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[43] Rhodanese
334
ENZYMES OF PROTEIN METABOLISM
[43]
[43] Rhodanese C N - + $203-- -o CNS- + SO3--
By B. H. SSR~O
Assay Method Principle. All published methods are based on the colorimetric determination of the thiocyanate formed in the reaction. The red color obtained in the presence of ferric ions is conveniently used for this purpose. The appearance of an interfering blue color (due to an iron-thiosulfate complex) is prevented in the present method 1 by the presence of formaldehyde. The method given here is applicable to rhodanese preparations of any degree of purity. Reagents 0.125 M Na2S203. 0.5% bovine serum albumin (stock solution). 0.20 M KH2P04. O.25 M KCN. 38% formaldehyde. Ferric nitrate reagent. 100 g. of Fe(NO3)~-9H20 + 200 ml. of 65% HN03 per 1000 ml. Enzyme. Dilute the enzyme in the presence of 0.0125 M thiosulfate and 0.025 % albumin (which protect the enzyme against inactivation by dilution) to obtain 0.3 to 1.6 R.U./ml. (See definition below.)
Procedure. One milliliter of Na~S~03, 0.5 ml. of KH2PO, and 0.5 ml. of KCN are mixed in a 50-ml. Erlenmeyer flask. One-half milliliter of the enzyme is added, and the reaction is stopped after 5 minutes at 20 ° by the addition of 0.5 ml. of 38% formaldehyde. Then 2.5 ml. of ferric nitrate reagent and 25 ml. of distilled water are added, and the optical density at 460 m~ is determined. One microequivalent of thiocyanate in the test gives the optical density, 0.104. The color is stable for at least 1 hour. A blank determination is always carried out by adding the formaldehyde to the test before the enzyme. With crude extracts or tissue homogenates it is necessary to remove the interfering turbidity by centrifugation or filtration before the optical density is determined. Definition of Unit and Specific Activity. One rhodanese unit (R.U.) is defined as that amount of enzyme which forms 10 microequivalents of 1B. H. SSrbo, Acta Chem. Scand. 7, 1129 (1953).
[43]
RHODANESE
335
thiocyanate under the above conditions. Specific activity is expressed as rhodanese units per milligram dry weight. With purified enzyme preparations protein is determined according to Bticher 2 instead of the dry weight. Purification Procedure
Crystalline rhodanese has repeatedly been obtained in this laboratory by the following procedure.1 Step 1. Extraction. Beef liver (fresh or frozen material can be used) is homogenized in a blendor with 2.5 1. of tap water per kilogram of liver, and 100 ml. of basic lead acetate solution (20%) is then added. The obtained precipitate is centrifuged off and discarded. Step 2. Fractionation with Ammonium Sulfate at pH 8.8. The following step is carried out in the cold room (+4°). Ammonium sulfate is added to 0.40 M concentration, and the pH is adjusted to 3.8 with 1 M HC1. The inactive precipitate is removed (by centrifugation or by suction filtration with the aid of Hyflo Super-Cel) and discarded. The ammonium sulfate concentration in the filtrate is raised to 1.33 M, and the obtained precipitate is removed after 3 hours and discarded. The enzyme is precipitated by raising the ammonium sulfate concentration to 1.91 M and is removed as before after 3 hours. The precipitate is suspended in a solution 0.05 M with respect to thiosulfate and Na2HPO4, with 60 ml./1. of fraction 1. Step 8. Fractionation with Ammoniacal Ammonium Sulfate. To fraction 2 is added 1.3 vol. of 3.25 M ammonium sulfate, adjusted to pH 7.9 with ammonia. After 3 hours at room temperature the precipitate is centrifuged off and discarded, and the enzyme is then precipitated from the supernatant by the addition of 100 g. of ammonium sulfate per liter. The precipitate is left in the cold room overnight and is then removed by centrifugation and dissolved in 0.01 M NaAc (1/~ vol. of fraction 2). Step 3. Dialysis. The remaining ammonium sulfate in fraction 3 is removed by dialysis against 0.01 M NaAc in the cold room. Step 5. Acetone Fractionation. The following step is carried out at - 5 °. Fraction 4 is adjusted with 0.1 M HAc to pH 4.9, and acetone is then gradually added to 35% by volume. The precipitate is removed by centrifugation, and the enzyme is precipitated from the supernatant by raising the acetone concentration to 50 %. The active precipitate is centrifuged off and dissolved in 0.01 M NaAc (1/~ vol. of fraction 4). If the first acetone precipitate contains more than 20% of the activity in fraction 4, it should be refractionated with acetone. 2 T. Biicher, Biochim. et Biophys. Acta 1, 292 (1947).
336
ENZYMES OF PROTEIN METABOLISM
[43]
Step 6. Dialysis. The remaining acetone in fraction 5 is removed by dialysis in the cold room against 0.01 M NaAc. Step 7. Ammonium Sulfate Fractionation at pH 4.5. The pH of fraction 6 is adjusted to 4.5 with 0.1 M HAc, and the enzyme is precipitated by the addition of an equal volume of 3.78 M ammonium sulfate. After 3 hours at room temperature the precipitate is centrifuged off and dissolved in 1 M ammonium sulfate of pH 7.8 (0.1 vol. of fraction 6). Step 8. Crystallization. The enzyme in fraction 7 is ready for crystallization if it has a specific activity of 200 R.U./mg. or better. Fraction 7 is adjusted to pH 7.8, and the enzyme is brought to crystallization by gradually increasing the concentration of ammonium sulfate. The best way to crystallize the enzyme is, however, to precipitate it with ammonium sulfate at pH 7.8 and then dissolve the amorphous precipitate in a small volume of 1 M ammonium sulfate, pH 7.8, at which point the enzyme immediately crystallizes. It can be recrystallized in the same way. The enzyme crystallizes in the form of rectangular plates or elongated prisms.
Properties Physicochemical Properties. When crystalline rhodanese was investigated in the Spinco ultracentrifuge, only one homogenous peak was observed. The molecular weight of 37,000 was obtained from the following data: sedimentation constant S, 3.0; diffusion constant, 7.5 × 10-7; and partial specific volume, 0.74. The enzyme showed a single peak and migrated anodically on electrophoresis in phosphate buffer at pH 7.4. At pH 6.5 and 5.5 partial denaturation occurred and the enzyme was no longer homogenous. The absorption spectrum of the crystalline enzyme showed only the usual protein band at 280 m~, with an extinction coefficient of 1.75/mg./ml./cm. at this wavelength. Specificity. The purified enzyme can use thiosulfate and different thiosulfonates 3 as sulfur donors but not colloidal sulfur 4 or other sulfur-containing compounds. Effect of Substrate Concentration, pH and Temperature. The optimal concentration of both thiosulfate and cyanide in the enzymatic reaction is 0.05 M in the case of the crystalline beef liver enzyme. 8 The optimal pH is 8.6, and the optimal temperature 50°. The apparent heat of activation is 7900 cal. These results differ somewhat from those reported by Saunders and Himwich 5 for rhodanese from other sources. 3 B. H. SSrbo, Acta Chem. Scand. 7, 1137 (1953). 4 B. H. SSrbo, Acta Chem. Scand. 7~ 32 (1953). 5 j . p. Saunders a n d W. A. Himwich, Am. J. Physiol. 163, 404 (1950).
[44]
GLUTAMINE
SYNTHESIS
337
Inhibitors. As rhodanese is strongly inhibited by sulfite and by incubation with cyanide in the absence of thiosulfate, it was suggested that the active group in the enzyme is a disulfide bond. 6 The enzyme is not inhibited by thiosemicarbazide, which makes the assumption of an active carbonyl group unlikely. Heavy metal enzyme inhibitors (except cyanide) do not inhibit the enzyme. 7 The enzyme is inhibited by some sulfhydryl reagents 5,7 but only incompletely and at rather high concerttrations of the inhibitors (0.001 M). SUMMARY OF PURIFICATION t)ROCEDURE a
Fraction 1. 2. 3. 4. 5. 6. 7. 8. 9.
Extract (NH4)2S04, p H 3.8 (NH4)2SO4, alc. Dialyzed Acetone Dialyzed (NH4)~SO4, pH 4.5 Crystals Recrystallized
Total volume, ml.
Total units, thousands
Specific activity, R.U./mg.
Yield, %
16,000 875 218 245 110 122 17.3 6.2 6.5
396 177 130 130 86.7 82.7 58.1 45.8 33.5
1.07 16.8 34.2 34.2 82.7 -201 257 267
100 45 33 33 22 21 14.7 11.5 8.5
B. H. SOrbo, Acta Chem. Scand. 7, 1129 (1953). e B. H. SSrbo, Acta Chem. Scan& 5, 1218 (1951). 7 B. H. SSrbo, Acta Chem. Scand. 5, 724 (1951).
[44] G l u t a m i n e S y n t h e s i s Glutamic acid W ATP + Ammonia--* Glutamine -t- ADP ~- Phosphate
By W. H. ELLIOTT The enzyme catalyzing the above reaction is called glutamine synthetase.
Assay Method Principle. The method devised by Speck I and Elliott 2 is based on the fact that the enzyme is capable of forming ~,-glutamyl hydroxamic acid ' J. F. Speck, J. Biol. Chem. 179, 1405 (1949). 2 W. H. Elliott, Biochem. J. 49, 106 (1951).
ENZYMES OF PROTEIN METABOLISM
[43]
[43] Rhodanese C N - + $203-- -o CNS- + SO3--
By B. H. SSR~O
Assay Method Principle. All published methods are based on the colorimetric determination of the thiocyanate formed in the reaction. The red color obtained in the presence of ferric ions is conveniently used for this purpose. The appearance of an interfering blue color (due to an iron-thiosulfate complex) is prevented in the present method 1 by the presence of formaldehyde. The method given here is applicable to rhodanese preparations of any degree of purity. Reagents 0.125 M Na2S203. 0.5% bovine serum albumin (stock solution). 0.20 M KH2P04. O.25 M KCN. 38% formaldehyde. Ferric nitrate reagent. 100 g. of Fe(NO3)~-9H20 + 200 ml. of 65% HN03 per 1000 ml. Enzyme. Dilute the enzyme in the presence of 0.0125 M thiosulfate and 0.025 % albumin (which protect the enzyme against inactivation by dilution) to obtain 0.3 to 1.6 R.U./ml. (See definition below.)
Procedure. One milliliter of Na~S~03, 0.5 ml. of KH2PO, and 0.5 ml. of KCN are mixed in a 50-ml. Erlenmeyer flask. One-half milliliter of the enzyme is added, and the reaction is stopped after 5 minutes at 20 ° by the addition of 0.5 ml. of 38% formaldehyde. Then 2.5 ml. of ferric nitrate reagent and 25 ml. of distilled water are added, and the optical density at 460 m~ is determined. One microequivalent of thiocyanate in the test gives the optical density, 0.104. The color is stable for at least 1 hour. A blank determination is always carried out by adding the formaldehyde to the test before the enzyme. With crude extracts or tissue homogenates it is necessary to remove the interfering turbidity by centrifugation or filtration before the optical density is determined. Definition of Unit and Specific Activity. One rhodanese unit (R.U.) is defined as that amount of enzyme which forms 10 microequivalents of 1B. H. SSrbo, Acta Chem. Scand. 7, 1129 (1953).
[43]
RHODANESE
335
thiocyanate under the above conditions. Specific activity is expressed as rhodanese units per milligram dry weight. With purified enzyme preparations protein is determined according to Bticher 2 instead of the dry weight. Purification Procedure
Crystalline rhodanese has repeatedly been obtained in this laboratory by the following procedure.1 Step 1. Extraction. Beef liver (fresh or frozen material can be used) is homogenized in a blendor with 2.5 1. of tap water per kilogram of liver, and 100 ml. of basic lead acetate solution (20%) is then added. The obtained precipitate is centrifuged off and discarded. Step 2. Fractionation with Ammonium Sulfate at pH 8.8. The following step is carried out in the cold room (+4°). Ammonium sulfate is added to 0.40 M concentration, and the pH is adjusted to 3.8 with 1 M HC1. The inactive precipitate is removed (by centrifugation or by suction filtration with the aid of Hyflo Super-Cel) and discarded. The ammonium sulfate concentration in the filtrate is raised to 1.33 M, and the obtained precipitate is removed after 3 hours and discarded. The enzyme is precipitated by raising the ammonium sulfate concentration to 1.91 M and is removed as before after 3 hours. The precipitate is suspended in a solution 0.05 M with respect to thiosulfate and Na2HPO4, with 60 ml./1. of fraction 1. Step 8. Fractionation with Ammoniacal Ammonium Sulfate. To fraction 2 is added 1.3 vol. of 3.25 M ammonium sulfate, adjusted to pH 7.9 with ammonia. After 3 hours at room temperature the precipitate is centrifuged off and discarded, and the enzyme is then precipitated from the supernatant by the addition of 100 g. of ammonium sulfate per liter. The precipitate is left in the cold room overnight and is then removed by centrifugation and dissolved in 0.01 M NaAc (1/~ vol. of fraction 2). Step 3. Dialysis. The remaining ammonium sulfate in fraction 3 is removed by dialysis against 0.01 M NaAc in the cold room. Step 5. Acetone Fractionation. The following step is carried out at - 5 °. Fraction 4 is adjusted with 0.1 M HAc to pH 4.9, and acetone is then gradually added to 35% by volume. The precipitate is removed by centrifugation, and the enzyme is precipitated from the supernatant by raising the acetone concentration to 50 %. The active precipitate is centrifuged off and dissolved in 0.01 M NaAc (1/~ vol. of fraction 4). If the first acetone precipitate contains more than 20% of the activity in fraction 4, it should be refractionated with acetone. 2 T. Biicher, Biochim. et Biophys. Acta 1, 292 (1947).
336
ENZYMES OF PROTEIN METABOLISM
[43]
Step 6. Dialysis. The remaining acetone in fraction 5 is removed by dialysis in the cold room against 0.01 M NaAc. Step 7. Ammonium Sulfate Fractionation at pH 4.5. The pH of fraction 6 is adjusted to 4.5 with 0.1 M HAc, and the enzyme is precipitated by the addition of an equal volume of 3.78 M ammonium sulfate. After 3 hours at room temperature the precipitate is centrifuged off and dissolved in 1 M ammonium sulfate of pH 7.8 (0.1 vol. of fraction 6). Step 8. Crystallization. The enzyme in fraction 7 is ready for crystallization if it has a specific activity of 200 R.U./mg. or better. Fraction 7 is adjusted to pH 7.8, and the enzyme is brought to crystallization by gradually increasing the concentration of ammonium sulfate. The best way to crystallize the enzyme is, however, to precipitate it with ammonium sulfate at pH 7.8 and then dissolve the amorphous precipitate in a small volume of 1 M ammonium sulfate, pH 7.8, at which point the enzyme immediately crystallizes. It can be recrystallized in the same way. The enzyme crystallizes in the form of rectangular plates or elongated prisms.
Properties Physicochemical Properties. When crystalline rhodanese was investigated in the Spinco ultracentrifuge, only one homogenous peak was observed. The molecular weight of 37,000 was obtained from the following data: sedimentation constant S, 3.0; diffusion constant, 7.5 × 10-7; and partial specific volume, 0.74. The enzyme showed a single peak and migrated anodically on electrophoresis in phosphate buffer at pH 7.4. At pH 6.5 and 5.5 partial denaturation occurred and the enzyme was no longer homogenous. The absorption spectrum of the crystalline enzyme showed only the usual protein band at 280 m~, with an extinction coefficient of 1.75/mg./ml./cm. at this wavelength. Specificity. The purified enzyme can use thiosulfate and different thiosulfonates 3 as sulfur donors but not colloidal sulfur 4 or other sulfur-containing compounds. Effect of Substrate Concentration, pH and Temperature. The optimal concentration of both thiosulfate and cyanide in the enzymatic reaction is 0.05 M in the case of the crystalline beef liver enzyme. 8 The optimal pH is 8.6, and the optimal temperature 50°. The apparent heat of activation is 7900 cal. These results differ somewhat from those reported by Saunders and Himwich 5 for rhodanese from other sources. 3 B. H. SSrbo, Acta Chem. Scand. 7, 1137 (1953). 4 B. H. SSrbo, Acta Chem. Scand. 7~ 32 (1953). 5 j . p. Saunders a n d W. A. Himwich, Am. J. Physiol. 163, 404 (1950).
[44]
GLUTAMINE
SYNTHESIS
337
Inhibitors. As rhodanese is strongly inhibited by sulfite and by incubation with cyanide in the absence of thiosulfate, it was suggested that the active group in the enzyme is a disulfide bond. 6 The enzyme is not inhibited by thiosemicarbazide, which makes the assumption of an active carbonyl group unlikely. Heavy metal enzyme inhibitors (except cyanide) do not inhibit the enzyme. 7 The enzyme is inhibited by some sulfhydryl reagents 5,7 but only incompletely and at rather high concerttrations of the inhibitors (0.001 M). SUMMARY OF PURIFICATION t)ROCEDURE a
Fraction 1. 2. 3. 4. 5. 6. 7. 8. 9.
Extract (NH4)2S04, p H 3.8 (NH4)2SO4, alc. Dialyzed Acetone Dialyzed (NH4)~SO4, pH 4.5 Crystals Recrystallized
Total volume, ml.
Total units, thousands
Specific activity, R.U./mg.
Yield, %
16,000 875 218 245 110 122 17.3 6.2 6.5
396 177 130 130 86.7 82.7 58.1 45.8 33.5
1.07 16.8 34.2 34.2 82.7 -201 257 267
100 45 33 33 22 21 14.7 11.5 8.5
B. H. SOrbo, Acta Chem. Scand. 7, 1129 (1953). e B. H. SSrbo, Acta Chem. Scan& 5, 1218 (1951). 7 B. H. SSrbo, Acta Chem. Scand. 5, 724 (1951).
[44] G l u t a m i n e S y n t h e s i s Glutamic acid W ATP + Ammonia--* Glutamine -t- ADP ~- Phosphate
By W. H. ELLIOTT The enzyme catalyzing the above reaction is called glutamine synthetase.
Assay Method Principle. The method devised by Speck I and Elliott 2 is based on the fact that the enzyme is capable of forming ~,-glutamyl hydroxamic acid ' J. F. Speck, J. Biol. Chem. 179, 1405 (1949). 2 W. H. Elliott, Biochem. J. 49, 106 (1951).