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Sensitive assays for trypsin, elastase, and chymotrypsin using new fluorogenic substrates
ANALYTICAL
78, 47-51 (1977)
BIOCHEMISTRY
Sensitive Assays for Trypsin, Elastase, and Chymotrypsin Using New Fluorogenic Substrates M. ZIMMERMAN, Merck
Institute
B. ASHE, E. C. YUREWICZ, for
Therapeutic
Research,
Rahway,
AND G.PATEL New
Jersey
07065
Received April 14, 1976; accepted October 21, 1976 Sensitive fluorogenic substrates for trypsin, chymotrypsin, and elastase were prepared. These substrates are amides of an acyl amino acid or peptide with 7-amino-4-methyicoumarin (AMC). The substrates with their respective K,,,/K, ratios given in parentheses are: for chymotrypsin, glutaryl-Phe-AMC (78) and Ala-Ala-Phe-AMC TFA (1660); for trypsin, benzoyl-DL-Arg-AMC (800) and Carbobenzoxy (Cbz)-L-Arg-AMC (5300); for elastase, N-acetyl-Ala-Ala-ProAla-AMC (15,000). The detection limits obtained by using the best substrates and short incubation times are: chymotrypsin, 25 ng; trypsin, 5 ng; and elastase, 2 ng.
We recently reported (1) that the fluorogenic substrate, 7-glutarylphenylalaninamido-4-methylcoumarin was a stable amide substrate allowing the sensitive determination of chymotrypsin. The sensitivity is due to the fact that the leaving group, 7-amino-4-methylcoumarin, is highly fluorescent. Esters of the oxy analog of this amine, 4-methylumbelliferone, have been used as active site titrants of proteases (2) but the instability of these esters in water would preclude extensive use of such compounds as substrates. The amides are extremely stable in solution and are, in addition, more closely related to the natural peptide substrates. In this paper, we describe the preparation and use of fluorogenic amide substrates for trypsin and elastase and an improved chymotrypsin substrate. EXPERIMENTAL Materials
Chymotrypsin and trypsin were purchased from Worthington Biochemical Corp., elastase (chromatographically purified) was obtained from Miles, and human granulocyte elastase was prepared according to the method of Taylor and Crawford (3). 7-Glutarylphenylalaninamido-4-methylcoumarin (7-glutaryl-Phe-AMC) and 7-amino-4-methylcoumarin. (AMC) were synthesized as described previously (1). Amino acid derivatives were purchased from Bachem Labs. N-Tris-(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES) was purchased from Calbiochem. N-Methylmorpholine was obtained from Matheson, Coleman & Bell, and isobutyl chloroformate 47 Copyright 0 1977 by Academic Press, Inc. AI! rights of reproduction m any form reserved.
ISSN ooO3-2697
48
ZIMMERMANET
AL.
from Aldrich Chem. Co. All solvents used in the coupling were dried over molecular sieves.
reactions
Synthesis of Substrates 7-(N-Benzoyl-DL-argininamido)+methylcoumarin. N-Benzoyl-DLarginine, 4.7 g (0.017 mol), was dissolved in dilute HCl and concentrated to dryness in vacua. The resulting hydrochloride was dissolved in dimethylformamide (DMF), and to this solution was added 3.0 g (0.017 mol) of AMC and 3.5 g (0.02 mol) of N, N’-dicyclohexylcarbodiimide (DCC), both in DMF. After standing 48 h at 25°C the reaction mixture was filtered to remove dicyclohexylurea, and the filtrate was concentrated to dryness in vacua. The residue was crystallized from hot methanol. The yield was 240 mg (0.55 mmol) of the desired product, mp 158- 159°C. 7-(N-Cbz-L-argininamido)-4-methylcoumarin. N-Cbz-L-arginine (0.53 g, 1.7 mmol) was suspended in 250 ml of tetrahydrofuran (THF) and treated with HCl gas until all the solid had dissolved. The solvent was removed in vacua, and the hydrochloride was washed successfully with THF and anhydrous ethyl ether. The hydrochloride was dissolved in DMF and cooled to -5°C in a dry ice-methanol bath. To this was added triethylamine (0.24 ml, 1.7 mmol) and isobutyl chloroformate (0.22 ml, 1.17 mmol). The reaction was stirred at -5°C for 25 min to allow formation of the mixed anhydride. To this was added a solution of AMC (0.2 g, 1.14 mmol) in 50 ml of DMF. Reaction was allowed to proceed overnight at 25°C. The solvent was removed from the reaction mixture in vacua. The residue was dissolved in chloroformmethanol (80:20) and applied to a silica (Baker) column packed in the same solvent; elution with this solvent was allowed to proceed. Fractions containing the product, as determined by analytical tic on silica GF plates, were concentrated, and the product was crystallized from methanol:ether; yield: 70 mg (0.15 mmol), 13%, mp 217-219°C. 7-(Alanylalanylphenylalaninamido)-4-methylcoltmarin
trijkoroacetate.
AMC, 2.0 g (11.4 mmol), was dissolved in 150 ml of THF. N-t-butoxycarbonyl (Boc)-phenylalanine (4.5 g, 17.1 mmol) was dissolved in THF and cooled to -5°C. Triethylamine 2.4 ml (17.1 mmol) was added followed by 2.34 ml (17.1 mmol) of isobutyl chloroformate. This solution was allowed to react at -5°C for 30 min to form the anhydride after which time the AMC solution was added and allowed to react overnight at room temperature. The solution was filtered to remove the triethylamine . HCl, and solvent was removed in vacua. The residue was taken up in ethyl acetate and washed twice with cold 1 N HCl and 5% NaHC03. The solvent was dried with anhydrous Na,SO, and removed in vacua to yield 4 g (9.5 mmol) of a white solid, N-t-Boc-Phe-AMC. This was deblocked with HCl in ethyl acetate to give Phe-AMC . HCl, concentrated to dryness in vacua, neutralized with triethylamine, and dissolved in THF.
FLUOROMETRIC
SERINE
PROTEASE
ASSAYS
49
N-t-Boc-alanine, 1.8 g (9.5 mmol), was reacted with isobutyl chloroformate (1.3 ml, 9.5 mmol) in the presence of triethylamine (1.32 ml, 9.5 mmol) in THF at -5°C for 30 min. To this was added the Phe-AMC solution, and the reaction mixture was allowed to stand overnight at room temperature. Treatment as previously described yielded 4.5 g of a pale yellow solid, N-t-Boc-Ala-Phe-AMC. The above procedure was repeated to yield 3.9 g (6.9 mmol) of N-t-Boc-Ala-Ala-Phe-AMC; overall yield, 73%. This was deblocked with trifluoroacetic acid (TFA) and crystallized from ether to yield 3.13 g of Ala-Ala-Phe-AMC * TFA, mp 140- 143°C. 7-(N-Acetylalanylalanylprolylalaninamido~-4-methylcoumarin. N-tBoc-Ala-AMC was prepared by mixed anhydride coupling of N-tBoc-Ala with AMC as described before for the phenylalanine derivative. The product was deblocked with HCI in ethyl acetate and coupled to N-acetyl-Ala-Ala-Pro, prepared essentially according to the procedure of Thompson and Blout (4) via the mixed anhydride procedure. Starting with 2 g of the amino coumarin one obtains 510 mg (0.97 mmol), 9% yield, of N-acetyl-Ala-Ala-Pro-Ala-AMC, mp 145- 146”C, crystallized from methylene chloride/cyclohexane. All structures were checked by tic and nmr. Due to the high fluorescence of the amino coumarin starting material which could contaminate AMC peptides, the last two peptides were purified on preparative tic silica plates (2000 pu) using CHCl,:MeOH (70:30) as the solvent. Compounds were recovered from the plates by extraction of the silica with methanol. Assays
Enzyme assays were conducted at 24°C using 0.2 mM substrate in 50 mM TES buffer, pH 8.0, containing 10 mM CaCI, and 1% DMSO; the final volume was 1.0 ml. 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 PM solution of quinine sulfate in 0.1 N sulfuric acid gave 1.O relative fluorescence unit. Protein content of the granulocyte elastase was determined spectrophotometrically by the method of Warburg and Christian (5) using an extinction coefficient of 9.85 (1% solution, 280 nm) reported by Baugh and Travis (6). RESULTS
AND DISCUSSION
All ofthe substrates reported here and their common hydrolysis product, 7-amino-4-methylcoumarin, are highly fluorescent, but their excitation and
50
ZIMMERMAN
ET AL.
emission maxima are distinctly different (1). With the excitation and emission wavelengths of the spectrofluorometer set at 380 and 460 nm, respectively, 7-amino-4-methylcoumarin retained 22% of its maximal fluorescence but now possessed a relative fluorescence at least 500-fold greater than any of the amide substrates. Because of the insolubility of these substrates in water, the compounds were initially dissolved in DMSO and diluted with buffer to the appropriate concentration. Neither TES buffer nor DMSO at concentrations up to 10% has inhibitory or stimulatory effect on the enzymes concerned using the conventional substrates: N-t-Boc-Ala-Ala-Pro-Ala-p-nitroanilide for elastase, glutarylphenylalanine-p-nitroanilide for chymotrypsin, and cr-N-benzoyl-DL-arginine-p-nitroanilide for trypsin. The fluorogenic substrates are water stable, showing no detectable spontaneous hydrolysis after a S-hr incubation under assay conditions. Each substrate is specific for its particular enzyme. No detectable hydrolysis was observed after 5-hr incubations of Ala-Ala-PheAMC-TFA with 1 pg/ml of trypsin or hog pancreatic elastase, of N-Cbz-Arg-AMC with 1 pg/ml of chymotrypsin or hog pancreatic elastase, or of N-acetyl-Ala-Ala-Pro-Ala-AMC with 1 pg/ml of trypsin or chymotrypsin. From Table I it is apparent that the use of the specific fluorogenic substrates allows sensitive detection of the amidase activity of the proteases studied using relatively short incubation times (5 min). All of the detection limits are in nanograms, and, in each case, the rate of TABLE FLUOROGENIC SUBSTRATES
1 FOR VARIOUS
PROTEASES
Enzyme
Detection= limit (ng)
K, (mM)
K eat (see-I)
KcadKm
Glutaryl-Phe-AMC
Chymotrypsin
500
0.67
0.052
78
Ala-Ala-Phe:AMC ,TFA
Chymotrypsin
25
0.5
0.83
1,660
N-Benzoyl-ArgAMC
Trypsin
50
0.25
0.2
800
N-Cbz-Arg-AMC
Trypsin
5
0.25
1.3
5,300
N-Acetyl-Ala-Ala-ProAla-AMC
Hog pancreatic elastase
2
0.5
7.5
15,ooo
13
0.63
2
3,200
Substrate
Granulocyte elastase
o Initial velocity = change of 0.001 relative fluorescence unit/min at 25°C.
FLUOROMETRIC
SERINE PROTEASE
ASSAYS
51
hydrolysis is proportional to enzyme concentration over at least a lOO-fold range. Such sensitivity should be highly desirable in assaying tissue extracts for neutral proteases. Elastase, which is present in human PMN in very low concentrations, can be determined easily in granule extracts from these cells. The K,s for the substrates with their respective enzymes do not differ markedly. The increased sensitivity of the peptide for chymotrypsin in contrast to the simple acyl amino acid amide is consistent with the observations of Baumann et al. (7) on the effect of extending the peptide chain on the K,,, for substrates of chymotrypsin. However, these authors observed a large effect of such structural alteration on K,, not observed here. REFERENCES 1. Zimmerman, M., Yurewicz, E. C., and Pate], G. (1976) Anal. Biochem. 70, 258-262. 2. Jameson, G. W., Roberts, D. V., Adams, R. W., Kyle, W. S. A., and Elmore, D. T. (1973) Biochem. J. 131, 107. 3. Taylor, J., and Crawford, I. (1975) Arch. Biochem. Biophys. 169, 91- 101. 4. Thompson, R. C. and Blout, E. R. (1973) Biochemistry 12, 57-65. 5. Warburg, 0. and Christian, W. (1942) Biochem. Z. 310, 384. 6. Baugh, R. J. and Travis J. (1976) Biochemistry 15, 836-841. 7. Baumann, W. K., Bizzozero, S. A., and Dutler, H. (1973)Eur. J. Biochem. 39,381-391.
78, 47-51 (1977)
BIOCHEMISTRY
Sensitive Assays for Trypsin, Elastase, and Chymotrypsin Using New Fluorogenic Substrates M. ZIMMERMAN, Merck
Institute
B. ASHE, E. C. YUREWICZ, for
Therapeutic
Research,
Rahway,
AND G.PATEL New
Jersey
07065
Received April 14, 1976; accepted October 21, 1976 Sensitive fluorogenic substrates for trypsin, chymotrypsin, and elastase were prepared. These substrates are amides of an acyl amino acid or peptide with 7-amino-4-methyicoumarin (AMC). The substrates with their respective K,,,/K, ratios given in parentheses are: for chymotrypsin, glutaryl-Phe-AMC (78) and Ala-Ala-Phe-AMC TFA (1660); for trypsin, benzoyl-DL-Arg-AMC (800) and Carbobenzoxy (Cbz)-L-Arg-AMC (5300); for elastase, N-acetyl-Ala-Ala-ProAla-AMC (15,000). The detection limits obtained by using the best substrates and short incubation times are: chymotrypsin, 25 ng; trypsin, 5 ng; and elastase, 2 ng.
We recently reported (1) that the fluorogenic substrate, 7-glutarylphenylalaninamido-4-methylcoumarin was a stable amide substrate allowing the sensitive determination of chymotrypsin. The sensitivity is due to the fact that the leaving group, 7-amino-4-methylcoumarin, is highly fluorescent. Esters of the oxy analog of this amine, 4-methylumbelliferone, have been used as active site titrants of proteases (2) but the instability of these esters in water would preclude extensive use of such compounds as substrates. The amides are extremely stable in solution and are, in addition, more closely related to the natural peptide substrates. In this paper, we describe the preparation and use of fluorogenic amide substrates for trypsin and elastase and an improved chymotrypsin substrate. EXPERIMENTAL Materials
Chymotrypsin and trypsin were purchased from Worthington Biochemical Corp., elastase (chromatographically purified) was obtained from Miles, and human granulocyte elastase was prepared according to the method of Taylor and Crawford (3). 7-Glutarylphenylalaninamido-4-methylcoumarin (7-glutaryl-Phe-AMC) and 7-amino-4-methylcoumarin. (AMC) were synthesized as described previously (1). Amino acid derivatives were purchased from Bachem Labs. N-Tris-(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES) was purchased from Calbiochem. N-Methylmorpholine was obtained from Matheson, Coleman & Bell, and isobutyl chloroformate 47 Copyright 0 1977 by Academic Press, Inc. AI! rights of reproduction m any form reserved.
ISSN ooO3-2697
48
ZIMMERMANET
AL.
from Aldrich Chem. Co. All solvents used in the coupling were dried over molecular sieves.
reactions
Synthesis of Substrates 7-(N-Benzoyl-DL-argininamido)+methylcoumarin. N-Benzoyl-DLarginine, 4.7 g (0.017 mol), was dissolved in dilute HCl and concentrated to dryness in vacua. The resulting hydrochloride was dissolved in dimethylformamide (DMF), and to this solution was added 3.0 g (0.017 mol) of AMC and 3.5 g (0.02 mol) of N, N’-dicyclohexylcarbodiimide (DCC), both in DMF. After standing 48 h at 25°C the reaction mixture was filtered to remove dicyclohexylurea, and the filtrate was concentrated to dryness in vacua. The residue was crystallized from hot methanol. The yield was 240 mg (0.55 mmol) of the desired product, mp 158- 159°C. 7-(N-Cbz-L-argininamido)-4-methylcoumarin. N-Cbz-L-arginine (0.53 g, 1.7 mmol) was suspended in 250 ml of tetrahydrofuran (THF) and treated with HCl gas until all the solid had dissolved. The solvent was removed in vacua, and the hydrochloride was washed successfully with THF and anhydrous ethyl ether. The hydrochloride was dissolved in DMF and cooled to -5°C in a dry ice-methanol bath. To this was added triethylamine (0.24 ml, 1.7 mmol) and isobutyl chloroformate (0.22 ml, 1.17 mmol). The reaction was stirred at -5°C for 25 min to allow formation of the mixed anhydride. To this was added a solution of AMC (0.2 g, 1.14 mmol) in 50 ml of DMF. Reaction was allowed to proceed overnight at 25°C. The solvent was removed from the reaction mixture in vacua. The residue was dissolved in chloroformmethanol (80:20) and applied to a silica (Baker) column packed in the same solvent; elution with this solvent was allowed to proceed. Fractions containing the product, as determined by analytical tic on silica GF plates, were concentrated, and the product was crystallized from methanol:ether; yield: 70 mg (0.15 mmol), 13%, mp 217-219°C. 7-(Alanylalanylphenylalaninamido)-4-methylcoltmarin
trijkoroacetate.
AMC, 2.0 g (11.4 mmol), was dissolved in 150 ml of THF. N-t-butoxycarbonyl (Boc)-phenylalanine (4.5 g, 17.1 mmol) was dissolved in THF and cooled to -5°C. Triethylamine 2.4 ml (17.1 mmol) was added followed by 2.34 ml (17.1 mmol) of isobutyl chloroformate. This solution was allowed to react at -5°C for 30 min to form the anhydride after which time the AMC solution was added and allowed to react overnight at room temperature. The solution was filtered to remove the triethylamine . HCl, and solvent was removed in vacua. The residue was taken up in ethyl acetate and washed twice with cold 1 N HCl and 5% NaHC03. The solvent was dried with anhydrous Na,SO, and removed in vacua to yield 4 g (9.5 mmol) of a white solid, N-t-Boc-Phe-AMC. This was deblocked with HCl in ethyl acetate to give Phe-AMC . HCl, concentrated to dryness in vacua, neutralized with triethylamine, and dissolved in THF.
FLUOROMETRIC
SERINE
PROTEASE
ASSAYS
49
N-t-Boc-alanine, 1.8 g (9.5 mmol), was reacted with isobutyl chloroformate (1.3 ml, 9.5 mmol) in the presence of triethylamine (1.32 ml, 9.5 mmol) in THF at -5°C for 30 min. To this was added the Phe-AMC solution, and the reaction mixture was allowed to stand overnight at room temperature. Treatment as previously described yielded 4.5 g of a pale yellow solid, N-t-Boc-Ala-Phe-AMC. The above procedure was repeated to yield 3.9 g (6.9 mmol) of N-t-Boc-Ala-Ala-Phe-AMC; overall yield, 73%. This was deblocked with trifluoroacetic acid (TFA) and crystallized from ether to yield 3.13 g of Ala-Ala-Phe-AMC * TFA, mp 140- 143°C. 7-(N-Acetylalanylalanylprolylalaninamido~-4-methylcoumarin. N-tBoc-Ala-AMC was prepared by mixed anhydride coupling of N-tBoc-Ala with AMC as described before for the phenylalanine derivative. The product was deblocked with HCI in ethyl acetate and coupled to N-acetyl-Ala-Ala-Pro, prepared essentially according to the procedure of Thompson and Blout (4) via the mixed anhydride procedure. Starting with 2 g of the amino coumarin one obtains 510 mg (0.97 mmol), 9% yield, of N-acetyl-Ala-Ala-Pro-Ala-AMC, mp 145- 146”C, crystallized from methylene chloride/cyclohexane. All structures were checked by tic and nmr. Due to the high fluorescence of the amino coumarin starting material which could contaminate AMC peptides, the last two peptides were purified on preparative tic silica plates (2000 pu) using CHCl,:MeOH (70:30) as the solvent. Compounds were recovered from the plates by extraction of the silica with methanol. Assays
Enzyme assays were conducted at 24°C using 0.2 mM substrate in 50 mM TES buffer, pH 8.0, containing 10 mM CaCI, and 1% DMSO; the final volume was 1.0 ml. 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 PM solution of quinine sulfate in 0.1 N sulfuric acid gave 1.O relative fluorescence unit. Protein content of the granulocyte elastase was determined spectrophotometrically by the method of Warburg and Christian (5) using an extinction coefficient of 9.85 (1% solution, 280 nm) reported by Baugh and Travis (6). RESULTS
AND DISCUSSION
All ofthe substrates reported here and their common hydrolysis product, 7-amino-4-methylcoumarin, are highly fluorescent, but their excitation and
50
ZIMMERMAN
ET AL.
emission maxima are distinctly different (1). With the excitation and emission wavelengths of the spectrofluorometer set at 380 and 460 nm, respectively, 7-amino-4-methylcoumarin retained 22% of its maximal fluorescence but now possessed a relative fluorescence at least 500-fold greater than any of the amide substrates. Because of the insolubility of these substrates in water, the compounds were initially dissolved in DMSO and diluted with buffer to the appropriate concentration. Neither TES buffer nor DMSO at concentrations up to 10% has inhibitory or stimulatory effect on the enzymes concerned using the conventional substrates: N-t-Boc-Ala-Ala-Pro-Ala-p-nitroanilide for elastase, glutarylphenylalanine-p-nitroanilide for chymotrypsin, and cr-N-benzoyl-DL-arginine-p-nitroanilide for trypsin. The fluorogenic substrates are water stable, showing no detectable spontaneous hydrolysis after a S-hr incubation under assay conditions. Each substrate is specific for its particular enzyme. No detectable hydrolysis was observed after 5-hr incubations of Ala-Ala-PheAMC-TFA with 1 pg/ml of trypsin or hog pancreatic elastase, of N-Cbz-Arg-AMC with 1 pg/ml of chymotrypsin or hog pancreatic elastase, or of N-acetyl-Ala-Ala-Pro-Ala-AMC with 1 pg/ml of trypsin or chymotrypsin. From Table I it is apparent that the use of the specific fluorogenic substrates allows sensitive detection of the amidase activity of the proteases studied using relatively short incubation times (5 min). All of the detection limits are in nanograms, and, in each case, the rate of TABLE FLUOROGENIC SUBSTRATES
1 FOR VARIOUS
PROTEASES
Enzyme
Detection= limit (ng)
K, (mM)
K eat (see-I)
KcadKm
Glutaryl-Phe-AMC
Chymotrypsin
500
0.67
0.052
78
Ala-Ala-Phe:AMC ,TFA
Chymotrypsin
25
0.5
0.83
1,660
N-Benzoyl-ArgAMC
Trypsin
50
0.25
0.2
800
N-Cbz-Arg-AMC
Trypsin
5
0.25
1.3
5,300
N-Acetyl-Ala-Ala-ProAla-AMC
Hog pancreatic elastase
2
0.5
7.5
15,ooo
13
0.63
2
3,200
Substrate
Granulocyte elastase
o Initial velocity = change of 0.001 relative fluorescence unit/min at 25°C.
FLUOROMETRIC
SERINE PROTEASE
ASSAYS
51
hydrolysis is proportional to enzyme concentration over at least a lOO-fold range. Such sensitivity should be highly desirable in assaying tissue extracts for neutral proteases. Elastase, which is present in human PMN in very low concentrations, can be determined easily in granule extracts from these cells. The K,s for the substrates with their respective enzymes do not differ markedly. The increased sensitivity of the peptide for chymotrypsin in contrast to the simple acyl amino acid amide is consistent with the observations of Baumann et al. (7) on the effect of extending the peptide chain on the K,,, for substrates of chymotrypsin. However, these authors observed a large effect of such structural alteration on K,, not observed here. REFERENCES 1. Zimmerman, M., Yurewicz, E. C., and Pate], G. (1976) Anal. Biochem. 70, 258-262. 2. Jameson, G. W., Roberts, D. V., Adams, R. W., Kyle, W. S. A., and Elmore, D. T. (1973) Biochem. J. 131, 107. 3. Taylor, J., and Crawford, I. (1975) Arch. Biochem. Biophys. 169, 91- 101. 4. Thompson, R. C. and Blout, E. R. (1973) Biochemistry 12, 57-65. 5. Warburg, 0. and Christian, W. (1942) Biochem. Z. 310, 384. 6. Baugh, R. J. and Travis J. (1976) Biochemistry 15, 836-841. 7. Baumann, W. K., Bizzozero, S. A., and Dutler, H. (1973)Eur. J. Biochem. 39,381-391.