phosphate buffer (pH 7.0), and 10 pmoles of EDTA in a final volume of 1.2 ml. Thioredoxin was added to one of the cuvettes which was used as a blank in the spectrophotometer.
After a zero time reading at 340 rnp, 1 to 2 pg of thioredoxin reductase (after gel electrophoresis) were added to each cuvette, and the apparent increase in the absorbance at 340 rnp was recorded at different time intervals.
A constant reading should be obtained after 2 min. From the difference between

Comparison of Methods
Method 1 could be used at all stages of purification. Its major drawback was the amount of time involved.
Method 2 is not very accurate and could not be used during the early stages of purification.
It is very rapid and can be made very sensitive. It is the method of choice for locating thioredoxin during column chromatography.
Method 3 is the only method which gives the absolute amount of thioredoxin present in a sample. However, reliable values are only obtained with quite pure samples of thioredoxin, since other disulfide-containing compounds may give rise to recycling of thioredoxin and thus interfere with the assay.

Thioredoxin Reductase Assay
The enzyme was measured by Method 2 described above for thioredoxin.
About 20 pg of thioredoxin after DEAE-cellulose chromatography were added instead of thioredoxin reductase to each of two cuvettes and the sample containing this enzyme activity was added to one of them. One unit of thioredoxin reductase is defined as the amount of enzyme which under the above conditions produced an increase in the absorbance at 412 rnp of 1 per min.
Specific activity is defined as units per mg of protein.

Pur$cation of Thioredoxin
Unless stated otherwise, all manipulations were carried out around 4'; centrifugations were performed at 15,000 x g. All Tris buffers were neutralized with HCI and t,he molarities refer to the Tris ion.
All buffers contained 0.001 M EDTA.
Extraction-Disintegrated yeast (2176 g) was stirred with 6500 ml of 0.05 M Tris, pH 7.5, for 30 min. Insoluble material was removed by centrifugation (20 min The precipitate was allowed to settle overnight and was collected by centrifugation for 45 min. The supernatant solution was discarded. The precipitate was dissolved in about 600 ml of 0.05 M Tris, pH 7.5, and ammonium sulfate was removed by gel filtration on a column (14 x 30 cm) of Sephadex G-25, equilibrated with the same buffer. The pH of the solution was lowered to 4.6 by addition of 1.0 M acetic acid (100 ml).
The precipitate was immediately removed by centrifugation and the supernatant solution was neutralized to pH 7.  When the solution had reached a temperature of 60" it was cooled in an ice-NaCl bath (-loo).
The precipitate was discarded after centrifugation.
Chromatography on Sephadex G-50-Thioredoxin was precipitated from the supernatant solution from the previous step (4600 ml) by addition of ammonium sulfate (0.612 g per ml); the precipitate was allowed to settle overnight, collected by centrifugation for 90 min, dissolved in about 300 ml of 0.01 M ammonium acetate (pH 8.6), and chromatographed on a column of Sephadex G-50 (11 X 140 cm) equilibrated with the same buffer. Elution (70. to SO-ml fractions) was performed at a rate of 350 ml per hour with the same buffer. Thioredoxin activity appeared in the latter part of the chromatogram (Fig. 1) well separated from the main protein peak which contained thioredoxin reductase activity.
First DEAE-cellulose Chromatography-A column of DEAEcellulose (5 X 24 cm) was washed first with 3 liters of 0.2 M ammonium acetate, pH 8.6, and subsequently with 3 liters of 0.01 hf ammonium acetate, p1-I 8.6. The washing procedure was carried out with a hydrostatic pressure of 2 m of water. The pooled thioredoxin fractions from the Sephadex G-50 step (1670 ml) were directly adsorbed to the top of the column (0.8 ml of DEAE-cellulose packed under the above conditions was used per mg of protein).
The DEAE-cellulose was then washed with 3 column volumes (1500 ml in total) of 0.01 M acetate buffer. After that, chromatography was started with a linear gradient of ammonium acetate, pH 8.6 (0.01 M to 0.08 M, 3750 ml of each). Fractions of about 70 ml were collected at 20-min intervals. Thioredoxin activity appeared as two peaks around 0.047 M (thioredoxin I) and 0.056 M (thioredoxin II) acetate, respectively (Fig. 2). The activities were pooled separately and lyophilized in presence of n-mannitol (5 mg of n-mannitol per ~4280 unit of protein).
The dry material was dissolved in water and then desalted by filtration through a column of Sephadex G-25 equilibrated with 0.01 M ammonium acetate, pH 8.6. Second DEAE-cellulose Chromatography--Both thioredoxins The ordinate gives the logarithm of the protein concentration (in fringes), and the abscissa represents the square of the distance from the center of rotation.
The liquid column was 3 mm.
were rechromatographed on columns of DEAE-cellulose (3 x 30 cm, 5 ml of DIME-cellulose per mg of protein). Both thioredoxin activities coincided with the main protein peaks appearing at acetate concentrations of 0.046 M (thiorcdoxin I) and 0.057 M (thioredoxin II).
The active fractions were pooled and lyophilized as described above. Before gel electrophoresis both thioredoxins were equilibrated on short Sephadex columns with the "upper gel buffer" (5).
Preparative Polyacrylamide Gel Electrophoresis-Thioredoxin I (16 mg) or thioredoxin II (12 mg) from the previous steps were introduced in about 10 ml of the upper gel buffer containing 12% sucrose and layered carefully on top of the gel. The final separation was done at 85 ma which gave a potential of about 1000 volts over the apparatus.
Seven-milliliter fractions were collected at 4-min intervals and analyzed for protein and thioredoxin activity (Fig. 3, A and B). The fractions containing thioredoxin were pooled and lyophilized with mannitol.
Finally, thioredoxin was equilibrated with 0.1 M potassium buffer, pH 7.0, on a short column of Sephadex G-25.
A summary of the complete purification procedure is given in Table I. During the purification the largest difficulties arose from the necessit,y of reducing the volumes of t#he very dilute solutions obtained after the chromatographic steps. Both thioredoxins showed a tendency to aggregate, particularly at acid pH values. This difficulty, which was more pronounced with thioredoxin I, was in part prevented by using mannitol during the lyophilizations.

Properties of Thioredoxins
Evidence is presented below that thioredoxin II was obtained as a homogeneous protein, while our best preparations of thioredoxin I still contained some minor impurities.
For this reason, a more detailed characterization is only given for thioredoxin II. Molecular Weight-Sedimentation equilibrium runs were done with both thioredoxins and a plot of In c against x2 for t,hioredoxin II is given in Fig. 4

Tryptic peptide pattern of carboxymethylated
thioredoxin II. Electrophoresis was run at 60 volts per cm for 50 min and chromatography was carried out with I-butanol-acetic acidwater-pyridine (30:6:24:20, by volume) overnight. Peptides were located with ninhydrin.
with respect to molecular weight. 011 the other hand, a similar experiment with thioredoxin I showed a quite sudden increase of the slope at the bottom of the cell, indicating contamination with heavy material.
The partial specific volume of thioredoxin II, calculated (25) from the amino acid composition (see below), was 0.736 ml per g. With this value a molecular weight of 12,600 could be calculated (24).
Amino Acid Composition and Peptide Map-The amino acid composition of tllioredoxin II is given in Table II. The cnlculations were based on a molecular weight of 12,600. The mole- cule contained 1 single residue of tryptophan and 1 or 2 residues of arginine; 2 half-cystines and 2 methionines were found.
Thioredoxin II contained a total of 11 to 13 trypsin-sensitive peptide bonds (10 to 11 lysine + one to two arginine).
A peptide map after trypsin digestion showed the presence of 12 ninhydrin-positive spots (Fig. 5). Xpectrum-Spectra of thioredoxin II at. neutral and alkaline pH are given in Fig. 6. The presence of chromophores other than tryptophan and tyrosine is not indicat'ed.
Similar spectra were found with thioredoxin I but are not shown here.

Purijcation of Thioredoxin Reductase
This enzyme was purified together with thioredoxin up to the Sephadex G-50 step. At this point (cf. Fig. 1) thioredoxin reductase was eluted close to the void volume.
For further purification, the materials from two chromatograms were pooled, precipitated with ammonium sulfate (0.612 g per ml), and centrifuged.
The precipitate was dissolved in a minimal volume of 0.02 M potassium phosphate buffer, pH 7.0, and passed through a column of Sephadex G-25 equilibrated with the same buffer.
Preparative Polyacrylam.ide Gel Electrophoresis-This step was carried out as described for thioredoxin.
The resulting purification is illustrated by Fig. 8. The active fractions were pooled and concentrated by ultrafiltration to a final volume of 2 ml. A summary of the latter part of the purification procedure is given in Table III.

Properties of Thioredoxin Reductase
Spectrum and FAD Content-The spectrum of thioredoxin reductase in 0.1 11 phosphate buffer, pH 7.0, is shown in Fig. 9 (Curve il).
It had three maxima around 276, 380, and 460 rnp and thus resembled the spectrum of t,he E. coli enzyme. When a solution of the yeast reductase was boiled for 5 min, the spectrum of the supernatant solution after centrifugation showed two maxima at 373 and 450 rnp, respectively (Fig. 9, Curve B). This spectrum was then similar to that of free FAD (Fig. 9, Curve C). Furthermore, after boiling of the enzyme, FAD was identified directly in the supernatant solut'ion by thin layer chromatography on polyethyleneimine cellulose (5,27). The amount of FAD in the enzyme could be estimated from the absorbance at 450 mp of the boiled enzyme solution and the known molar extinction coefficient (11.3 x 103) of the nucleotide. According to such a calculation 1 Aao unit of enzyme corresponded to about 7 mpmoles of FAD.
UZtracentr$ugation-In sedimentation velocity experiments thioredoxin reductase (4 mg per ml) sedimented as a single peak with an SZO,~ value of 5.0 S. On low speed sedimentation equilibrium centrifugation it was apparent, however, that the enzyme was not homogeneous, since plots of In c against x2 showed an upward curvature. An approximate molecular weight of the enzyme could be obtained from the data by using the two extreme slopes of the plot at the meniscus and t,he bottom of the cell. In this way limiting values of 64,000 and 80,000 were obtained.

Properties of Thioredoxin System
Stoichiometry-The stoichiometry of reaction (1) was established with both thioredoxin I and II by comparing the appearance of -SH groups with the disappearance of NADPH.
In a first experiment, 0.149 mg of thioredoxin I was incubated with NADPH and thioredoxin reductase under the conditions of Method 3. From the decrease in absorbance at 340 rnp, it could be calculated that 10.8 mpmoles of NADPH had been oxidized when the reaction had gone to completion.
At this point the sample was boiled for 1 min and the amount of -SH groups was determined with 5,5'-dithiobis ( In a similar experiment, 0.143 mg of thioredoxin II consumed 11.8 mpmoles of NADPH and produced 21.4 m,umoles of -SH groups.
In both experiments thus almost 2 moles of SH were produced for each mole of NADPH consumed. The slightly low figures are probably explained by reoxidation of some -SH groups during the boiling process.
From the above data we can calculate t,hat 13.700 g of thioredoxin I and 12.200 g of thioredoxin II, respectively, were required for the oxidation of 1 mole of NADPH.
pH Optimum- Fig.  10 shows that the reduction of both thioredoxin I and II had a pH optimum of about 6.5. The reaction appeared to be inhibited in Tris buffers as compared to acetate or phosphate buffers.

K, Values
for Thioredoxin-Lineweaver-Burk plots for both thioredoxins at saturating concentrations of NADPH are given in Fig. 11 been reached a measured excess of NADP was added. This resulted in the establishment of new equilibria (Fig. 12). From the known starting concentrations of the reactants, the stoichiometry of Reaction 1, and the changes in absorbance at 340 mp, the different equilibrium concentrations of the components could be determined and used for the calculation of K,, In this way, values of 2.1 x log (pH 7.85) and 4.0 x log (pH 8.8) were obtained.
From the average value of K,, = 3 x log and the known oxidation-reduction potential of the NADPH-NADP+ couple C-0.305 voIt at PI-I 7 and 200), the oxidation-reduction potential for the couple thioredoxin-(SH) 2-thioredoxin-Sz from yeast could be calculated to be -0.24 volt. This value is Pedro Gonzalez Porqué, Astor Baldesten and Peter Reichard