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A useful spectrophotometric rate assay for pepsin
.\N.\LYPIC’.\L
41, 36G-364
IIlOCHEl\lIdTliT
A Useful T. PETER
(1971)
Spectrophotometric STEIN,l
The
TED
W. REID,”
Rate
Assay
for
AND
DAVID
FAHRNEY3
of
Department Los
Chemistry, UGuersity Angeles, Califo?nia 9002/t
Received
August
of
Pepsin
California,
15, 1969
Studies of relations between structure and function of the enzyme pepsin have been hampered by lack of suitable assays. Although a number of synthetic peptide substrates have been described, they have not been widely used. The most convenient assay previously available uses denatured hemoglobin as substrate (1) and is not particularly satisfactory because the kinetic parameters cannot be evaluated. The recent discovery that certain sulfite esters are excellent substrates for pepsin offers a new, facile method of determining the specific activity of a solution of pepsin, as well as the kinetic parameters k,,, and & (2). This paper describes the preparation of phenyl sulfite and its use as a specific substrate for spectrophotometric assays for pepsin. The reaction is believed to proceed by t,he following general scheme: 0
0 s + HO’ b-
+ H o 2 MATERIALS
AND
METHODS
Twice-crystallized swine pepsin was purchased from Worthington Biochemical Corporation. A standard solution of “pure enzyme” in 0.1 M ’ Present of Surgery, *Present (Ophthalmology Conn. 3 Present Collins, Cal.
address: University of Pennsylvania, Philadelphia, Pa. address: Yale University School of Section), Molecular Biophysics
Medicine, Deparbments and Biochemistry,
address
Department
: Colorado
State
University, 360
School
of
Medicine,
Department of Surgery New Haven,
of Biochemistry,
Fort
glycine/0.5 M lithium perchlorate buffer, pH 3.5, is stored in a refrigerator and is stable for several days. The buffer is prepared by acidification of a 0.1 M glycine/0.5 M LiClO, solution with 70% perchloric acid at a Corning pH meter model 7 standardized against Beckman pH 4 standard buffer. The concentration of pepsin is estimated from the absorbance at 270 nm, assuming a molar absorptivity of 50,900 M-l cm-’ as determined by Perlmann (3). For several experiments a sample of homogeneous pepsin was prepared from crystalline swine pepsinogen (l’l’orthington Biochemical Corp.) by the method of Rajogapalan, Stein, and Moore (4). Amino acid analysis of this preparation gave excellent agreement with that reported by these researchers. Phenyl sulfite is readily prepared but care must be taken to remove impurities which markedly inhibit pepsin. Reagent-grade chemicals were used. Phenol (18.8 gm, 0.2 mole), anhydrous pyridine (16.2 ml, 0.2 mole), and dry ether (50 ml) were placed in a 250-ml 3-neck flask immersed in a salt-ice bath and outfitted with a drying tube, dropping funnel, and magnetic stirrer. Thionyl chloride (7.2 ml, 0.1 mole) in 25 ml of dry ether was added with vigorous stirring over a period of 30 min. Stirring was continued for an additional 30 nun at 0” and the mixture was filtered through a sintered-glass filter. The ethereal filtrate was washed with 0.01 M HCI, water, 5% aqueous Na,CO:,, and water at 0”, then dried over MgSO,. The product, was isolated by diat’illation through a short Vigreaux column at reduced pressure (b.p. 130”, 0.4 mm Hg) and was purified by crystallization. The ester was dissolved in dry ether at, room t’emperature, pentane (spectral grade) was added until the solution became cloudy, and the mixture was cooled at -20”. Further recrystallization from ether/pentane afforded a 60% yield of phenyl sulfite, m.p. 4-5”. Analysis. S 13.98.
C&d.
for
CxzHxi0.S:
C 61.52,
H 4.30, S 13.68.
Found:
C 61.73,
H 4.34,
The compound is kept in a deep freeze and is stable for months, as judged by the absorbance at 270 nm. A 1k3 M solution in spectral-grade acetonitrile should have an absorbance between 0.035 and 0.045, due to the S=O absorption at this wavelength (the Ed,, of methyl sulfite is about 35 in acetonitrile) . A saturated stock solution of phenyl sulfite (about 0.15 mdl) is prepared by adding 0.2 ml of phenyl sulfite to 125 ml of 0.1 M glycine/0.5 M LiClO, buffer, pH 3.7. The mixture is stirred vigorously for 10 min and filtered twice through three layers of \Vhatman ISo. 1 filter paper. A fresh stock solution is prepared each day for careful work. If relative enzyme activities are satisfactory for a particular experiment, then the substrate
362
STEIiTj
REID,
AND
I’AHRNEY
concentration need not be determined. However, the concentration of phenyl sulfite can be estimated from the amount of phenol that is released upon acid-catalyzed hydrolysis. One milliliter of 6M HCl is added to 5 ml of stock solution, and the absorbance at 270 nm is read after 1 hr at room temperature. The ac.,T0o f hydrolysis was 2.5 + 0.1 X 1CP M-l cm-l. The assay is run by pipetting 3.00 ml of phenyl sulfite stock solution into a cuvet (l-cm light path). The reaction is started by adding 0.100 ml of pepsin solution containing 30 to 300 pg of enzyme, and the production of phenol is followed at 270 nm. RESULTS
AND
DISCUSSION
The pepsin-catalyzed hydrolysis of phenyl sulfite can be monitored at 270 nm. As shown in Fig. 1, the rate of increase of absorbance at 270 nm is a linear function of enzyme concentration. A final concentration of (0.3-3) x 10e6M pepsin is optimal for 5 min assay periods with a Gilford recording spectrophotometer. At 3 X 1O-6M enzyme about 10% of the substrate is hydrolyzed in 5 min. Duplicate assays agree well within 1%. Glycine was chosen as the buffer because the nonenzymic rate of hydrolysis of phenyl sulfite is much slower in this buffer than in other buffers. Lithium perchlorate was added to the buffer solution because it exerts a salting-in effect on sulfite esters, resulting in quicker attainment of a homogeneous solution and higher solubility of the ester. The observed first-order rate constant is 1.2 X 1P mill-l in 0.1 M glycine, as against 5 x 10;” mine1 in 0.1 M acetate, both buffers 0.5 M in LiClO,, pH 3.7, 25”. Thus, the nonenzymic rate of hydrolysis is negligible in the glycine/LiClO, buffer, and no blank correction is necessary. The enzyme
FIG. 1. A plot of rate of increase of absorbance at 270 mm against concentration of pepsin. Initial phenyl sulfite concentration was 1.52 X 10” M.
SPECTROPHOTOMETRIC
ASSAY
FOR
353
PEPSIN
exhibits maximum sulfite esterase activity in the range pH 2 to 4. Below pH 2, enzyme activity decreases rapidly and the acid-catalyzed hydrolysis of sulfite esters becomes measurable. Initial rates of hydrolysis conform to the Michaelis-Rlenten equation:4
uo = E,,,[Slo[E~l,‘(h’m
+ 1%).
A plot of ~,)-l against [S] 0-1 is present’ed in Fig. 2. The steady-state kinetic parameters were calculated by means of a computer program written for
OL
2 [s];’
FIG.
2. vO-’ versus
[SIG
at 25”,
pH
2.71.
4 x lo4 (M-l) Pepsin
6
concentration
was
1.37 X 10.“M.
the Wilkinson (5) weighted form of the Michaelis-Menten equation: values observed for the pepsin used in this work are kcat = 1.3 min-l and Km = 1.0 X 10e4M at 25”, pH 3.7. These values compare well with peptide and depsipeptide substrates for pepsin: lccat = 4.2 min-I, K, = 8.4 x 10e4 M for N-acetyl-L-phenylalanyl-L-diiodotyrosine at pH 4.5 and 37” (6) ; and lCeat= 46 min-I, li,, = 4 x 1tP M for benzyloxycarbonyl-nhistidyl-p-nitro-~-phenylalanyI-~-pl~enyl-~-Iactic acid methyl ester at pH 4.0 and 37” (7). The above values of the steady-state kinet,ic parameters for sulfite ester hydrolysis were obtained with a homogeneous sample of pepsin prepared in this laboratory and represent the highest specific activity observed to date. Several reports indicate that the kinet’ic properties of commercial pepsin differ little from those of homogeneous pepsin. This has been our experience, except that several commercial pepsin lots have had markedly lower specific activities toward ljhenyl sulfite, ranging down to 25%. This variability is generally reflected in Ic,.,,,. ’ [ET1 is the total concentration of enzyme, ester at zero time, k,;,, is the catalytic constant Michaelis constant. The initial velocity v. (dA/dt)oAe.
[Sl, is the or turnover is -(d[Sl/dt)0
concentration of sulfite number. and Km is the = 0.5 (d[Pl/cZt)0 =
364
STEIN,
REID,
AND
FAHRNEY
SUMMARY
Phenyl sulfite is a specific substrate for pepsin, whose hydrolysis can be followed spectrophotometrically. This compound is superior to peptide substrates in its easeof synthesis and its ability to be monitered spectrophotometrically. The sulfite assay is therefore the method of choice for both routine and detailed assays of pepsin. ACKNOWLEDGMENT This work was supported in part by grant BM 13446 and a Postdoctoral (R.P.S.), U. S. Public Health Service.
Fellowship
REFERENCES 1. ANSON. M. L., J. Gen. Phgsiol. 22, 79 (1938). 2. REID, T. W., .~ND FAHRNEY. D., J. Am. Chem. sot. 89, 3941 (1967). 3. PERLMANN, G. E., J. Bid. Chem. 241, 153 (1966). 4. RAJAGOPALAN, R. G., MOORE. S.,, AND STEIN, W. H., J. Bid. Chem. 241, 4295 (1966) 5. WILKINSON, G. N., Biochem. J. 80, 324 (1961). 6. JACKSON, W, T.. SCHLAMOTVITZ, M.. ASD SHAW, A., Biochemistry 4, 1537 (1965) 7. INOUYE, K., AND FRUTON, J. S., J. Am. Chem. Sot. 89, 187 (1967).
41, 36G-364
IIlOCHEl\lIdTliT
A Useful T. PETER
(1971)
Spectrophotometric STEIN,l
The
TED
W. REID,”
Rate
Assay
for
AND
DAVID
FAHRNEY3
of
Department Los
Chemistry, UGuersity Angeles, Califo?nia 9002/t
Received
August
of
Pepsin
California,
15, 1969
Studies of relations between structure and function of the enzyme pepsin have been hampered by lack of suitable assays. Although a number of synthetic peptide substrates have been described, they have not been widely used. The most convenient assay previously available uses denatured hemoglobin as substrate (1) and is not particularly satisfactory because the kinetic parameters cannot be evaluated. The recent discovery that certain sulfite esters are excellent substrates for pepsin offers a new, facile method of determining the specific activity of a solution of pepsin, as well as the kinetic parameters k,,, and & (2). This paper describes the preparation of phenyl sulfite and its use as a specific substrate for spectrophotometric assays for pepsin. The reaction is believed to proceed by t,he following general scheme: 0
0 s + HO’ b-
+ H o 2 MATERIALS
AND
METHODS
Twice-crystallized swine pepsin was purchased from Worthington Biochemical Corporation. A standard solution of “pure enzyme” in 0.1 M ’ Present of Surgery, *Present (Ophthalmology Conn. 3 Present Collins, Cal.
address: University of Pennsylvania, Philadelphia, Pa. address: Yale University School of Section), Molecular Biophysics
Medicine, Deparbments and Biochemistry,
address
Department
: Colorado
State
University, 360
School
of
Medicine,
Department of Surgery New Haven,
of Biochemistry,
Fort
glycine/0.5 M lithium perchlorate buffer, pH 3.5, is stored in a refrigerator and is stable for several days. The buffer is prepared by acidification of a 0.1 M glycine/0.5 M LiClO, solution with 70% perchloric acid at a Corning pH meter model 7 standardized against Beckman pH 4 standard buffer. The concentration of pepsin is estimated from the absorbance at 270 nm, assuming a molar absorptivity of 50,900 M-l cm-’ as determined by Perlmann (3). For several experiments a sample of homogeneous pepsin was prepared from crystalline swine pepsinogen (l’l’orthington Biochemical Corp.) by the method of Rajogapalan, Stein, and Moore (4). Amino acid analysis of this preparation gave excellent agreement with that reported by these researchers. Phenyl sulfite is readily prepared but care must be taken to remove impurities which markedly inhibit pepsin. Reagent-grade chemicals were used. Phenol (18.8 gm, 0.2 mole), anhydrous pyridine (16.2 ml, 0.2 mole), and dry ether (50 ml) were placed in a 250-ml 3-neck flask immersed in a salt-ice bath and outfitted with a drying tube, dropping funnel, and magnetic stirrer. Thionyl chloride (7.2 ml, 0.1 mole) in 25 ml of dry ether was added with vigorous stirring over a period of 30 min. Stirring was continued for an additional 30 nun at 0” and the mixture was filtered through a sintered-glass filter. The ethereal filtrate was washed with 0.01 M HCI, water, 5% aqueous Na,CO:,, and water at 0”, then dried over MgSO,. The product, was isolated by diat’illation through a short Vigreaux column at reduced pressure (b.p. 130”, 0.4 mm Hg) and was purified by crystallization. The ester was dissolved in dry ether at, room t’emperature, pentane (spectral grade) was added until the solution became cloudy, and the mixture was cooled at -20”. Further recrystallization from ether/pentane afforded a 60% yield of phenyl sulfite, m.p. 4-5”. Analysis. S 13.98.
C&d.
for
CxzHxi0.S:
C 61.52,
H 4.30, S 13.68.
Found:
C 61.73,
H 4.34,
The compound is kept in a deep freeze and is stable for months, as judged by the absorbance at 270 nm. A 1k3 M solution in spectral-grade acetonitrile should have an absorbance between 0.035 and 0.045, due to the S=O absorption at this wavelength (the Ed,, of methyl sulfite is about 35 in acetonitrile) . A saturated stock solution of phenyl sulfite (about 0.15 mdl) is prepared by adding 0.2 ml of phenyl sulfite to 125 ml of 0.1 M glycine/0.5 M LiClO, buffer, pH 3.7. The mixture is stirred vigorously for 10 min and filtered twice through three layers of \Vhatman ISo. 1 filter paper. A fresh stock solution is prepared each day for careful work. If relative enzyme activities are satisfactory for a particular experiment, then the substrate
362
STEIiTj
REID,
AND
I’AHRNEY
concentration need not be determined. However, the concentration of phenyl sulfite can be estimated from the amount of phenol that is released upon acid-catalyzed hydrolysis. One milliliter of 6M HCl is added to 5 ml of stock solution, and the absorbance at 270 nm is read after 1 hr at room temperature. The ac.,T0o f hydrolysis was 2.5 + 0.1 X 1CP M-l cm-l. The assay is run by pipetting 3.00 ml of phenyl sulfite stock solution into a cuvet (l-cm light path). The reaction is started by adding 0.100 ml of pepsin solution containing 30 to 300 pg of enzyme, and the production of phenol is followed at 270 nm. RESULTS
AND
DISCUSSION
The pepsin-catalyzed hydrolysis of phenyl sulfite can be monitored at 270 nm. As shown in Fig. 1, the rate of increase of absorbance at 270 nm is a linear function of enzyme concentration. A final concentration of (0.3-3) x 10e6M pepsin is optimal for 5 min assay periods with a Gilford recording spectrophotometer. At 3 X 1O-6M enzyme about 10% of the substrate is hydrolyzed in 5 min. Duplicate assays agree well within 1%. Glycine was chosen as the buffer because the nonenzymic rate of hydrolysis of phenyl sulfite is much slower in this buffer than in other buffers. Lithium perchlorate was added to the buffer solution because it exerts a salting-in effect on sulfite esters, resulting in quicker attainment of a homogeneous solution and higher solubility of the ester. The observed first-order rate constant is 1.2 X 1P mill-l in 0.1 M glycine, as against 5 x 10;” mine1 in 0.1 M acetate, both buffers 0.5 M in LiClO,, pH 3.7, 25”. Thus, the nonenzymic rate of hydrolysis is negligible in the glycine/LiClO, buffer, and no blank correction is necessary. The enzyme
FIG. 1. A plot of rate of increase of absorbance at 270 mm against concentration of pepsin. Initial phenyl sulfite concentration was 1.52 X 10” M.
SPECTROPHOTOMETRIC
ASSAY
FOR
353
PEPSIN
exhibits maximum sulfite esterase activity in the range pH 2 to 4. Below pH 2, enzyme activity decreases rapidly and the acid-catalyzed hydrolysis of sulfite esters becomes measurable. Initial rates of hydrolysis conform to the Michaelis-Rlenten equation:4
uo = E,,,[Slo[E~l,‘(h’m
+ 1%).
A plot of ~,)-l against [S] 0-1 is present’ed in Fig. 2. The steady-state kinetic parameters were calculated by means of a computer program written for
OL
2 [s];’
FIG.
2. vO-’ versus
[SIG
at 25”,
pH
2.71.
4 x lo4 (M-l) Pepsin
6
concentration
was
1.37 X 10.“M.
the Wilkinson (5) weighted form of the Michaelis-Menten equation: values observed for the pepsin used in this work are kcat = 1.3 min-l and Km = 1.0 X 10e4M at 25”, pH 3.7. These values compare well with peptide and depsipeptide substrates for pepsin: lccat = 4.2 min-I, K, = 8.4 x 10e4 M for N-acetyl-L-phenylalanyl-L-diiodotyrosine at pH 4.5 and 37” (6) ; and lCeat= 46 min-I, li,, = 4 x 1tP M for benzyloxycarbonyl-nhistidyl-p-nitro-~-phenylalanyI-~-pl~enyl-~-Iactic acid methyl ester at pH 4.0 and 37” (7). The above values of the steady-state kinet,ic parameters for sulfite ester hydrolysis were obtained with a homogeneous sample of pepsin prepared in this laboratory and represent the highest specific activity observed to date. Several reports indicate that the kinet’ic properties of commercial pepsin differ little from those of homogeneous pepsin. This has been our experience, except that several commercial pepsin lots have had markedly lower specific activities toward ljhenyl sulfite, ranging down to 25%. This variability is generally reflected in Ic,.,,,. ’ [ET1 is the total concentration of enzyme, ester at zero time, k,;,, is the catalytic constant Michaelis constant. The initial velocity v. (dA/dt)oAe.
[Sl, is the or turnover is -(d[Sl/dt)0
concentration of sulfite number. and Km is the = 0.5 (d[Pl/cZt)0 =
364
STEIN,
REID,
AND
FAHRNEY
SUMMARY
Phenyl sulfite is a specific substrate for pepsin, whose hydrolysis can be followed spectrophotometrically. This compound is superior to peptide substrates in its easeof synthesis and its ability to be monitered spectrophotometrically. The sulfite assay is therefore the method of choice for both routine and detailed assays of pepsin. ACKNOWLEDGMENT This work was supported in part by grant BM 13446 and a Postdoctoral (R.P.S.), U. S. Public Health Service.
Fellowship
REFERENCES 1. ANSON. M. L., J. Gen. Phgsiol. 22, 79 (1938). 2. REID, T. W., .~ND FAHRNEY. D., J. Am. Chem. sot. 89, 3941 (1967). 3. PERLMANN, G. E., J. Bid. Chem. 241, 153 (1966). 4. RAJAGOPALAN, R. G., MOORE. S.,, AND STEIN, W. H., J. Bid. Chem. 241, 4295 (1966) 5. WILKINSON, G. N., Biochem. J. 80, 324 (1961). 6. JACKSON, W, T.. SCHLAMOTVITZ, M.. ASD SHAW, A., Biochemistry 4, 1537 (1965) 7. INOUYE, K., AND FRUTON, J. S., J. Am. Chem. Sot. 89, 187 (1967).