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[68] Phenoxazinone synthase (Streptomyces antibioticus)—Syntheses of cinnabarinic acid and actinocin
[68]
PHENOXAZINONE SYNTHASE
549
dialyzed against this buffer overnight. T h e dialyzed material, in a volume of 35 ml, is placed on a 2.4 × 42 cm DEAE-cellulose column that has been equilibrated with this buffer. T h e enzyme is eluted with this buffer; no gradient is applied./3-Ketoadipate enol-lactone hydrolase is retarded on such columns for several hold-up volumes; accordingly, a large purification can be achieved. Peak fractions are pooled and dialyzed against dry commercial sucrose until the volume is less than 30 ml. T h e preparation is then dialyzed against buffer A overnight. Second Ammonium Sulfate Fractionation. T h e dialyzed material is separated into fractions which precipitate between 50 and 60%, 60 and 70%, and 70 and 75% saturated a m m o n i u m sulfate by addition of the powdered salt; fractions are dialyzed against buffer A overnight. T h e 70-75% saturated a m m o n i u m sulfate fraction, which contains 400-fold purified/3-ketoadipate enol-lactone hydrolase, is quite heterogeneous. Table V shows a typical purification.
Properties ~-Ketoadipate enol-lactone hydrolase exhibits optimal activity from pH 7.5 to pH 10; the activity is substantially lower in buffers below neutrality. T h e Michaelis constant of the enzyme is 1.2 × 10 -s M; its molecular weight has been estimated to be about 33,000.
[68] Phenoxazinone Synthase
(Streptomyces
antibioticus)-Syntheses of Cinnabarinic Acid and Actinocin 1 By EDWARD KATZ a n d HERBERT WEISSBACH RI
+1 1/202 R2
R1
Rx
~
~
0 R2
+3tt20 R2
Phenoxazinones have been found in various insect pigments la (ommochromes), in certain mold metabolites, 2 e.g., cinnabarin and cin1See article [69] for cinnabarinic acid synthase from Tecona stans and from rat liver. laA. Butenandt, Angew. Chem.69, 16 (1957). 2j. Gripenberg, Acta. Chem. Scand. 12,603 (1958).
550
AROMATIC AMINO ACIDS
[68]
nabarinic acid, and in the family of chromopeptide antibiotics, the actinomycins? Phenoxazinone synthase is very likely involved in the synthesis of actinocin (2-amino-4,6-dimethyl-3-phenoxazinone- 1,9-dicarboxylic acid, the chromophore of actinomycin. The enzyme catalyzes the oxidative condensation of 2 moles of an 0-aminophenol to form 1 mole of a phenoxazinone.
Assay Method. The enzyme assay is based on the increase in optical density in the 430-455 m/~ region due to the formation of the phenoxazinone pigment. 4 Incubations are performed in a Beckman DU spectrophotometer in which the cuvette chamber is maintained at 37 °. The reaction mixture contains 2 micromoles of substrate, 250 micromoles of sodium acetate buffer, pH 5.3, enzyme, and water to a total volume of 3 ml. Because of the availability of 3-hydroxyanthranilic acid, it is used as the substrate for routine studies, and the corresponding phenoxazinone (cinnabarinic acid) formed is measured at 452 m/~ (•452 = 18 × 103). Readings generally are taken at 1- to 2-minute intervals over a 10-minute period; a unit of activity is expressed as an optical density change of 1.0 at 452 mt~ over a period of 5 minutes. Because of an initial lag period in the rate of the reaction, the increase between 5 and 10 minutes is used. A control cuvette containing boiled enzyme is employed to correct for the small nonenzymatic oxidation of the substrate.
Growth of the Organism and Purification of the Enzyme. Streptomyces antibioticus ATCC 14888 is cultured as described previously. 4-6 The organism is inoculated into 100 ml of a medium containing 2.5% N-Z Amine A, (Sheffield Chemical Co., Norwich, New York) arid 1% Difco beef extract in tap water (pH 7.0) and incubated at 30 ° on a New Brunswick reciprocating shaking machine for 48 hours at 240-280 rpm. The mycelium is harvested by centrifugation at 9000 rpm for 10 minutes. After decanting, the mycelium is washed twice in 100 ml of sterile isotonic saline and recentrifuged. A final suspension of the mycelium is made in 50 ml of saline and 2 ml of the suspension are inoculated into 100-ml aliquots of the galactose-glutamic acid-mineral salts medium containing L-glutamic acid.HCl, 2.5 g; galactose, 10 g; K2HPO4, 1.0 g; MgSO4" 7 H20, 25 mg; FeSO4.7 H20, 25 mg; ZnSO4.7 H20, 25 mg; CAC12"2 H20, 25 mg in 1000 ml of distilled water (pH 7.!-7.2). After 2-4 days' growth in the glutamic acid-galactose-mineral salts medium, 3H. Brockmann, Ann. N. Y. Acad. Sci. 89, 323 (1960). 4E. Katz and H. Weissbach, J. Biol. Chem. 237, 882 (1962). 5E. Katz, P. Pienta, and A. Sivak, Appl. Microbiol. 6, 236 (1958). eE. Katz and W. A. Goss, Biochem.J. 73, 458 (1959).
[68]
PHENOXAZINONE SYNTHASE
551
the mycelium is harvested by centrifugation and washed twice with isotonic saline. T h e antibiotic titer of the culture medium at the time of harvesting is generally between 40 and 60 ~g of actinomycin per milliliter. T h e washed mycelium can be kept in .the cold (4°) for a period of 1 month with no appreciable loss of enzymatic activity. The mycelium (wet weight) is suspended in one to two volumes of cold deionized water and disrupted either by sonic treatment at 10 kc for 15 minutes or by passage through an Aminco French pressure cell. The resulting suspension is then centrifuged at 20,000 g for 20 minutes to remove intact cells and cellular debris. A m m o n i u m sulfate (19.7 g) is added slowly to 150 ml of sonic extract of S. antibioticus, and the precipitate obtained after centrifugation is discarded. An additional 13.2 g of ammonium sulfate are then added to the supernatant fraction, and the resulting precipitate is collected by centrifugation and dissolved in 7 ml of cold deionized water. This fraction is dialyzed for 18 hours against 0.001 M sodium phosphate buffer, pH 6.0. The dialyzed preparation (8.5 ml) is placed on a diethylaminoethyl (DEAE) cellulose column (1 x 18 cm) which had been equilibrated with 0.001 M sodium phosphate buffer, pH 6.0. The column is washed first with 200 ml of 0.1 M NaCI, and the enzyme is then eluted with 0.25 M NaCI. The major fraction of activity is present in the first 200-300 ml. The active fractions are combined and then dialyzed for 16 hours against 0.001 M sodium phosphate buffer, pH 6.0. T h e dialyzed eluate contains about 0.035 mg of protein per milliliter. Calcium phosphate gel (80 mg) is added, and the gel supernatant fluid is discarded. After the gel is washed with water, the enzymatic activity is removed by two elutions with 6 ml of 0.2 M phosphate buffer, pH 7.4. The gel eluate, approximately 25-fold purified, is then dialyzed against phosphate buffer as described above. The enzyme can be purified further by adsorbing the activity in the dialyzed calcium phosphate eluate on alumina C~ gel (8 mg of gel per milligram of protein); the activity is eluted with 0.2 M sodium phosphate buffer, pH 7.4 (Table I). TABLE I PURIFICATION OF PHENOXAZINONESYNTHASEa Fraction Sonic extract Ammonium sulfate (18-32%) DEAE-cellulose eluate CaPO4 eluate Alumina Cz, eluate
Volume (ml)
Units/ml
Protein (mg/ml)
150 8.5
18.4 256
2.45 14.3
202 11.2 19.5
3.6 51 12.5
0.035 0.27 0.057
aDetails o f the purification procedure are described in the text.
Specific activity 7.5 17.4 103 189 220
Recovery (%) 78 26 21 9
552
AROMATIC AMINO ACIDS
[68]
The sonic extract, kept frozen, loses little activity over a period of 2-4 weeks, and extracts assayed as much as 5 months later still have appreciable activity. The purified preparation loses less than 20% of its activity in 1 week. Properties oJ Reaction. With both the sonic extract and the purified enzyme preparation, a significant lag period is observed in phenoxazinone formation during the initial 2 minutes of incubation. After this lag phase, the rate of reaction is linear; enzyme activity units are based on the optical density change during the 5- to 10-minute period. An initial lag phase is observed with all the 0-aminophenols tested as substrates. The reaction proceeds best at pH 5.0-5.2. T h e reaction is inhibited by a variety of metal chelating agents, but a definite metal requirement for the enzyme from S. antibioticus has not been shown. Mn 2+ and Ca 2+ ions stimulate the rate of the reaction 30-50% with both the crude and purified enzyme. A more pronounced metal effect is observed with extracts from another actinomycin-producing organism (Streptomyces strain 3723, Institute of Microbiology Culture Collection, Rutgers University). Upon the addition of calcium an 8- to 10-fold increase in activity is obtained; Mn 2+ ions stimulate 3- to 5-fold. Substrate Specificity. All the 0-aminophenols tested are converted to compounds having the spectral characteristics of phenoxazinones. 4"7 The compounds tested and the spectral maxima of the phenoxazinones formed are listed in Table II. Anthranilic acid, m-aminophenol, and m-hydroxybenzoic acid are not converted to colored derivatives. The apparent Michaelis-Menten constant for 4-methyl-3-hydroxyanthranilic acid, 3-hydroxyanthranilic acid, and the anthraniloyl peptides is calculated to be 1 to 2 × 10 -4 M. Inhibitors of the Reaction. A number of compounds related to an 0aminophenol are inhibitors of the enzymatic reaction. 7 For example, 0-, m-, and p-anisidine, 0-, m-, and p-aminobenzoic acid, m.-aminophenol, and o-aminobenzenethiol are good to excellent inhibitors of enzyme activity. Inhibition of enzymatic activity is observed when an equimolar concentration of 3-amino-4-hydroxybiphenol (53%), 2-amino-4-tertbutylphenol (45%) or 3-amino-4-naphthol (81%) is used. Anthranilic acid (o-aminobenzoic acid) and m-aminophenol are competitive inhibitors, whereas 0- and m-anisidine are noncompetitive inhibitors of the enzymatic reaction. Use of Phenoxazinone Synthesis as an Assay for 3-Hydroxyanthranilic Acid. Although the enzymatic reaction with 3-hydroxyanthranilic acid proceeds to about 70% of completion, it is still possible to use this reaction 7E. Katz and H. Weissbach, in "Developments in Industrial Microbiology" (C. Koda, ed.), Vol. 8, p. 67. Am. Inst. Biol. Sci., 1967.
[68]
PHENOXAZINONE SYNTHASE
553
TABLE II SUBSTRATE SPECIFICITY OF PHENOXAZINONE SYNTHASE a
Substrate
Relative rate (OD/5 min)
3-Hydroxyanthranilic acid 4-Methyl-3-hydroxyanthranilic acid 3-Hydroxykynurenine 4-Methyl-3-hydroxyanthranilic acid methyl ester 0-Aminophenol 4-Methyl-3-hydroxyanthraniioyl-L-threonine 4-Methyl-3-hydroxyanthraniloyI-L-threonyl-D-valine 4-Methyl-3-hydroxyanthraniloyl-L-threonyl-D-valyl-L-proline 4-Methyl-3-hydroxyanthraniloyl-L-threonyl-D-valylL-prolyl-sarcosine 4-Methyl-3-hydroxyanthraniloyl-L-threonyl-D-valylL-prolyl-sarcosyl-N-methyl-L-valine
Maximal wave length of product (mt~)
100 105 30 110 90 110 118 111
452 458 425 -435 432 420 450 448 448
115
448
122
448
aSpectra were recorded at pH 5.2 in 0.083 M sodium acetate buffer. The 4-methyl-3hydroxy anthraniloyl peptides were obtained from Dr. Leon Goodman, Stanford Research Institute. All compounds were employed at a concentration of 6.7 x 10-4 M. as an assay f o r this substance. U n d e r conditions in which the substrate is limiting the a m o u n t o f substrate reacting is p r o p o r t i o n a l to the conc e n t r a t i o n e m p l o y e d . T h e sensitivity o f the assay can be increased by c a r r y i n g out the reaction in a 1 ml v o l u m e , c o n t a i n i n g 0.05 ml o f sonic extract, substrate ( 3 - h y d r o x y a n t h r a n i l i c acid), a n d acetate buffer, p H 5.3. U n d e r these conditions, it is possible to assay as little as 10 millim i c r o m o l e s o f 3 - h y d r o x y a n t h r a n i l i c acid as m e a s u r e d by p h e n o x a z i n o n e f o r m a t i o n . A blank c o n t a i n i n g boiled e n z y m e is e m p l o y e d , a n d r e a d i n g s are t a k e n at zero time a n d at 5 - m i n u t e intervals until the reaction stops, usually a f t e r 15 m i n u t e s with a c r u d e sonic extract o f S. antibioticus a n d low levels o f 3 - h y d r o x y a n t h r a n i l i c acid.
PHENOXAZINONE SYNTHASE
549
dialyzed against this buffer overnight. T h e dialyzed material, in a volume of 35 ml, is placed on a 2.4 × 42 cm DEAE-cellulose column that has been equilibrated with this buffer. T h e enzyme is eluted with this buffer; no gradient is applied./3-Ketoadipate enol-lactone hydrolase is retarded on such columns for several hold-up volumes; accordingly, a large purification can be achieved. Peak fractions are pooled and dialyzed against dry commercial sucrose until the volume is less than 30 ml. T h e preparation is then dialyzed against buffer A overnight. Second Ammonium Sulfate Fractionation. T h e dialyzed material is separated into fractions which precipitate between 50 and 60%, 60 and 70%, and 70 and 75% saturated a m m o n i u m sulfate by addition of the powdered salt; fractions are dialyzed against buffer A overnight. T h e 70-75% saturated a m m o n i u m sulfate fraction, which contains 400-fold purified/3-ketoadipate enol-lactone hydrolase, is quite heterogeneous. Table V shows a typical purification.
Properties ~-Ketoadipate enol-lactone hydrolase exhibits optimal activity from pH 7.5 to pH 10; the activity is substantially lower in buffers below neutrality. T h e Michaelis constant of the enzyme is 1.2 × 10 -s M; its molecular weight has been estimated to be about 33,000.
[68] Phenoxazinone Synthase
(Streptomyces
antibioticus)-Syntheses of Cinnabarinic Acid and Actinocin 1 By EDWARD KATZ a n d HERBERT WEISSBACH RI
+1 1/202 R2
R1
Rx
~
~
0 R2
+3tt20 R2
Phenoxazinones have been found in various insect pigments la (ommochromes), in certain mold metabolites, 2 e.g., cinnabarin and cin1See article [69] for cinnabarinic acid synthase from Tecona stans and from rat liver. laA. Butenandt, Angew. Chem.69, 16 (1957). 2j. Gripenberg, Acta. Chem. Scand. 12,603 (1958).
550
AROMATIC AMINO ACIDS
[68]
nabarinic acid, and in the family of chromopeptide antibiotics, the actinomycins? Phenoxazinone synthase is very likely involved in the synthesis of actinocin (2-amino-4,6-dimethyl-3-phenoxazinone- 1,9-dicarboxylic acid, the chromophore of actinomycin. The enzyme catalyzes the oxidative condensation of 2 moles of an 0-aminophenol to form 1 mole of a phenoxazinone.
Assay Method. The enzyme assay is based on the increase in optical density in the 430-455 m/~ region due to the formation of the phenoxazinone pigment. 4 Incubations are performed in a Beckman DU spectrophotometer in which the cuvette chamber is maintained at 37 °. The reaction mixture contains 2 micromoles of substrate, 250 micromoles of sodium acetate buffer, pH 5.3, enzyme, and water to a total volume of 3 ml. Because of the availability of 3-hydroxyanthranilic acid, it is used as the substrate for routine studies, and the corresponding phenoxazinone (cinnabarinic acid) formed is measured at 452 m/~ (•452 = 18 × 103). Readings generally are taken at 1- to 2-minute intervals over a 10-minute period; a unit of activity is expressed as an optical density change of 1.0 at 452 mt~ over a period of 5 minutes. Because of an initial lag period in the rate of the reaction, the increase between 5 and 10 minutes is used. A control cuvette containing boiled enzyme is employed to correct for the small nonenzymatic oxidation of the substrate.
Growth of the Organism and Purification of the Enzyme. Streptomyces antibioticus ATCC 14888 is cultured as described previously. 4-6 The organism is inoculated into 100 ml of a medium containing 2.5% N-Z Amine A, (Sheffield Chemical Co., Norwich, New York) arid 1% Difco beef extract in tap water (pH 7.0) and incubated at 30 ° on a New Brunswick reciprocating shaking machine for 48 hours at 240-280 rpm. The mycelium is harvested by centrifugation at 9000 rpm for 10 minutes. After decanting, the mycelium is washed twice in 100 ml of sterile isotonic saline and recentrifuged. A final suspension of the mycelium is made in 50 ml of saline and 2 ml of the suspension are inoculated into 100-ml aliquots of the galactose-glutamic acid-mineral salts medium containing L-glutamic acid.HCl, 2.5 g; galactose, 10 g; K2HPO4, 1.0 g; MgSO4" 7 H20, 25 mg; FeSO4.7 H20, 25 mg; ZnSO4.7 H20, 25 mg; CAC12"2 H20, 25 mg in 1000 ml of distilled water (pH 7.!-7.2). After 2-4 days' growth in the glutamic acid-galactose-mineral salts medium, 3H. Brockmann, Ann. N. Y. Acad. Sci. 89, 323 (1960). 4E. Katz and H. Weissbach, J. Biol. Chem. 237, 882 (1962). 5E. Katz, P. Pienta, and A. Sivak, Appl. Microbiol. 6, 236 (1958). eE. Katz and W. A. Goss, Biochem.J. 73, 458 (1959).
[68]
PHENOXAZINONE SYNTHASE
551
the mycelium is harvested by centrifugation and washed twice with isotonic saline. T h e antibiotic titer of the culture medium at the time of harvesting is generally between 40 and 60 ~g of actinomycin per milliliter. T h e washed mycelium can be kept in .the cold (4°) for a period of 1 month with no appreciable loss of enzymatic activity. The mycelium (wet weight) is suspended in one to two volumes of cold deionized water and disrupted either by sonic treatment at 10 kc for 15 minutes or by passage through an Aminco French pressure cell. The resulting suspension is then centrifuged at 20,000 g for 20 minutes to remove intact cells and cellular debris. A m m o n i u m sulfate (19.7 g) is added slowly to 150 ml of sonic extract of S. antibioticus, and the precipitate obtained after centrifugation is discarded. An additional 13.2 g of ammonium sulfate are then added to the supernatant fraction, and the resulting precipitate is collected by centrifugation and dissolved in 7 ml of cold deionized water. This fraction is dialyzed for 18 hours against 0.001 M sodium phosphate buffer, pH 6.0. The dialyzed preparation (8.5 ml) is placed on a diethylaminoethyl (DEAE) cellulose column (1 x 18 cm) which had been equilibrated with 0.001 M sodium phosphate buffer, pH 6.0. The column is washed first with 200 ml of 0.1 M NaCI, and the enzyme is then eluted with 0.25 M NaCI. The major fraction of activity is present in the first 200-300 ml. The active fractions are combined and then dialyzed for 16 hours against 0.001 M sodium phosphate buffer, pH 6.0. T h e dialyzed eluate contains about 0.035 mg of protein per milliliter. Calcium phosphate gel (80 mg) is added, and the gel supernatant fluid is discarded. After the gel is washed with water, the enzymatic activity is removed by two elutions with 6 ml of 0.2 M phosphate buffer, pH 7.4. The gel eluate, approximately 25-fold purified, is then dialyzed against phosphate buffer as described above. The enzyme can be purified further by adsorbing the activity in the dialyzed calcium phosphate eluate on alumina C~ gel (8 mg of gel per milligram of protein); the activity is eluted with 0.2 M sodium phosphate buffer, pH 7.4 (Table I). TABLE I PURIFICATION OF PHENOXAZINONESYNTHASEa Fraction Sonic extract Ammonium sulfate (18-32%) DEAE-cellulose eluate CaPO4 eluate Alumina Cz, eluate
Volume (ml)
Units/ml
Protein (mg/ml)
150 8.5
18.4 256
2.45 14.3
202 11.2 19.5
3.6 51 12.5
0.035 0.27 0.057
aDetails o f the purification procedure are described in the text.
Specific activity 7.5 17.4 103 189 220
Recovery (%) 78 26 21 9
552
AROMATIC AMINO ACIDS
[68]
The sonic extract, kept frozen, loses little activity over a period of 2-4 weeks, and extracts assayed as much as 5 months later still have appreciable activity. The purified preparation loses less than 20% of its activity in 1 week. Properties oJ Reaction. With both the sonic extract and the purified enzyme preparation, a significant lag period is observed in phenoxazinone formation during the initial 2 minutes of incubation. After this lag phase, the rate of reaction is linear; enzyme activity units are based on the optical density change during the 5- to 10-minute period. An initial lag phase is observed with all the 0-aminophenols tested as substrates. The reaction proceeds best at pH 5.0-5.2. T h e reaction is inhibited by a variety of metal chelating agents, but a definite metal requirement for the enzyme from S. antibioticus has not been shown. Mn 2+ and Ca 2+ ions stimulate the rate of the reaction 30-50% with both the crude and purified enzyme. A more pronounced metal effect is observed with extracts from another actinomycin-producing organism (Streptomyces strain 3723, Institute of Microbiology Culture Collection, Rutgers University). Upon the addition of calcium an 8- to 10-fold increase in activity is obtained; Mn 2+ ions stimulate 3- to 5-fold. Substrate Specificity. All the 0-aminophenols tested are converted to compounds having the spectral characteristics of phenoxazinones. 4"7 The compounds tested and the spectral maxima of the phenoxazinones formed are listed in Table II. Anthranilic acid, m-aminophenol, and m-hydroxybenzoic acid are not converted to colored derivatives. The apparent Michaelis-Menten constant for 4-methyl-3-hydroxyanthranilic acid, 3-hydroxyanthranilic acid, and the anthraniloyl peptides is calculated to be 1 to 2 × 10 -4 M. Inhibitors of the Reaction. A number of compounds related to an 0aminophenol are inhibitors of the enzymatic reaction. 7 For example, 0-, m-, and p-anisidine, 0-, m-, and p-aminobenzoic acid, m.-aminophenol, and o-aminobenzenethiol are good to excellent inhibitors of enzyme activity. Inhibition of enzymatic activity is observed when an equimolar concentration of 3-amino-4-hydroxybiphenol (53%), 2-amino-4-tertbutylphenol (45%) or 3-amino-4-naphthol (81%) is used. Anthranilic acid (o-aminobenzoic acid) and m-aminophenol are competitive inhibitors, whereas 0- and m-anisidine are noncompetitive inhibitors of the enzymatic reaction. Use of Phenoxazinone Synthesis as an Assay for 3-Hydroxyanthranilic Acid. Although the enzymatic reaction with 3-hydroxyanthranilic acid proceeds to about 70% of completion, it is still possible to use this reaction 7E. Katz and H. Weissbach, in "Developments in Industrial Microbiology" (C. Koda, ed.), Vol. 8, p. 67. Am. Inst. Biol. Sci., 1967.
[68]
PHENOXAZINONE SYNTHASE
553
TABLE II SUBSTRATE SPECIFICITY OF PHENOXAZINONE SYNTHASE a
Substrate
Relative rate (OD/5 min)
3-Hydroxyanthranilic acid 4-Methyl-3-hydroxyanthranilic acid 3-Hydroxykynurenine 4-Methyl-3-hydroxyanthranilic acid methyl ester 0-Aminophenol 4-Methyl-3-hydroxyanthraniioyl-L-threonine 4-Methyl-3-hydroxyanthraniloyI-L-threonyl-D-valine 4-Methyl-3-hydroxyanthraniloyl-L-threonyl-D-valyl-L-proline 4-Methyl-3-hydroxyanthraniloyl-L-threonyl-D-valylL-prolyl-sarcosine 4-Methyl-3-hydroxyanthraniloyl-L-threonyl-D-valylL-prolyl-sarcosyl-N-methyl-L-valine
Maximal wave length of product (mt~)
100 105 30 110 90 110 118 111
452 458 425 -435 432 420 450 448 448
115
448
122
448
aSpectra were recorded at pH 5.2 in 0.083 M sodium acetate buffer. The 4-methyl-3hydroxy anthraniloyl peptides were obtained from Dr. Leon Goodman, Stanford Research Institute. All compounds were employed at a concentration of 6.7 x 10-4 M. as an assay f o r this substance. U n d e r conditions in which the substrate is limiting the a m o u n t o f substrate reacting is p r o p o r t i o n a l to the conc e n t r a t i o n e m p l o y e d . T h e sensitivity o f the assay can be increased by c a r r y i n g out the reaction in a 1 ml v o l u m e , c o n t a i n i n g 0.05 ml o f sonic extract, substrate ( 3 - h y d r o x y a n t h r a n i l i c acid), a n d acetate buffer, p H 5.3. U n d e r these conditions, it is possible to assay as little as 10 millim i c r o m o l e s o f 3 - h y d r o x y a n t h r a n i l i c acid as m e a s u r e d by p h e n o x a z i n o n e f o r m a t i o n . A blank c o n t a i n i n g boiled e n z y m e is e m p l o y e d , a n d r e a d i n g s are t a k e n at zero time a n d at 5 - m i n u t e intervals until the reaction stops, usually a f t e r 15 m i n u t e s with a c r u d e sonic extract o f S. antibioticus a n d low levels o f 3 - h y d r o x y a n t h r a n i l i c acid.