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A new chromogenic substrate for subtilisin
ANALYTICAL
62, 371-376 (1974)
BIOCHEMISTRY
A New
Chromogenic
Substrate
for Subtilisin
L. A. LYUBLINSKAYA, S. V. BELYAEV, A. YA. STRONGIN, L. I?. MATYASH, E. D. LEVIN, AND V. M. STEPANOV Institute
of Genetics and Selection USSR,
Mosco~u~,
D-182,
of Industrial Post
Ofice
Box
Microorganisms, 379
Received October 10, 1973; accepted June 10, 1974 A new chromogenic substrate, benzyloxycarbonyl-glycyl-glycyl-L-leucine p-nitroanilide, was synthesized allowing convenient spectrophotometric assay of subtilisin activity. This substrate has been applied to the determination of subtilisin in solution as well as for the identification of subtilisin after gel electrophoresis or isoelectrophocusing. Catalytic parameters for the new substrate were determined.
Subtilisins, serine proteinases produced by Bacillus subtilis, are widely used for research as well as for applied purposes. The assay of these enzymes has been based on the measurement of protein hydrolysis (1) or on the splitting of peptide substrates followed by ninhydrin reaction (2). Specific chromogenic substrates proved to be very convenient for the assay of proteolytic enzymes were unknown for subtilisins. To close the gap we synthesized benzyloxycarbonyl-glycyl-glycyl-L-leucine p-nitroanilide-ZGGLNA-which turned out to be an effective chromogenic substrate for subtilisin. The choice of this structure was based on the following considerations. p-Nitroanilides proved themselves as useful chromogenic substrates for various proteinases (3,4). According to Morihara and co-workers (5,6), subtilisin exhibits a distinct preference for n-leucine at the carboxyl side of susceptible amide bond. The binding site of subtilisin is known to contain the subsites interacting with the amino acids which precede the residue participating in the peptide bond to be cleaved (5,6). Therefore, the nitroaniline moiety was attached to the acylated tripeptide with leucine as the C-terminal residue to create favourable conditions for enzyme-substrate interaction. It turned out that ZGGLNA satisfied the requirements for a chromogenic substrate and might be used not only for the assay of subtilisin and related enzymes but also for the localization of subtilisin bands after gel electrophoresis of electrofocusing. 371 Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
372
LYUBLINSKAY.4
MATERIALS
ET
AND
AL.
METHODS
Subtilisins. Subtilisin A (Subtilopeptidase A) was purchased from Serva. Enzyme concentration was estimated using the absorbance at 280 nm (A’,8 = 8.6) (7). Commercial preparation of subtilisin-SPlO Bioprase (Nagarse, Japan) has been used in some electrophoresis experiments. Reagents for disc-electrophoresis. Acrylamide was purchased from Koch-Light; N,N’-methylenebisacrylamide, N,N,N’,N’-tetramethylethylenediamine (TEMED) , riboflavine, and glycine from Reanal; Coomassie Blue GL from Serva; Tris from Merck; sucrose from BDH. All these reagents were used without further purification. Benzyloxycarbonyl-glycyl-glycyl-L-leudne p-&roan&de (ZGGLNA). To the solution of 15.7 g (5.9 mmoles) of benzyloxycarbonyl-glycylglycine in 15 ml of dry dimethylformamide 0.84 ml (6 mmoles) of triethylamine were added at -20” followed after 10 min by 0.84 ml (6 mmoles) of isobutylchloroformate. After 30 min a solution of n-leucine p-nitroanilide was added to this mixture at -15”. The solution of n-leucine p-nitroanilide was prepared by the addition of 0.84 ml (6 mmoles) of triethylamine to 1.96 g (5.9 mmoles) of L-leucine p-nitroanilide hydrobromide (8) in 5 ml of dimethylformamide. The reaction was allowed to proceed under stirring for 1 hr at -15”, then 1 hr at 20” after which the mixture was kept overnight in the refrigerator. The precipitate was removed by filtration and the solution was evaporated at reduced pressure giving an oil which was dissolved in 75 ml of ethyl acetate and washed successively with water (15 ml), 0.5 N NaHC03 (3 X 15 ml), water (15 ml), 0.5 N HCl (3 X 15 ml), and water (2 X 15 ml). The ethyl acetate layer was dried over Na2S04 and evaporated to dryness. The residue after recrystallisation from ethyl acetate-light petroleum gave 2.1 g of ZGGLNA (yield 71.5%), mp 152-154”, [,a] go - 5.9 (c 1, dimethylformamide) , Rf 0.97 (n-butanol-pyridine-wateracetic acid 15: 12: 10: 3 by vol) . Anal: Calcd for C,,H,,N,O, (499.51) ; C 57.72; H 5.84; N 14.02. Found: C 57.75; H 5.87; N 13.70. Kinetic measurements. 3 ml of the substrate solution in 0.05 M TrisHCl buffer, pH 8.5 containing 7.5% CH,CN, were kept for 6-S min in the cell of Cary 15 spectrophotometer, thermostated at 37”. After the temperature equilibration, 0.01 ml of enzyme solution in the same buffer was added and the release of p-nitroaniline was monitored at 410 nm. Usually it took 3-5 min to measure the initial rate of hydrolysis, the conversion of the substrate being 2-6s. The molar absorbance of p-nitroaniline at 410 nm measured under the same conditions was equal to 8900 M-km-‘.
JCBTILISIN
SUBSTRATE
373
ACTIVITY
Disc-electrophoresis. The experiments were performed in CanalcoEurope device in Tris-glycine buffer, pH 9.5 (9) using 6 X 70 mm glass tubes at 7.5% concn of polyacrylamide. 0.10-0.15 ml of 20% sucrose solution containing l-100 /:g of subtilisin A was placed on the top of concentrating gel. Electrophoresis was performed at 4-5 mA/tube for l-l.5 hr at 4”. The gels were stained with 0.3% solution of Coomassie Blue GL in 7.5% acetic acid and destained in 7.5% acetic acid. The localization of subtilisin activity. Immediately after electrophoresis, the gel was immersed for 30 min at 4” in a mixture containing 0.8 ml of 0.05 M Tris-HCl-CaCl, buffer, pH 8.5, and 0.2 ml of 8 mM ZGGLNA solution in 75% dimethylformamide. This operation, aimed to saturate the gel with the substrate, was followed by incubation at 40” for 30 min. Light yellow band turning black when the gel was examined in ultraviolet light (max intensity at 360 nm) showed the localization of the active enzyme. RESULTS
AND DISCUSSION
Benzyloxycarbonyl-glycyl-glycyl-L-leucine p-nitroanilide was prepared with good yield by the coupling of benzyloxycarbonyl-glycyl-glycine and L-leucine p-nitroanilide according to the mixed anhydride procedure (10). Subtilisin catalyzes the hydrolysis of this substrate cleaving the bond bet’ween the leucine residue and the p-nitroaniline moiety. This pattern of the reaction was affirmed by paper electrophoresis of the digest. The release of the coloured reaction product, p-nitroaniline, can be easily followed spectrophotometrically at 410 nm. This wavelength, being slightly shifted from the absorption maximum of p-nitroaniline (380 nm) , has been chosen for the measurement since the substrate absorption (h,X = 317 nm) is negligible at 410 nm (Fig. 1). The kinetic parameters for subtilisin-catalyzed hydrolysis of ZGGLNA at pH 8.5 and 37” were determined in the presence of a large excess of substrate. The substrate concentration ranged from 4 to 20.10m3 M, that of the enzyme from 6 to 6O.KV M. As shown in Fig. 2, the initial velocity of the substrate hydrolysis is a linear function of the enzyme concentration. The initial rate of the substrate hydrolysis follows the MichaelisMenten equation : dA
J’o = dt
1 x -Q
=
koat . Eo . So
Km+&,
’
where dA/dt is the rate alteration of the absorption at 410 nm; &,--the substrate concentration in the reaction mixture at zero time; EO--the total concentration of the enzyme; k ,,t-the catalytic constant; Km-the Michaelis constant.
374
LYUBLINSKAYA
FIG.
aniline
1. Absorption (2).
spectra
of
ET
AL.
Z-Gly-Gly-L-Leu-p-nitroanilide
(1)
and
p-nitro-
The high extinction coefficient of p-nitroaniline at 410 nm allows the determination of the initial rate of ZGGLNA hydrolysis with satisfactory accuracy, an average error being about 1%. The Michaelis constant, K,, and V were calculated from a LineweaverBurk plot (Fig. 3). lCcat was calculated from the V value taking into account that the preparation of subtilisin contained 80% of the active enzyme. This has been shown by affinity chromatography on a sorbent prepared by the attachment of benzyloxycarbonyl-glycyl-glycyl-n-
2. The initial rate of hydrolysis of Z-Gly-Gly-L-Leu-p-nitroanilide of subtilisin concentration (So = 6.14 X 10d M).
FIG.
tion
aa a func-
SUBTILISIN
SUBSTRATE
ACTIVITY
375
FIG. 3. Lineweaver-Burk plot for subtilisin-catalyzed hydrolysis of Z-Gly-GIy-LLeu-p-nitroanilide at the following enzyme concentrations: E0 = 6.08 X lo-’ M (1) ; E, = 7.75 X lo-’ M (2).
leucine to Sepharose 4B through a hexamethylenediamine moiety serving as an “arm.” The parameters obtained (pH 8.5, 37”) : Km = 8 X lo-* M; lc,,, = 0.6 set-I; Ic,,,/K, = 7.5 X lo2 see-%-* are comparable with those found by Morihara and co-workers (5) for subtilisin BPN’-catalyzed hydrolysis of benzyloxycarbonyl-glycyl-glycyl-L-leucine amide (pH 8.5, 40”, 15% dimethylformamide) : Km = 33.3 X 1O-3 M; JCcat = 14.9 see-I; k,,,/ Ii, = 4.5 X lo* sec-lM-l. It has to be noted that this comparison is somewhat limited by the difference in experimental conditions as well as by the use of different types of subtilisin. ZGGLNA turned out to be useful not only for subtilisin assay in solutions but also for detecting of the enzyme in polyacrylamide gels after disc-electrophoresis. Thus, upon disc-electrophoresis of a commercial subtilisin preparation it was possible to localize less than 1 pg of the enzyme by incubating the gel with the substrate. This procedure was applied to the identification of subtilisin in culture filtrates of various strains of Bacillus subtilis. REFERENCES 1. MORIHARA, 2. YEMM,
3. BUNDY, 4. NISHI, 5. MORIHARA, 515.
Ii., E. W.,
H. (1969) Arch. Biochem. Biophgs. 129, 620. E. C. (1955) Analyst 80, 209. H. F. (1962) Anal. Biochem. 3, 431. N., TOHURA, S.? ASD NOGUCHI, I. (1970) Bull. Chem. Sot. Jap. 43, 2900. K., OKA, T., AND TSUZUKI, H. (1970) Arch. B&hem. Biophys. 138, AND AND
TSUZUI~I,
COCKING,
376
LYUBLINSKAYA
ET
AL.
6. MORIHARA, K., TSUZUKI, H., AND OKA, T. (1971) Biochem. Biophys. Res. Commun. 42, 1000. 7. GUNTELBERG, A. V., AND OTTESEN, M. (1954) Comt. Rend. Trav. Lab. Carlsberg 29, 36. 8. TUPPY, H., WIESBAUER, U., AND WINTERBERGER, E. (1962) Z. Physiol. Chem. 329, 278. 9. DAVIS, B. I. (1964) Ann. N. Y. Acad. Sci. USA 121, 404. 10. LYUBLINSKAYA, L. A. (1973) in Khimiya Proteoliticheskikh Fermentov, Vilnius. str. 73.
62, 371-376 (1974)
BIOCHEMISTRY
A New
Chromogenic
Substrate
for Subtilisin
L. A. LYUBLINSKAYA, S. V. BELYAEV, A. YA. STRONGIN, L. I?. MATYASH, E. D. LEVIN, AND V. M. STEPANOV Institute
of Genetics and Selection USSR,
Mosco~u~,
D-182,
of Industrial Post
Ofice
Box
Microorganisms, 379
Received October 10, 1973; accepted June 10, 1974 A new chromogenic substrate, benzyloxycarbonyl-glycyl-glycyl-L-leucine p-nitroanilide, was synthesized allowing convenient spectrophotometric assay of subtilisin activity. This substrate has been applied to the determination of subtilisin in solution as well as for the identification of subtilisin after gel electrophoresis or isoelectrophocusing. Catalytic parameters for the new substrate were determined.
Subtilisins, serine proteinases produced by Bacillus subtilis, are widely used for research as well as for applied purposes. The assay of these enzymes has been based on the measurement of protein hydrolysis (1) or on the splitting of peptide substrates followed by ninhydrin reaction (2). Specific chromogenic substrates proved to be very convenient for the assay of proteolytic enzymes were unknown for subtilisins. To close the gap we synthesized benzyloxycarbonyl-glycyl-glycyl-L-leucine p-nitroanilide-ZGGLNA-which turned out to be an effective chromogenic substrate for subtilisin. The choice of this structure was based on the following considerations. p-Nitroanilides proved themselves as useful chromogenic substrates for various proteinases (3,4). According to Morihara and co-workers (5,6), subtilisin exhibits a distinct preference for n-leucine at the carboxyl side of susceptible amide bond. The binding site of subtilisin is known to contain the subsites interacting with the amino acids which precede the residue participating in the peptide bond to be cleaved (5,6). Therefore, the nitroaniline moiety was attached to the acylated tripeptide with leucine as the C-terminal residue to create favourable conditions for enzyme-substrate interaction. It turned out that ZGGLNA satisfied the requirements for a chromogenic substrate and might be used not only for the assay of subtilisin and related enzymes but also for the localization of subtilisin bands after gel electrophoresis of electrofocusing. 371 Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
372
LYUBLINSKAY.4
MATERIALS
ET
AND
AL.
METHODS
Subtilisins. Subtilisin A (Subtilopeptidase A) was purchased from Serva. Enzyme concentration was estimated using the absorbance at 280 nm (A’,8 = 8.6) (7). Commercial preparation of subtilisin-SPlO Bioprase (Nagarse, Japan) has been used in some electrophoresis experiments. Reagents for disc-electrophoresis. Acrylamide was purchased from Koch-Light; N,N’-methylenebisacrylamide, N,N,N’,N’-tetramethylethylenediamine (TEMED) , riboflavine, and glycine from Reanal; Coomassie Blue GL from Serva; Tris from Merck; sucrose from BDH. All these reagents were used without further purification. Benzyloxycarbonyl-glycyl-glycyl-L-leudne p-&roan&de (ZGGLNA). To the solution of 15.7 g (5.9 mmoles) of benzyloxycarbonyl-glycylglycine in 15 ml of dry dimethylformamide 0.84 ml (6 mmoles) of triethylamine were added at -20” followed after 10 min by 0.84 ml (6 mmoles) of isobutylchloroformate. After 30 min a solution of n-leucine p-nitroanilide was added to this mixture at -15”. The solution of n-leucine p-nitroanilide was prepared by the addition of 0.84 ml (6 mmoles) of triethylamine to 1.96 g (5.9 mmoles) of L-leucine p-nitroanilide hydrobromide (8) in 5 ml of dimethylformamide. The reaction was allowed to proceed under stirring for 1 hr at -15”, then 1 hr at 20” after which the mixture was kept overnight in the refrigerator. The precipitate was removed by filtration and the solution was evaporated at reduced pressure giving an oil which was dissolved in 75 ml of ethyl acetate and washed successively with water (15 ml), 0.5 N NaHC03 (3 X 15 ml), water (15 ml), 0.5 N HCl (3 X 15 ml), and water (2 X 15 ml). The ethyl acetate layer was dried over Na2S04 and evaporated to dryness. The residue after recrystallisation from ethyl acetate-light petroleum gave 2.1 g of ZGGLNA (yield 71.5%), mp 152-154”, [,a] go - 5.9 (c 1, dimethylformamide) , Rf 0.97 (n-butanol-pyridine-wateracetic acid 15: 12: 10: 3 by vol) . Anal: Calcd for C,,H,,N,O, (499.51) ; C 57.72; H 5.84; N 14.02. Found: C 57.75; H 5.87; N 13.70. Kinetic measurements. 3 ml of the substrate solution in 0.05 M TrisHCl buffer, pH 8.5 containing 7.5% CH,CN, were kept for 6-S min in the cell of Cary 15 spectrophotometer, thermostated at 37”. After the temperature equilibration, 0.01 ml of enzyme solution in the same buffer was added and the release of p-nitroaniline was monitored at 410 nm. Usually it took 3-5 min to measure the initial rate of hydrolysis, the conversion of the substrate being 2-6s. The molar absorbance of p-nitroaniline at 410 nm measured under the same conditions was equal to 8900 M-km-‘.
JCBTILISIN
SUBSTRATE
373
ACTIVITY
Disc-electrophoresis. The experiments were performed in CanalcoEurope device in Tris-glycine buffer, pH 9.5 (9) using 6 X 70 mm glass tubes at 7.5% concn of polyacrylamide. 0.10-0.15 ml of 20% sucrose solution containing l-100 /:g of subtilisin A was placed on the top of concentrating gel. Electrophoresis was performed at 4-5 mA/tube for l-l.5 hr at 4”. The gels were stained with 0.3% solution of Coomassie Blue GL in 7.5% acetic acid and destained in 7.5% acetic acid. The localization of subtilisin activity. Immediately after electrophoresis, the gel was immersed for 30 min at 4” in a mixture containing 0.8 ml of 0.05 M Tris-HCl-CaCl, buffer, pH 8.5, and 0.2 ml of 8 mM ZGGLNA solution in 75% dimethylformamide. This operation, aimed to saturate the gel with the substrate, was followed by incubation at 40” for 30 min. Light yellow band turning black when the gel was examined in ultraviolet light (max intensity at 360 nm) showed the localization of the active enzyme. RESULTS
AND DISCUSSION
Benzyloxycarbonyl-glycyl-glycyl-L-leucine p-nitroanilide was prepared with good yield by the coupling of benzyloxycarbonyl-glycyl-glycine and L-leucine p-nitroanilide according to the mixed anhydride procedure (10). Subtilisin catalyzes the hydrolysis of this substrate cleaving the bond bet’ween the leucine residue and the p-nitroaniline moiety. This pattern of the reaction was affirmed by paper electrophoresis of the digest. The release of the coloured reaction product, p-nitroaniline, can be easily followed spectrophotometrically at 410 nm. This wavelength, being slightly shifted from the absorption maximum of p-nitroaniline (380 nm) , has been chosen for the measurement since the substrate absorption (h,X = 317 nm) is negligible at 410 nm (Fig. 1). The kinetic parameters for subtilisin-catalyzed hydrolysis of ZGGLNA at pH 8.5 and 37” were determined in the presence of a large excess of substrate. The substrate concentration ranged from 4 to 20.10m3 M, that of the enzyme from 6 to 6O.KV M. As shown in Fig. 2, the initial velocity of the substrate hydrolysis is a linear function of the enzyme concentration. The initial rate of the substrate hydrolysis follows the MichaelisMenten equation : dA
J’o = dt
1 x -Q
=
koat . Eo . So
Km+&,
’
where dA/dt is the rate alteration of the absorption at 410 nm; &,--the substrate concentration in the reaction mixture at zero time; EO--the total concentration of the enzyme; k ,,t-the catalytic constant; Km-the Michaelis constant.
374
LYUBLINSKAYA
FIG.
aniline
1. Absorption (2).
spectra
of
ET
AL.
Z-Gly-Gly-L-Leu-p-nitroanilide
(1)
and
p-nitro-
The high extinction coefficient of p-nitroaniline at 410 nm allows the determination of the initial rate of ZGGLNA hydrolysis with satisfactory accuracy, an average error being about 1%. The Michaelis constant, K,, and V were calculated from a LineweaverBurk plot (Fig. 3). lCcat was calculated from the V value taking into account that the preparation of subtilisin contained 80% of the active enzyme. This has been shown by affinity chromatography on a sorbent prepared by the attachment of benzyloxycarbonyl-glycyl-glycyl-n-
2. The initial rate of hydrolysis of Z-Gly-Gly-L-Leu-p-nitroanilide of subtilisin concentration (So = 6.14 X 10d M).
FIG.
tion
aa a func-
SUBTILISIN
SUBSTRATE
ACTIVITY
375
FIG. 3. Lineweaver-Burk plot for subtilisin-catalyzed hydrolysis of Z-Gly-GIy-LLeu-p-nitroanilide at the following enzyme concentrations: E0 = 6.08 X lo-’ M (1) ; E, = 7.75 X lo-’ M (2).
leucine to Sepharose 4B through a hexamethylenediamine moiety serving as an “arm.” The parameters obtained (pH 8.5, 37”) : Km = 8 X lo-* M; lc,,, = 0.6 set-I; Ic,,,/K, = 7.5 X lo2 see-%-* are comparable with those found by Morihara and co-workers (5) for subtilisin BPN’-catalyzed hydrolysis of benzyloxycarbonyl-glycyl-glycyl-L-leucine amide (pH 8.5, 40”, 15% dimethylformamide) : Km = 33.3 X 1O-3 M; JCcat = 14.9 see-I; k,,,/ Ii, = 4.5 X lo* sec-lM-l. It has to be noted that this comparison is somewhat limited by the difference in experimental conditions as well as by the use of different types of subtilisin. ZGGLNA turned out to be useful not only for subtilisin assay in solutions but also for detecting of the enzyme in polyacrylamide gels after disc-electrophoresis. Thus, upon disc-electrophoresis of a commercial subtilisin preparation it was possible to localize less than 1 pg of the enzyme by incubating the gel with the substrate. This procedure was applied to the identification of subtilisin in culture filtrates of various strains of Bacillus subtilis. REFERENCES 1. MORIHARA, 2. YEMM,
3. BUNDY, 4. NISHI, 5. MORIHARA, 515.
Ii., E. W.,
H. (1969) Arch. Biochem. Biophgs. 129, 620. E. C. (1955) Analyst 80, 209. H. F. (1962) Anal. Biochem. 3, 431. N., TOHURA, S.? ASD NOGUCHI, I. (1970) Bull. Chem. Sot. Jap. 43, 2900. K., OKA, T., AND TSUZUKI, H. (1970) Arch. B&hem. Biophys. 138, AND AND
TSUZUI~I,
COCKING,
376
LYUBLINSKAYA
ET
AL.
6. MORIHARA, K., TSUZUKI, H., AND OKA, T. (1971) Biochem. Biophys. Res. Commun. 42, 1000. 7. GUNTELBERG, A. V., AND OTTESEN, M. (1954) Comt. Rend. Trav. Lab. Carlsberg 29, 36. 8. TUPPY, H., WIESBAUER, U., AND WINTERBERGER, E. (1962) Z. Physiol. Chem. 329, 278. 9. DAVIS, B. I. (1964) Ann. N. Y. Acad. Sci. USA 121, 404. 10. LYUBLINSKAYA, L. A. (1973) in Khimiya Proteoliticheskikh Fermentov, Vilnius. str. 73.