J. Biochem., 81, 71-78 (1977)
Tsuneyuki NAGAOKA,1 Akira HACHIMORI, Atsushi TAKEDA, and Tatsuya SAMEJIMA2 Department of Chemistry, College of Science and Engineering, Aoyama Gakuin University, Setagaya-ku, Tokyo 157 Received for publication, June 18, 1976
A new method of affinity chromatography using blue dextran-Sepharose 4B resin was established to purify NADP+-dependent isocitrate dehydrogenase [EC 1.1 1 42] from Bacillus stearothermophilus in high yield. The purified preparation was found to be homogeneous on disc gel electrophoresis. The SH groups of the enzyme were modified with 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) to determine the number of SH groups per molecule and their contribution to the enzyme activity. One SH group was titrated with DTNB per subunit (the native enzyme consisted of two subunits) and after complete denaturation with 4 M guanidineHC1 the number of titratable SH groups remained unchanged. ORD and CD measurements showed that the a-helical conformation of the polypeptide backbone was unaffected by DTNB modification, though the near ultraviolet CD spectrum was evidently altered. The fluorescence derived from tryptophanyl residue(s) was quenched by the modification to 30% of the native level, which may indicate the presence of SH in the vicinity of tryptophanyl residues). A remarkable decrease of the enzyme activity was detected upon modification with DTNB, but there was some discrepancy between the rate of inactivation and that of modification of SH groups. The presence of substrate and Mgs+ gave partial protection against modification of the SH groups by DTNB. Complete protection of the native enzyme activity against heating at 65° was observed in the presence of substrate and MgI+, but the thermostabihty of the enzyme was markedly reduced by modification of the SH groups.
Although purification of NADP+-dependent isocitrate dehydrogenase [//ireo-D.-isocitrate : NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42]
from Bacillus stearothermophilus has been already reported by Howard and Becker (J) and Hibino et al. (2), the yield in their purifications was fairly low. Using blue dextran affinity chromatography, we have established a new method of preparation of the
T~Z " ~T. _,. , , T.. _ , 1 Present address: Shinotest Labs. Ltd., Onodai, Sagamihara, Kanagawa 229. • To whom correspondence and reprint requests should be addressed. Abbreviations- DTNB, 5, 5'-dithiobis(2-nitrobenzoic acid); DTT, dithiothreitol; SDS, sodium dodecyl sulfate. Vol. 81, No. 1, 1977
enz
>' m e
wlth a Slmpler
Procedure and better yield. y b e e n reported that blue dextranSepharose 4B affinity column chromatography is useful in some cases for the purification of enzymes which have a specific binding site for nucleotide or II h a s alread
71
Downloaded from https://academic.oup.com/jb/article-abstract/81/1/71/836012 by university of winnipeg user on 18 January 2019
DTNB Modification of SH Groups of Isocitrate Dehydrogenase from Bacillus stearothermophilus Purified by Affinity Chromatography
72
T. NAGAOKA, A. HACHIMORI, A. TAKEDA, and T. SAMEJ1MA
were determined by Lowry's method using bovine serum albumin as a standard (11). The protein concentrations of the purified enzymes were estimated spectrophotometncally using an absorbance of A%= 10.9 (1,2). Modification of Enzyme with DTNB—Modification of the SH groups of the enzyme was carried out by reacting 10 /JI of lOmM DTNB solution (about a 20-fold molar excess) at room temperature with 0 6 ml of enzyme solution (0.807 mg/ml) in 80 mM phosphate buffer, pH 8 0, containing 1 mM EDTA, which had been dialyzed previously against the same buffer solution for 24 h The number of SH groups was estimated from the increase of absorbance at 410 nm using a molar extinction coefficient of 13,600 M"1-cm"1 for thionitrobenzoate ions liberated (12). Electrophoresis—Disc electrophoresis in 7.5% polyacrylamide gel with Tris-glycine buffer, pH 8.5, was carried out by the method of Ornstein (13) and Davis (14). The sample (50 //g) was charged on a gel column and a current of 5 mA was applied per gel tube. The protein in the gel was stained with 1.0% amide black in 7% acetic acid. SDS-polyacrylamide gel electrophoresis of the EXPERIMENTAL enzymes in the presence of 1 % SDS was performed Cells—Bacillus stearothermophilus (NCA No. essentially according to the method of Weber and 2184) was grown at 65° and the cells were harvested Osborn (75), using 10% gel at room temperature as described in the previous paper (2). Some of for 5 h at 8 mA per tube. The enzymes were kept the mass cultures used in the present study was overnight in 1 % SDS and 1 % 2-mercaptoethanol kindly supplied by the Central Research Laborato- containing 10 mM phosphate buffer, pH 7.2, prior to electrophoresis. ries, Ajinomoto Co. Spectrophotometry—Absorption spectra were Assay of the Enzyme—The enzymatic activity was measured at room temperature in 45 mM measured with a Hitachi EPS-3T automatic recordTris-H,SO4 buffer, pH 8.0, containing 45 /JM ing spectrophotometer at room temperature, using NADP + , 220 fXM DL-isocitrate, and 3.3 mM MgSO4 a 1.0 cm cell. ORD and CD—ORD and CD were measured in a total volume of 3.0 ml. The reaction was initiated with 10 or 30 //I of enzyme solution which with a Jasco ORD-UV-5 recording spectropolarhad been appropriately diluted. The initial ve- imeter and a Jasco J-40 automatic recording locity was measured by following the rate of in- dichrograph at room temperature, respectively. crease in absorbance at 340 run over the first 3 min Cell lengths of 5, 2, 1, and 0.1 mm were used, of the reaction. One unit of specific activity is depinding on protein concentration. The absorbdefined as 0.01 absorbance increase at 340 nm per ance of the enzyme solution at each wavelength was mg of protein per min which corresponds to the always kept below 2. The ORD and CD data were expressed in terms of reduced mean residue rotation reduction of 4.8 x 10~3 /imole of NADP+. Protein Concentrations—The protein concen- [m'\ and mean residue ellipticity, [0], respectively; trations of crude extract were determined by the the mean residue molecular weight of the enzyme micro-biuret method (JO). For the partially was taken as 109 (2). purified enzyme, enzyme modified with DTNB and Fluorometry—Fluorescence spectra were measenzyme reduced with DTT, protein concentrations ured with a Hitachi MPF-4 spectrofluorometer at /. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/81/1/71/836012 by university of winnipeg user on 18 January 2019
derivatives such as ATP or NAD + (3-7). Isocitrate dehydrogenase [EC 1.1.1.42] from Azotobacter vinelandii (8) or pig heart (9) was reported to contain SH groups essential for the enzymatic activity. In addition, we reported in the previous paper (2) that p-chloromercuribenzoate completely inhibits the enzyme activity at low concentration, in agreement with the results of Howard and Becker (/). These observations are indicative of the presence of essential SH group(s) for the enzyme activity. No data are available on the number of SH groups or disulfide bonds in the thermophilic enzyme. Therefore, we have attempted to establish the number of SH groups in the molecule by modification with 5,5'-dithiobis(2-nitrobenzoic acid) and to determine the effect of the modification on the enzyme activity. The present paper describes the purification of the enzyme by blue dextran affinity chromatography, and the modification of SH groups with DTNB, and together with the changes in enzyme activity, absorption spectra, CD spectra, fluorescence, and thermostabihty accompanying the modification.
73
MODIFICATION OF THERMOPHILIC ISOCITRATE DEHYDROGENASE
Purification of the Enzyme by Blue DextranSepharose 4B Affinity Chromatography—Crude Extract: Bacterial cells (150 g wet weight) were suspended in 50 niM phosphate buffer, pH 7.5, containing 1 ITIM EDTA, and disrupted in a sonicator (20 kc, 3 min) at 0-4° after adding a small amount of DNase. Cell debris was removed by centnfugation at 14,000 x g for 35 min and crude extract of the enzyme was obtained by further centnfugation at 20,000 x g for 30 min. Ammonium Sulfate Fractionation—The crude extract thus obtained was 50% saturated with ammonium sulfate by adding the solid salt with stirring at 4°. After standing for 15 nun, the precipitate was removed by centnfugation at 10,000 x g for 10 min. The supernatant fraction was then 70% saturated by adding solid ammonium sulfate. After standing for 15 min, the precipitate was collected by centrifugation at 10,000 x g for 10 min and dissolved with 50 mM phosphate buffer, pH 7.0, followed by dialysis against 25 mM phosphate buffer, pH 7.0, at 4° overnight. DE-52 Column Chromatography—The above enzyme solution was applied to a DEAE-cellulose (DE-52) column (3x27 cm) equilibrated with 25
mM phosphate buffer, pH 7.0, and washed with the same buffer, followed by 50 mM phosphate buffer, pH 7.0. The enzyme fraction was eluted with 100 mM phosphate buffer, pH 7.0. The active fractions with specific activity above 50 were combined and dialyzed overnight against 90 mM phosphate buffer, pH 7.0, at 4°. DEAE-Sephadex A-50 Column Chromatography —The dialyzed enzyme sample was applied to a DEAE-Sephadex A-50 column (1.9x40 cm) previously equilibrated with 90 mM phosphate buffer, pH 7.0, and washed with the same buffer. The enzyme was eluted with a linear concentration gradient from 90 to 250 mM of the same buffer. The active effluents with specific activity above 100 were pooled and concentrated by ultrafiltration, followed by dialysis overnight against 10 mM TrisHC1 buffer, pH 8 0, containing 10 ITIM MgCl, at 4°. Blue Dextran Affinity Chromatography—The concentrated and dialyzed enzyme solution was charged onto a blue dextran-Sepharose 4B column (1.5x36 cm) previously equilibrated with 10 mM Tris-HCl buffer, pH 8.0, containing 10 mM MgCl,, and washed with the same buffer. Elution was performed with a linear concentration gradient system of 10 mM MgClj-25 mM EDTA in 10 mM Tris-HCl buffer, pH 8.0, i.e., buffer solution containing 10 mM MgCU was mixed with an equal volume of the same buffer containing 25 ITIM EDTA in a gradienter. All procedures were carried out at room temperature except for centrifugations (0-4°). The fractions containing the enzyme were pooled and concentrated by ultrafiltration for storage at —25°. The results of a typical purification are summarized in Table I. The enzyme thus purified was found to be homogeneous on disc gel electrophoresis; the specific activity was approximately
TABLE I. Summary of the purification procedure. Fraction Crude extract Ammonium Sulfate DEAE-Cellulose (DE-52) DEAE-Sephadex Blue dextran affinity chromatography
Vol. 81, No. 1, 1977
Volume (ml)
Total protein (mg)
Total units
610 260 405 205
22,300 6,910 910 210
213,200 145,600 121,500 81,500
20
11.6
45,800
Specific activity
9.5 21 125 385 3,950
Yield (100) 68.3 59.9 38.3 21.5
Downloaded from https://academic.oup.com/jb/article-abstract/81/1/71/836012 by university of winnipeg user on 18 January 2019
room temperature. Chemicals —DEAE- cellulose (DE-52) was purchased from Whatman Ltd. and blue dextran, Sepharose 4B and DEAE-Sephadex A-50 from Pharmacia Fine Chemicals. Blue dextran-Sepharose 4B resin was prepared according to the method of Ryan and Vestling (5). DNase [EC 3.1.4.5], DL-isocitrate acid (tnsodium salt), and NADP + were purchased from Sigma Chemical Co. Dithiothreitol and 5,5'-dithiobis(2-nitrobenzoic acid) were obtained from Seikagaku Kogyo Co. and Wako Pure Chemical Industries, respectively.
T. NAGAOKA, A. HACHIMORI, A. TAKEDA, and T. SAMEJIMA
74
420 times that of the crude extract. The yield was much higher than in the previously reported methods (/, 2).
Subunit Molecular Weight—SDS-gel electrophoresis gave a molecular weight in 1 % SDS solution of 45,000, as shown in Fig. 1; only a single band was detected. This indicates that the native enzyme, with a molecular weight of 92,500 (/), is composed of two suburuts, which agrees with the result obtained in 6 M guanidine-HCl solution by Howard and Becker (7). Modification of SH Groups and Inactivation of the Enzyme by DTNB—DTNB titrated only one SH group per subunit within 2.5 nun and no further modification was detected, as shown in Fig. 2. The rate of inactivation of the enzyme was rather slow, in contrast with the rapid modification of the SH group. About 60% activity was observed after 2.5 min and an asymptotic decrease of activity occurred up to about 20 min, after which the residual activity become constant (about 6%). It should be pointed out that there is a clear discrepancy between the rate of inactivation of the enzyme and that of modification of SH. On the other hand, the total number of SH groups was also estimated to be one per subunit after complete
Tsuneyuki NAGAOKA,1 Akira HACHIMORI, Atsushi TAKEDA, and Tatsuya SAMEJIMA2 Department of Chemistry, College of Science and Engineering, Aoyama Gakuin University, Setagaya-ku, Tokyo 157 Received for publication, June 18, 1976
A new method of affinity chromatography using blue dextran-Sepharose 4B resin was established to purify NADP+-dependent isocitrate dehydrogenase [EC 1.1 1 42] from Bacillus stearothermophilus in high yield. The purified preparation was found to be homogeneous on disc gel electrophoresis. The SH groups of the enzyme were modified with 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) to determine the number of SH groups per molecule and their contribution to the enzyme activity. One SH group was titrated with DTNB per subunit (the native enzyme consisted of two subunits) and after complete denaturation with 4 M guanidineHC1 the number of titratable SH groups remained unchanged. ORD and CD measurements showed that the a-helical conformation of the polypeptide backbone was unaffected by DTNB modification, though the near ultraviolet CD spectrum was evidently altered. The fluorescence derived from tryptophanyl residue(s) was quenched by the modification to 30% of the native level, which may indicate the presence of SH in the vicinity of tryptophanyl residues). A remarkable decrease of the enzyme activity was detected upon modification with DTNB, but there was some discrepancy between the rate of inactivation and that of modification of SH groups. The presence of substrate and Mgs+ gave partial protection against modification of the SH groups by DTNB. Complete protection of the native enzyme activity against heating at 65° was observed in the presence of substrate and MgI+, but the thermostabihty of the enzyme was markedly reduced by modification of the SH groups.
Although purification of NADP+-dependent isocitrate dehydrogenase [//ireo-D.-isocitrate : NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42]
from Bacillus stearothermophilus has been already reported by Howard and Becker (J) and Hibino et al. (2), the yield in their purifications was fairly low. Using blue dextran affinity chromatography, we have established a new method of preparation of the
T~Z " ~T. _,. , , T.. _ , 1 Present address: Shinotest Labs. Ltd., Onodai, Sagamihara, Kanagawa 229. • To whom correspondence and reprint requests should be addressed. Abbreviations- DTNB, 5, 5'-dithiobis(2-nitrobenzoic acid); DTT, dithiothreitol; SDS, sodium dodecyl sulfate. Vol. 81, No. 1, 1977
enz
>' m e
wlth a Slmpler
Procedure and better yield. y b e e n reported that blue dextranSepharose 4B affinity column chromatography is useful in some cases for the purification of enzymes which have a specific binding site for nucleotide or II h a s alread
71
Downloaded from https://academic.oup.com/jb/article-abstract/81/1/71/836012 by university of winnipeg user on 18 January 2019
DTNB Modification of SH Groups of Isocitrate Dehydrogenase from Bacillus stearothermophilus Purified by Affinity Chromatography
72
T. NAGAOKA, A. HACHIMORI, A. TAKEDA, and T. SAMEJ1MA
were determined by Lowry's method using bovine serum albumin as a standard (11). The protein concentrations of the purified enzymes were estimated spectrophotometncally using an absorbance of A%= 10.9 (1,2). Modification of Enzyme with DTNB—Modification of the SH groups of the enzyme was carried out by reacting 10 /JI of lOmM DTNB solution (about a 20-fold molar excess) at room temperature with 0 6 ml of enzyme solution (0.807 mg/ml) in 80 mM phosphate buffer, pH 8 0, containing 1 mM EDTA, which had been dialyzed previously against the same buffer solution for 24 h The number of SH groups was estimated from the increase of absorbance at 410 nm using a molar extinction coefficient of 13,600 M"1-cm"1 for thionitrobenzoate ions liberated (12). Electrophoresis—Disc electrophoresis in 7.5% polyacrylamide gel with Tris-glycine buffer, pH 8.5, was carried out by the method of Ornstein (13) and Davis (14). The sample (50 //g) was charged on a gel column and a current of 5 mA was applied per gel tube. The protein in the gel was stained with 1.0% amide black in 7% acetic acid. SDS-polyacrylamide gel electrophoresis of the EXPERIMENTAL enzymes in the presence of 1 % SDS was performed Cells—Bacillus stearothermophilus (NCA No. essentially according to the method of Weber and 2184) was grown at 65° and the cells were harvested Osborn (75), using 10% gel at room temperature as described in the previous paper (2). Some of for 5 h at 8 mA per tube. The enzymes were kept the mass cultures used in the present study was overnight in 1 % SDS and 1 % 2-mercaptoethanol kindly supplied by the Central Research Laborato- containing 10 mM phosphate buffer, pH 7.2, prior to electrophoresis. ries, Ajinomoto Co. Spectrophotometry—Absorption spectra were Assay of the Enzyme—The enzymatic activity was measured at room temperature in 45 mM measured with a Hitachi EPS-3T automatic recordTris-H,SO4 buffer, pH 8.0, containing 45 /JM ing spectrophotometer at room temperature, using NADP + , 220 fXM DL-isocitrate, and 3.3 mM MgSO4 a 1.0 cm cell. ORD and CD—ORD and CD were measured in a total volume of 3.0 ml. The reaction was initiated with 10 or 30 //I of enzyme solution which with a Jasco ORD-UV-5 recording spectropolarhad been appropriately diluted. The initial ve- imeter and a Jasco J-40 automatic recording locity was measured by following the rate of in- dichrograph at room temperature, respectively. crease in absorbance at 340 run over the first 3 min Cell lengths of 5, 2, 1, and 0.1 mm were used, of the reaction. One unit of specific activity is depinding on protein concentration. The absorbdefined as 0.01 absorbance increase at 340 nm per ance of the enzyme solution at each wavelength was mg of protein per min which corresponds to the always kept below 2. The ORD and CD data were expressed in terms of reduced mean residue rotation reduction of 4.8 x 10~3 /imole of NADP+. Protein Concentrations—The protein concen- [m'\ and mean residue ellipticity, [0], respectively; trations of crude extract were determined by the the mean residue molecular weight of the enzyme micro-biuret method (JO). For the partially was taken as 109 (2). purified enzyme, enzyme modified with DTNB and Fluorometry—Fluorescence spectra were measenzyme reduced with DTT, protein concentrations ured with a Hitachi MPF-4 spectrofluorometer at /. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/81/1/71/836012 by university of winnipeg user on 18 January 2019
derivatives such as ATP or NAD + (3-7). Isocitrate dehydrogenase [EC 1.1.1.42] from Azotobacter vinelandii (8) or pig heart (9) was reported to contain SH groups essential for the enzymatic activity. In addition, we reported in the previous paper (2) that p-chloromercuribenzoate completely inhibits the enzyme activity at low concentration, in agreement with the results of Howard and Becker (/). These observations are indicative of the presence of essential SH group(s) for the enzyme activity. No data are available on the number of SH groups or disulfide bonds in the thermophilic enzyme. Therefore, we have attempted to establish the number of SH groups in the molecule by modification with 5,5'-dithiobis(2-nitrobenzoic acid) and to determine the effect of the modification on the enzyme activity. The present paper describes the purification of the enzyme by blue dextran affinity chromatography, and the modification of SH groups with DTNB, and together with the changes in enzyme activity, absorption spectra, CD spectra, fluorescence, and thermostabihty accompanying the modification.
73
MODIFICATION OF THERMOPHILIC ISOCITRATE DEHYDROGENASE
Purification of the Enzyme by Blue DextranSepharose 4B Affinity Chromatography—Crude Extract: Bacterial cells (150 g wet weight) were suspended in 50 niM phosphate buffer, pH 7.5, containing 1 ITIM EDTA, and disrupted in a sonicator (20 kc, 3 min) at 0-4° after adding a small amount of DNase. Cell debris was removed by centnfugation at 14,000 x g for 35 min and crude extract of the enzyme was obtained by further centnfugation at 20,000 x g for 30 min. Ammonium Sulfate Fractionation—The crude extract thus obtained was 50% saturated with ammonium sulfate by adding the solid salt with stirring at 4°. After standing for 15 nun, the precipitate was removed by centnfugation at 10,000 x g for 10 min. The supernatant fraction was then 70% saturated by adding solid ammonium sulfate. After standing for 15 min, the precipitate was collected by centrifugation at 10,000 x g for 10 min and dissolved with 50 mM phosphate buffer, pH 7.0, followed by dialysis against 25 mM phosphate buffer, pH 7.0, at 4° overnight. DE-52 Column Chromatography—The above enzyme solution was applied to a DEAE-cellulose (DE-52) column (3x27 cm) equilibrated with 25
mM phosphate buffer, pH 7.0, and washed with the same buffer, followed by 50 mM phosphate buffer, pH 7.0. The enzyme fraction was eluted with 100 mM phosphate buffer, pH 7.0. The active fractions with specific activity above 50 were combined and dialyzed overnight against 90 mM phosphate buffer, pH 7.0, at 4°. DEAE-Sephadex A-50 Column Chromatography —The dialyzed enzyme sample was applied to a DEAE-Sephadex A-50 column (1.9x40 cm) previously equilibrated with 90 mM phosphate buffer, pH 7.0, and washed with the same buffer. The enzyme was eluted with a linear concentration gradient from 90 to 250 mM of the same buffer. The active effluents with specific activity above 100 were pooled and concentrated by ultrafiltration, followed by dialysis overnight against 10 mM TrisHC1 buffer, pH 8 0, containing 10 ITIM MgCl, at 4°. Blue Dextran Affinity Chromatography—The concentrated and dialyzed enzyme solution was charged onto a blue dextran-Sepharose 4B column (1.5x36 cm) previously equilibrated with 10 mM Tris-HCl buffer, pH 8.0, containing 10 mM MgCl,, and washed with the same buffer. Elution was performed with a linear concentration gradient system of 10 mM MgClj-25 mM EDTA in 10 mM Tris-HCl buffer, pH 8.0, i.e., buffer solution containing 10 mM MgCU was mixed with an equal volume of the same buffer containing 25 ITIM EDTA in a gradienter. All procedures were carried out at room temperature except for centrifugations (0-4°). The fractions containing the enzyme were pooled and concentrated by ultrafiltration for storage at —25°. The results of a typical purification are summarized in Table I. The enzyme thus purified was found to be homogeneous on disc gel electrophoresis; the specific activity was approximately
TABLE I. Summary of the purification procedure. Fraction Crude extract Ammonium Sulfate DEAE-Cellulose (DE-52) DEAE-Sephadex Blue dextran affinity chromatography
Vol. 81, No. 1, 1977
Volume (ml)
Total protein (mg)
Total units
610 260 405 205
22,300 6,910 910 210
213,200 145,600 121,500 81,500
20
11.6
45,800
Specific activity
9.5 21 125 385 3,950
Yield (100) 68.3 59.9 38.3 21.5
Downloaded from https://academic.oup.com/jb/article-abstract/81/1/71/836012 by university of winnipeg user on 18 January 2019
room temperature. Chemicals —DEAE- cellulose (DE-52) was purchased from Whatman Ltd. and blue dextran, Sepharose 4B and DEAE-Sephadex A-50 from Pharmacia Fine Chemicals. Blue dextran-Sepharose 4B resin was prepared according to the method of Ryan and Vestling (5). DNase [EC 3.1.4.5], DL-isocitrate acid (tnsodium salt), and NADP + were purchased from Sigma Chemical Co. Dithiothreitol and 5,5'-dithiobis(2-nitrobenzoic acid) were obtained from Seikagaku Kogyo Co. and Wako Pure Chemical Industries, respectively.
T. NAGAOKA, A. HACHIMORI, A. TAKEDA, and T. SAMEJIMA
74
420 times that of the crude extract. The yield was much higher than in the previously reported methods (/, 2).
Subunit Molecular Weight—SDS-gel electrophoresis gave a molecular weight in 1 % SDS solution of 45,000, as shown in Fig. 1; only a single band was detected. This indicates that the native enzyme, with a molecular weight of 92,500 (/), is composed of two suburuts, which agrees with the result obtained in 6 M guanidine-HCl solution by Howard and Becker (7). Modification of SH Groups and Inactivation of the Enzyme by DTNB—DTNB titrated only one SH group per subunit within 2.5 nun and no further modification was detected, as shown in Fig. 2. The rate of inactivation of the enzyme was rather slow, in contrast with the rapid modification of the SH group. About 60% activity was observed after 2.5 min and an asymptotic decrease of activity occurred up to about 20 min, after which the residual activity become constant (about 6%). It should be pointed out that there is a clear discrepancy between the rate of inactivation of the enzyme and that of modification of SH. On the other hand, the total number of SH groups was also estimated to be one per subunit after complete
