ANALYTICAL BIOCHEMISTRY 70, 258--262 (1976)
A New Fluorogenic Substrate for Chymotrypsin MORRIS Z I M M E R M A N , E D W A R D Y U R E W I C Z , AND G O O L PATEL
Merck Institute ,for Therapeutic Research, Rahway, New Jersey 07065 Received May 19, 1975; accepted August 20, 1975 A new sensitive assay for amidase activity of chymotrypsin has been developed using 7-glutarylphenylalaninamido-4-methylcoumarin as substrate. Release of 7-amino-4-methylcoumarin was determined fluorometrically. As little as 0.5/zg/ml of chymotrypsin could be detected; at pH 8.0, Km= 0.7 raM. The substrate was not hydrolyzed by either trypsin or elastase and was capable of measuring chymotrypsin-like activity in tissue extracts. Hydrolysis of the substrate by chymotrypsin was blocked by specific inhibltors of the enzyme.
Nitroanilides have been used as chromogenic substrates for the amidase activity of serine proteases: benzoylarginine-p-nitroanilide for trypsin (I); glutarylphenylalanine-p-nitroanilide for chymotrypsin (2); and N-acetylAla-Ala-Ala-p-nitroanilide (3) and N-t-Boc-Ala-Ala-Pro-Ala-p-nitroanilide I (4) for elastase. It appeared to us that the corresponding amides of a coumarin derivative might be useful and more sensitive substrates for these proteases because the product of hydrolysis would be a fluorescent compound. In this paper we describe the preparation of 7glutarylphenylalaninamido-4-methylcoumarin and its ability to serve as a sensitive fluorogenic substrate for chymotrypsin. Its properties are compared with the corresponding chromogenic substrate, glutarylphenylalanine-p-nitroanilide. EXPERIMENTAL PROCEDURE
Materials Trypsin and chymotrypsin were purchased from Worthington Biochemical Corp., and chromatographically purified elastase was obtained from Miles. Chymostatin, a highly specific peptide inhibitor of chymotrypsin (5,6), was kindly provided by Dr. Umezawa, Institute of Microbial Chemistry, Tokyo, Japan. 7-Amino-4-Methylcoumarin. The 7-amino-4-methylcoumarin was prepared by a modification of the von Pechmann procedure (7) suggested to us by Dr. R. Loeffelman of the American Cyanamid Corp. since the Abbreviations used: t-Boc, tert-butoxycarbonyl; TES, N-tris (hydroxymethyl)methyl2-aminoethane sulfonic acid; PIPES, piperazine-N, N-bis(2-ethanesulfonic acid); DMSO, dimethyl sulfoxide" R F U , relative fluorescence unit. 258 Copyright © 1976 by Academic Press, lnc All rights of reproduction m any form reserved
F E U O R O M E T R I C CHYMOTRYPSIN ASSAY
259
published procedure gave negligible amounts of product: 109 g (1 mol) of m-aminophenol (Aldrich) was dissolved in 750 ml of hot ethyl acetate; then 60 ml of ethylchlorocarbonate (Eastman, 0.6 mol) was added over a period of 30 min while refluxing. After an additional 10 min the mixture was cooled, the suspension was filtered, and the cake was washed with ethyl acetate. After allowing the filtrate to stand 24 hr, the solvent was removed in v a c u o . The solid was washed with petroleum ether and dried to yield 85 g (0.47 tool) of 3-carbethoxyaminophenol. The following was then stirred for 3 hr at 25°C: 325 ml of 75% sulfuric acid, 70 g (0.54 mol) of ethyl acetoacetate (Aldrich), and 81.5 g (0.45 mol) of 3-carbethoxyaminophenol. After adding 2 liters of water, the suspension was filtered and the cake was washed first until free of acid, then with 2 liters of 2% sodium carbonate solution, followed by water wash until the washings were neutral. The cake was washed finally with absolute ethanol and dried in v a c u o to yield 88 g (0.36 mol) of 7-carbethoxyamido-4methylcoumarin, mp 190-191°C. The carbethoxy group was removed as follows: 92 ml of glacial acetic acid, 82 ml of concentrated sulfuric acid, and 80 g (0.32 mol) of 7-carbethoxyamido-4-methylcoumarinwere refluxed for 3 hr. After cooling to 25°C, the mixture was poured into a 1200-ml ice-water mixture, then warmed to 70°C and treated with Norit A-Celite (1:1). After being filtered warm, the solution was brought to pH 8 with 20% sodium carbonate solution. The precipitate which formed was collected and washed first with water and then with ethanol. The 7-amino-4methylcoumarin was crystallized from hot isopropanol: yield = 38 g (0.22 mol), nap 222-223°C (dec). The yield from 3-carbethoxyaminophenol was 47%. All compounds were identified by their nmr spectra. 7-Glutarylphenylalaninamido-4-Methylcoumarin. 7-Amino-4-methylcoumarin (1.1 g, 6.2 mmol) was dissolved in 10 ml of hot pyridine and then cooled to 25°C. After addition of 1.38g (5.2 mmoi) of N-t-Bocphenylalanine (Bachem) and 1.32g (6.4 mmol) of dicyclohexylcarbodiimide, both in methylene chloride, the mixture was incubated at 25°C overnight. Dicyclohexylurea and solvents were removed by filtration and drying in v a c u o . The residue was taken up with ethyl acetate, the solvent layer washed rapidly with 200 ml of cold 1 N HCI and then immediately with 5% sodium carbonate solution. The ethyl acetate layer was dried with anhydrous magnesium sulfate and the solvents removed. The 7-(N-tBoc-phenylalaninamido)-4-methylcoumarinwas crystallized from methylene chloride-cyclohexane: yield -- 1.2 g (2.8 mmol), 44%. To remove the t-Boc blocking group, 750 mg (1.7 mmol) of the compound was dissolved in 3 ml of trifluoroacetic acid. After 15 min the trifluoroacetic acid was removed in v a c u o , and the residue was taken up in methylene chloride and neutralized with triethylamine. After addition of 220 mg (1.9 mmol) of glutaric anhydride in methylene chloride, the mixture was incubated overnight. The 7-glutarylphenylalaninamido-4-methylcoumarin
260
ZIMMERMAN.
YUREWICZ AND PATEL
was crystallized by addition of a little cyclohexane: yield (60%) = 444 mg (1.02 mmol), mp 162-164°C. Assays. Enzyme assays were conducted at 24°C using 0.2 mM substrate in 50 mM TESI(pH 8.0) containing 10 mM CaC12 and 1% DMSOI; final volume was 1.0 ml. With use of the fluorogenic substrate, the fluorescence of the 7-amino-4-methylcoumarin produced was monitored continuously using an Aminco-Bowman spectrofluorometer equipped with a chart recorder. Activation and emission wavelengths were 380 and 460 nm, respectively. The instrument was standardized daily such that a 1.35/,6M solution of quinine sulfate in 0.1 N sulfuric acid gave 1.0 RFU,~When using the chromogenic substrate, the absorbance of thep-nitroaniline produced was determined at 410 nm (E = 8800) with a Beckman DB spectrophotometer equipped with a chart recorder. Protein content of tissue extracts was determined fluorometrically with crystalline bovine serum albumin as standard. Activation and emission wavelengths were 280 and 340 nm, respectively. Pancreas homogenate. Fresh monkey pancreas was homogenized at 4°C in 350 mM sucrose-10 mM PIPES 1, pH 6.5. After centrifugation at 450g, granules were pelleted from the supernatant solution by centrifugation at 17,000g. The pellet was suspended in phosphate-buffered saline and heated for 15 min at 37°C with 0.1% Triton X-100 to release granule enzymes. After centrifugation at 17,000g, the supernatant fluid was retained and stored at 4°C.
RESULTS AND DISCUSSION As summarized in Table 1, both 7-glutarylphenylalaninamido-4methylcoumarin and its hydrolysis product, 7-amino-4-methylcoumarin, were highly fluorescent, but their excitation and emission maxima were distinctly different. When the excitation and emission wavelengths of the spectrofluorometer were set to 380 and 460 nm, respectively, 7-amino-4methylcoumarin retained 22% of its maximal fluorescence (1.0 /AM solution = 0.58 RFU) but now possessed a relative fluorescence approxiTABLE l FLUORESCENCE PROPERTIES OF SUBSTITUTED 4-METHYLCOUMAR1NS M a x i m a (nm) Substitution
Excitation
Emission
Relative fluorescence (1.0/d,M solution) a
7-NH2 7-Glutarylphenylalaninamido
345 325
445 395
2.72 1.13
a In 50 mM T E S , pH 8.0, containing 10 mM CaCI2.
F L U O R O M E T R I C C H Y M O T R Y P S I N ASSAY I000
//
261
////,// o~°
o,
,'
,~
,;0
Chymolrypsm Concentrohon (,~g/ml)
FIG. 1. Initial velocity vs. chymotrypsin concentration. Assays were performed as described under Experimental Procedure. (O), Substrate, 7-glutarylphenylalaninamido-4methylcoumarin. Initial velocity U'0) is expressed as the change in (RFU/min) x 10a. Chymotrypsin concentrations ranged from 0.5 to 50 ~g/ml. (O), Substrate, glutarylphenylalanine-p-nitroanilide. Initial velocity is expressed as the change in (OD/min) × 103. Chymotrypsin concentrations ranged from 10 to 250/zg/ml.
mately 700-fold greater than that of an equimolar amount of its 7-glutarylphenylalaninamido analogue (0.2 mM solution = 0.16 RFU). The fluorogenic substrate was specific for chymotrypsin, with no detectable hydrolysis being observed with 50/xg/ml of either trypsin or elastase. As shown in Fig. 1, the limit of detection of chymotrypsin using the fluorogenic substrate at 24°C and following the reaction for 1-3 rain was 0.5 /xg/ml, and the rate of hydrolysis was proportional to enzyme concentration over at least a 100-fold range. This is to be compared to a
s
/// Oil
~ - - - - ~ 6 ~ o Pr~eln Concentrqhon
3- -
(rag/m1)
FIG. 2. Hydrolysis of 7-glutarylphenylalaninamido-4-methylcoumarin by monkey pancreas granule extract. Assays were performed as described under Experimental Procedure, except that CaC12 was omitted from the buffer and 5/xg/ml of trypsin was included to activate the zymogen. Preparation of the pancreas granule extract, which contained 7.2 mg of protein/ml, was also as described under Experimental Procedure. Tissue extract added is expressed as final tissue protein concentration, 36-360/xg/ml, in the assay mixture.
262
Z I M M E R M A N , Y U R E W I C Z A N D PATEL
detection limit of 10 /zg/ml of chymotrypsin using the chromogenic substrate under identical conditions. The specific activity of chymotrypsin (micromole of substrate hydrolyzed per milligram of enzyme per minute) was similar with both the fluorogenic and chromogenic substrates, 0.025 and 0.018, respectively. The Km was 0.67 mM with the fluorogenic substrate and 0.42 mM with the chromogenic substrate. The action of chymotrypsin on either substrate was inhibited (90%) by addition of chymostatin (4/zg/ml). Assay of chymotrypsin-like activity in monkey pancreas granule extracts was attempted with use of the fluorogenic substrate. Quenching of fluorescence by tissue extract was first examined by using a 7-amino-4methylcoumarin standard. No quenching was observed. Hydrolysis of the substrate could be observed with the tissue extract, equivalent to that of 48 /~g of chymotrypsin/ml. Activity was proportional to the amount of tissue protein added (Fig. 2) and was abolished by chymostatin (4/xg/ml). These studies clearly demonstrate the utility of 7-amino-4methylcoumarin as a starting material for synthesis of a sensitive fluorogenic substrate for assay of the amidase activity of chymotrypsin. Further appropriate amino acid and peptide derivatives of this highly fluorescent amine are currently being synthesized in our laboratory to yield specific fluorogenic substrates for trypsin and elastase. REFERENCES 1. Erlanger, B. F., Kokowsky, N., and Cohen, W. (1961)Arch. Biochem. Biophys. 95, 271 - 278. 2. Erlanger, B. F., Cooper, A. G., and Bendich, A. J. (1964)Biochemistry 3, 1880-1883. 3. Bieth, J., and Wermuth, C. G. (1973) Biochem. Biophys. Res. Commun. 53, 383-390. 4. Yurewicz, E. C., and Zimmerman. M. (1975) Fed. Proc. 34, 234. 5. Umezawa, H., Aoyagi, T., Marishima, H., Kunimoto, S., Matsuzaki, M,, Hamada, M., and Takeuchi, T. (1970)J. Antibiotics 23, 425-427. 6. Tatsuta, K., Mikami, N., Fujirnoto, K., Umezawa, S., Umezawa, H., and Aoyagi, T. (1973) J. Antibiotics 26, 625-646. 7. Von Pechmann, H., and Schwarz, O. (1899)Berichte 32, 3696-3699.
A New Fluorogenic Substrate for Chymotrypsin MORRIS Z I M M E R M A N , E D W A R D Y U R E W I C Z , AND G O O L PATEL
Merck Institute ,for Therapeutic Research, Rahway, New Jersey 07065 Received May 19, 1975; accepted August 20, 1975 A new sensitive assay for amidase activity of chymotrypsin has been developed using 7-glutarylphenylalaninamido-4-methylcoumarin as substrate. Release of 7-amino-4-methylcoumarin was determined fluorometrically. As little as 0.5/zg/ml of chymotrypsin could be detected; at pH 8.0, Km= 0.7 raM. The substrate was not hydrolyzed by either trypsin or elastase and was capable of measuring chymotrypsin-like activity in tissue extracts. Hydrolysis of the substrate by chymotrypsin was blocked by specific inhibltors of the enzyme.
Nitroanilides have been used as chromogenic substrates for the amidase activity of serine proteases: benzoylarginine-p-nitroanilide for trypsin (I); glutarylphenylalanine-p-nitroanilide for chymotrypsin (2); and N-acetylAla-Ala-Ala-p-nitroanilide (3) and N-t-Boc-Ala-Ala-Pro-Ala-p-nitroanilide I (4) for elastase. It appeared to us that the corresponding amides of a coumarin derivative might be useful and more sensitive substrates for these proteases because the product of hydrolysis would be a fluorescent compound. In this paper we describe the preparation of 7glutarylphenylalaninamido-4-methylcoumarin and its ability to serve as a sensitive fluorogenic substrate for chymotrypsin. Its properties are compared with the corresponding chromogenic substrate, glutarylphenylalanine-p-nitroanilide. EXPERIMENTAL PROCEDURE
Materials Trypsin and chymotrypsin were purchased from Worthington Biochemical Corp., and chromatographically purified elastase was obtained from Miles. Chymostatin, a highly specific peptide inhibitor of chymotrypsin (5,6), was kindly provided by Dr. Umezawa, Institute of Microbial Chemistry, Tokyo, Japan. 7-Amino-4-Methylcoumarin. The 7-amino-4-methylcoumarin was prepared by a modification of the von Pechmann procedure (7) suggested to us by Dr. R. Loeffelman of the American Cyanamid Corp. since the Abbreviations used: t-Boc, tert-butoxycarbonyl; TES, N-tris (hydroxymethyl)methyl2-aminoethane sulfonic acid; PIPES, piperazine-N, N-bis(2-ethanesulfonic acid); DMSO, dimethyl sulfoxide" R F U , relative fluorescence unit. 258 Copyright © 1976 by Academic Press, lnc All rights of reproduction m any form reserved
F E U O R O M E T R I C CHYMOTRYPSIN ASSAY
259
published procedure gave negligible amounts of product: 109 g (1 mol) of m-aminophenol (Aldrich) was dissolved in 750 ml of hot ethyl acetate; then 60 ml of ethylchlorocarbonate (Eastman, 0.6 mol) was added over a period of 30 min while refluxing. After an additional 10 min the mixture was cooled, the suspension was filtered, and the cake was washed with ethyl acetate. After allowing the filtrate to stand 24 hr, the solvent was removed in v a c u o . The solid was washed with petroleum ether and dried to yield 85 g (0.47 tool) of 3-carbethoxyaminophenol. The following was then stirred for 3 hr at 25°C: 325 ml of 75% sulfuric acid, 70 g (0.54 mol) of ethyl acetoacetate (Aldrich), and 81.5 g (0.45 mol) of 3-carbethoxyaminophenol. After adding 2 liters of water, the suspension was filtered and the cake was washed first until free of acid, then with 2 liters of 2% sodium carbonate solution, followed by water wash until the washings were neutral. The cake was washed finally with absolute ethanol and dried in v a c u o to yield 88 g (0.36 mol) of 7-carbethoxyamido-4methylcoumarin, mp 190-191°C. The carbethoxy group was removed as follows: 92 ml of glacial acetic acid, 82 ml of concentrated sulfuric acid, and 80 g (0.32 mol) of 7-carbethoxyamido-4-methylcoumarinwere refluxed for 3 hr. After cooling to 25°C, the mixture was poured into a 1200-ml ice-water mixture, then warmed to 70°C and treated with Norit A-Celite (1:1). After being filtered warm, the solution was brought to pH 8 with 20% sodium carbonate solution. The precipitate which formed was collected and washed first with water and then with ethanol. The 7-amino-4methylcoumarin was crystallized from hot isopropanol: yield = 38 g (0.22 mol), nap 222-223°C (dec). The yield from 3-carbethoxyaminophenol was 47%. All compounds were identified by their nmr spectra. 7-Glutarylphenylalaninamido-4-Methylcoumarin. 7-Amino-4-methylcoumarin (1.1 g, 6.2 mmol) was dissolved in 10 ml of hot pyridine and then cooled to 25°C. After addition of 1.38g (5.2 mmoi) of N-t-Bocphenylalanine (Bachem) and 1.32g (6.4 mmol) of dicyclohexylcarbodiimide, both in methylene chloride, the mixture was incubated at 25°C overnight. Dicyclohexylurea and solvents were removed by filtration and drying in v a c u o . The residue was taken up with ethyl acetate, the solvent layer washed rapidly with 200 ml of cold 1 N HCI and then immediately with 5% sodium carbonate solution. The ethyl acetate layer was dried with anhydrous magnesium sulfate and the solvents removed. The 7-(N-tBoc-phenylalaninamido)-4-methylcoumarinwas crystallized from methylene chloride-cyclohexane: yield -- 1.2 g (2.8 mmol), 44%. To remove the t-Boc blocking group, 750 mg (1.7 mmol) of the compound was dissolved in 3 ml of trifluoroacetic acid. After 15 min the trifluoroacetic acid was removed in v a c u o , and the residue was taken up in methylene chloride and neutralized with triethylamine. After addition of 220 mg (1.9 mmol) of glutaric anhydride in methylene chloride, the mixture was incubated overnight. The 7-glutarylphenylalaninamido-4-methylcoumarin
260
ZIMMERMAN.
YUREWICZ AND PATEL
was crystallized by addition of a little cyclohexane: yield (60%) = 444 mg (1.02 mmol), mp 162-164°C. Assays. Enzyme assays were conducted at 24°C using 0.2 mM substrate in 50 mM TESI(pH 8.0) containing 10 mM CaC12 and 1% DMSOI; final volume was 1.0 ml. With use of the fluorogenic substrate, the fluorescence of the 7-amino-4-methylcoumarin produced was monitored continuously using an Aminco-Bowman spectrofluorometer equipped with a chart recorder. Activation and emission wavelengths were 380 and 460 nm, respectively. The instrument was standardized daily such that a 1.35/,6M solution of quinine sulfate in 0.1 N sulfuric acid gave 1.0 RFU,~When using the chromogenic substrate, the absorbance of thep-nitroaniline produced was determined at 410 nm (E = 8800) with a Beckman DB spectrophotometer equipped with a chart recorder. Protein content of tissue extracts was determined fluorometrically with crystalline bovine serum albumin as standard. Activation and emission wavelengths were 280 and 340 nm, respectively. Pancreas homogenate. Fresh monkey pancreas was homogenized at 4°C in 350 mM sucrose-10 mM PIPES 1, pH 6.5. After centrifugation at 450g, granules were pelleted from the supernatant solution by centrifugation at 17,000g. The pellet was suspended in phosphate-buffered saline and heated for 15 min at 37°C with 0.1% Triton X-100 to release granule enzymes. After centrifugation at 17,000g, the supernatant fluid was retained and stored at 4°C.
RESULTS AND DISCUSSION As summarized in Table 1, both 7-glutarylphenylalaninamido-4methylcoumarin and its hydrolysis product, 7-amino-4-methylcoumarin, were highly fluorescent, but their excitation and emission maxima were distinctly different. When the excitation and emission wavelengths of the spectrofluorometer were set to 380 and 460 nm, respectively, 7-amino-4methylcoumarin retained 22% of its maximal fluorescence (1.0 /AM solution = 0.58 RFU) but now possessed a relative fluorescence approxiTABLE l FLUORESCENCE PROPERTIES OF SUBSTITUTED 4-METHYLCOUMAR1NS M a x i m a (nm) Substitution
Excitation
Emission
Relative fluorescence (1.0/d,M solution) a
7-NH2 7-Glutarylphenylalaninamido
345 325
445 395
2.72 1.13
a In 50 mM T E S , pH 8.0, containing 10 mM CaCI2.
F L U O R O M E T R I C C H Y M O T R Y P S I N ASSAY I000
//
261
////,// o~°
o,
,'
,~
,;0
Chymolrypsm Concentrohon (,~g/ml)
FIG. 1. Initial velocity vs. chymotrypsin concentration. Assays were performed as described under Experimental Procedure. (O), Substrate, 7-glutarylphenylalaninamido-4methylcoumarin. Initial velocity U'0) is expressed as the change in (RFU/min) x 10a. Chymotrypsin concentrations ranged from 0.5 to 50 ~g/ml. (O), Substrate, glutarylphenylalanine-p-nitroanilide. Initial velocity is expressed as the change in (OD/min) × 103. Chymotrypsin concentrations ranged from 10 to 250/zg/ml.
mately 700-fold greater than that of an equimolar amount of its 7-glutarylphenylalaninamido analogue (0.2 mM solution = 0.16 RFU). The fluorogenic substrate was specific for chymotrypsin, with no detectable hydrolysis being observed with 50/xg/ml of either trypsin or elastase. As shown in Fig. 1, the limit of detection of chymotrypsin using the fluorogenic substrate at 24°C and following the reaction for 1-3 rain was 0.5 /xg/ml, and the rate of hydrolysis was proportional to enzyme concentration over at least a 100-fold range. This is to be compared to a
s
/// Oil
~ - - - - ~ 6 ~ o Pr~eln Concentrqhon
3- -
(rag/m1)
FIG. 2. Hydrolysis of 7-glutarylphenylalaninamido-4-methylcoumarin by monkey pancreas granule extract. Assays were performed as described under Experimental Procedure, except that CaC12 was omitted from the buffer and 5/xg/ml of trypsin was included to activate the zymogen. Preparation of the pancreas granule extract, which contained 7.2 mg of protein/ml, was also as described under Experimental Procedure. Tissue extract added is expressed as final tissue protein concentration, 36-360/xg/ml, in the assay mixture.
262
Z I M M E R M A N , Y U R E W I C Z A N D PATEL
detection limit of 10 /zg/ml of chymotrypsin using the chromogenic substrate under identical conditions. The specific activity of chymotrypsin (micromole of substrate hydrolyzed per milligram of enzyme per minute) was similar with both the fluorogenic and chromogenic substrates, 0.025 and 0.018, respectively. The Km was 0.67 mM with the fluorogenic substrate and 0.42 mM with the chromogenic substrate. The action of chymotrypsin on either substrate was inhibited (90%) by addition of chymostatin (4/zg/ml). Assay of chymotrypsin-like activity in monkey pancreas granule extracts was attempted with use of the fluorogenic substrate. Quenching of fluorescence by tissue extract was first examined by using a 7-amino-4methylcoumarin standard. No quenching was observed. Hydrolysis of the substrate could be observed with the tissue extract, equivalent to that of 48 /~g of chymotrypsin/ml. Activity was proportional to the amount of tissue protein added (Fig. 2) and was abolished by chymostatin (4/xg/ml). These studies clearly demonstrate the utility of 7-amino-4methylcoumarin as a starting material for synthesis of a sensitive fluorogenic substrate for assay of the amidase activity of chymotrypsin. Further appropriate amino acid and peptide derivatives of this highly fluorescent amine are currently being synthesized in our laboratory to yield specific fluorogenic substrates for trypsin and elastase. REFERENCES 1. Erlanger, B. F., Kokowsky, N., and Cohen, W. (1961)Arch. Biochem. Biophys. 95, 271 - 278. 2. Erlanger, B. F., Cooper, A. G., and Bendich, A. J. (1964)Biochemistry 3, 1880-1883. 3. Bieth, J., and Wermuth, C. G. (1973) Biochem. Biophys. Res. Commun. 53, 383-390. 4. Yurewicz, E. C., and Zimmerman. M. (1975) Fed. Proc. 34, 234. 5. Umezawa, H., Aoyagi, T., Marishima, H., Kunimoto, S., Matsuzaki, M,, Hamada, M., and Takeuchi, T. (1970)J. Antibiotics 23, 425-427. 6. Tatsuta, K., Mikami, N., Fujirnoto, K., Umezawa, S., Umezawa, H., and Aoyagi, T. (1973) J. Antibiotics 26, 625-646. 7. Von Pechmann, H., and Schwarz, O. (1899)Berichte 32, 3696-3699.
