In the Laboratory The Effect of Organic Solvents and Other Parameters on Trypsin-Catalyzed Hydrolysis of N␣-Benzoyl-arginine-p-nitroanilide
A Project-Oriented Biochemical Experiment
L. C. Correia, A. C. Bocewicz, S. A. Esteves, M. G. Pontes, L. M. Versieux, S. M. R. Teixeira, M. M. Santoro, and M. P. Bemquerer* Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 6627 Belo Horizonte-M.G. 31270-910, Brazil; *[email protected]
Trypsin is a serine protease that has both amidase and
esterase activity (1–3). Its mechanism of action is based on a
Kinetic assays were performed through continuous mea-
nucleophilic catalysis with acid–base assistance and depends
surement of p-nitroaniline release from the hydrolysis of
on the presence of a catalytic triad (3). Since the enzyme is
BApNA in a Shimadzu UV160A spectrophotometer with
also relatively inexpensive, it can be used as a tool for teaching
controlled cell temperature. The initial velocities were ob-
kinetic and specificity properties of enzymes in undergraduate
tained from the slopes of the absorbance versus time plot.
Enzymatic reaction rates (mmol min᎑1 L᎑1) were obtained
It is known that organic solvents affect the catalytic
after dividing the absorbance slopes by the product molar
activity of enzymes (4–6 ), including proteases (7, 8). On the
other hand, the use of organic solvents may be advantageous
Parameter Effects on Substrate Hydrolysis Rate
to improve the solubility of hydrophobic substrates or tochange the equilibrium constant of hydrolytic reactions (7–9).
Furthermore, some large-scale enzymatic processes such as
the production of aspartame and other peptides (10) are per-
in 40 mmol L᎑1 Tris-HCl buffer, pH 7.4 (containing 20 mmol
L᎑1 CaCl2 and 25% DMSO solution). One milliliter of the
A number of protocols describing enzyme kinetic experi-
buffer and 15 µL of a trypsin solution (1.0 mg mL᎑1 in HCl
ments for undergraduate students have been published. Here,
1.0 × 10᎑3 mol L᎑1) were added to the spectrophotometer
we suggest a different approach that includes the investigation
cuvette. After temperature equilibration (5 min, 37 °C),
of effects of a series of alcohols on enzyme catalysis. We also
different volumes of the BApNA solution [(400 – x) µL)] and
investigated other parameters such as enzyme and substrate
DMSO solution (25%, x µL) were added to keep co-solvent
concentration, pH, and temperature. Johnson studied catalase
concentration constant. Absorbance increments at 410 nm
activity, employing an ingenious method for measuring enzyme
were recorded for 10 min. Reaction rates were obtained as
kinetics (11). Cornely et al. proposed an experiment for inves-
described previously. Data were plotted according to the equa-
tigating the hydrolysis of Nα-benzoyl-arginine-p-nitroanilide
tion of Michaelis–Menten, as the double reciprocal plot
(BApNA) using papain (12). Other kinetic experiments with
(Lineweaver–Burk plot), and as the Hanes plot ([S]/v vs [S]).
trypsin have also been proposed (13). With our experimental
protocol, the students are challenged with questions regarding
After the addition of buffer (1.0 mL of 40 mmol L᎑1 Tris-
basic concepts of enzyme kinetics and spectrophotometric
HCl, pH 7.4, containing 20 mmol L᎑1 CaCl
analyses. They will also be able to discuss the effect of non-
solution (15 µL of 1.0 mg mL᎑1 in HCl 1.0 ×
aqueous media on enzyme catalysis, which has technological
the temperature was equilibrated (15 to 70 °C). Then 200
implications. As a project-oriented approach, the protocol
provides minimal tutoring and students are encouraged to find
10᎑3 mol L᎑1 BApNA solution (in the buffer
containing 25% DMSO) was added and absorbance values
the solution for each problem for themselves. The project takes
were recorded at 410 nm up to 15 min. Reaction rates were
about four months with four hours per week spent in the
Materials and Methods
To avoid specific effects due to buffer salts, the same
buffer composition (0.2 mol L᎑1 glycine, 0.2 mol L᎑1 acetate,
and 0.2 mol L᎑1 Tris) was employed for the pH range studied,
Nα-benzoyl-DL-arginine-p-nitroanilide and p-nitroaniline
3.0 to 9.0. The pH values were adjusted with HCl or NaOH.
were purchased from Sigma and Merck. Tris-HCl, glycine
After addition of buffer and enzyme solution (15 µL of 1.0
hydrochloride, and sodium acetate were analytical grade salts.
mg mL᎑1 in HCl 1.0 × 10᎑3 mol L᎑1) and after temperature
Milli-Q water was employed. Pancreatic porcine trypsin (E.C.
equilibration, we added 200 µL of the 4.05 × 10᎑3 mol L᎑1
126.96.36.199) was a Sigma product (13,700 units/mg protein for
BApNA solution (prepared in the respective buffers containing
Nα-benzoyl-arginine ethyl ester hydrolysis). The organic solvents
25% DMSO). Absorbance values at 410 nm were recorded
In the Laboratory
Solutions of methanol, ethanol, 1-propanol, and 2-
propanol at 35% in 40 mmol L᎑1 Tris-HCl buffer, pH 7.4(with 20 mmol L᎑1 CaCl
BApNA solutions (4.05 × 10᎑3 mol L᎑1) were prepared in these
alcoholic solvents. One milliliter of alcohol solution and 15
µL of trypsin solution (1.0 mg mL᎑1 in HCl 1.0 × 10᎑3 mol
L᎑1) were added to the cuvette. The reactions were started byaddition of 200 µL of the 4.05 × 10᎑3 mol L᎑1 BApNA stock
solution. Enzymatic reaction rates (mmol min᎑1 mL᎑1) were
obtained after dividing the absorbance slopes by the respec-
tive molar extinction coefficient (Table 1). Error propagation
[N-benzoyl-arginine-p-nitroanilide] / (mmol L᎑1)
calculations were performed (14).
Determination of Molar Extinction Coefficient
A p-nitroaniline solution (4.5 × 10᎑4 mol L᎑1) was prepared
in 40 mmol L᎑1 Tris-HCl buffer, pH 7.4, containing 20 mmolL᎑1 CaCl
2 and 4% DMSO, or in 35% aqueous alcohol (con-
taining 4% DMSO). Aliquots of this solution (10–200 µL)
were added to the spectrophotometer cell and the volumemade up to 1.4 mL with the same buffer or alcohol solu-
tion. Absorbance values were recorded at 410 nm to furnishthe ε ([1/mol L᎑1] cm᎑1) values. Each experiment was repeated
(1/[N-benzoyl-arginine-p-nitroanilide]) / (L mmol᎑1)
1-Propanol is mildly irritating to eyes and mucous
membranes. Ingestion or inhalation of large quantities of
2-propanol may cause flushing, headache, dizziness, mentaldepression, nausea, vomiting, narcosis, anaesthesia, and coma.
Methanol is very toxic from ingestion and can lead to visual
impairment or complete blindness. Dimethyl sulfoxide is
Results and Discussion
In the first experiment we determined the enzyme con-
centration to be used in the following investigations. The
value of 1.2 × 10᎑2 mg mL᎑1 was chosen because it resulted
(1/[N-benzoyl-arginine-p-nitroanilide]) / (L mmol᎑1)
in a linear relationship of the reaction rate with time for 5 mineven for low BApNA concentration. To simplify the procedure,
Figure 1. A: Michaelis–Menten, B: Lineweaver–Burk, and C: Hanes
active-site titration was not performed (15).
plots of hydrolysis of BApNA catalyzed by trypsin.
The dependence of reaction rate on substrate concen-
tration was analyzed and the values of Km (1.20 ± 0.41 mmol
L᎑1) and Vmax (1.05 ± 0.23 mmol L᎑1 min᎑1) were calculated
by nonlinear regression of the Michaelis–Menten graph shown
in Figure 1A. These values can be compared to results obtained
with the Lineweaver–Burk plot, which are 1.31 ±
L᎑1 and 1.12 ± 0.35 mmol L᎑1 min᎑1 for K
tively. The Hanes plot provided values of 1.05 ± 0.24 mmol L᎑1
and 0.96 ± 0.18 mmol L᎑1 min᎑1 for Km and Vmax, respectively
(Figs. 1B and 1C). The source of the differences among
the three approaches may be discussed with the students. Forinstance, the distribution of errors is more uniform in the
Hanes plot than in the Lineweaver–Burk plot. In the Laboratory
The optimum pH value determined was 8.0, which
Three. Commercial trypsin is a mixture of enzyme mol-
corresponds to the expected value (1, 2). The optimum
ecules. β-Trypsin, for instance, can be purified in one step
temperature was approximately 40 °C. As pointed out by
by ion-exchange chromatography (20) and then kinetic data
Jaenicke (16 ), the optimum temperature for enzyme catalysis
can be obtained using a molecularly defined catalyst.
depends on the thermodynamic stability of the enzyme andnot on the physiological temperature. Data from our group
reveal that the denaturation temperature (Tm) for β-trypsin
is 54 °C at pH 3.0 (17). Thus, the enzyme is expected to be
Some further experimental observations and data are
available in this issue of JCE Online.
To investigate the effect of alcohols on trypsin activity,
the final co-solvent concentration was kept constant at 35%
(by volume), since higher concentration of the alcohols maycause enzyme inactivation (18). The enzyme maintained its
1. Beynon, R. J.; Bond, J. S. Proteolytic Enzymes: a Practical Approach;
catalytic activity in the presence of all four alcohols (Table 1).
Oxford University Press: Oxford, 1989.
Nevertheless, the reaction rate was reduced in the presence
2. Johnson, A.; Gautham, N.; Pattabhi, V. Biochim. Biophys. Acta
of methanol and further reduced by ethanol, 1-propanol, and
2-propanol. Since polar organic solvents are usually harmful
3. Dodson, G.; Wlodawer, A. Trends Biochem. Sci.1998,23,
to protein structure (4–6 ), it was surprising to find that
methanol had the least effect on enzyme activity. The dena-
4. Halling, P. J. Enzyme Microb. Technol. 1994,16, 178–206.
turing effect of alcohols on trypsin has been reported by others
5. Klibanov, A. M. Trends Biotechnol.1997,15, 97–101.
in the following order: methanol < ethanol < 2-propanol <
6. Carrea, G.; Riva, S. Angew. Chem., Int. Ed. Engl.2000,22,
1-propanol (18 ). According to these studies, at 35% alcohol
volume, only 1-propanol causes denaturation of trypsin.
7. Bemquerer, M. P.; Adlercreutz, P.; Tominaga, M. Int. J. Pept.
Simon showed that trypsin activity is not significantly affected
Prot. Res. 1994, 44, 448–456.
by the presence of organic co-solvents in volume percentages
8. Wangikar, P. P.; Rich, J. O.; Clark, D. S.; Dordick, J. S. J. Am.
up to 80% (19). Thus, the students will learn that enzyme
Chem. Soc.1995,34, 12302–12310.
studies in organic media are not straightforward and that
9. Partridge, J.; Moore, B. D.; Halling, P. J. J. Mol. Catal. B 1999,
controversial data have been reported.
To obtain correct rate values, the molar extinction coef-
10. Gill, I.; López-Fandiño, R.; Jorba, X.; Vulfson, E. N. Enzyme
ficients were calculated in the presence of each alcohol as
Microb. Technol.1996,18, 162–183.
shown in Table 1. The students may be asked which param-
11. Johnson, A. K. A. J. Chem. Educ.
eters may affect the molar extinction coefficient. The ε val-
12. Cornely, K.; Crespo, E.; Earley, M.; Kloter, R.; Levesque, A.;
ues seem to be higher in the presence of linear-chain alkyl
alcohols than in buffer. Values were recorded after temperature
13. Anderson, J.; Byrne, T.; Woelfel, K. J.; Meany, J. E.;
equilibration because the ε value of p-nitroaniline varied with
Spyridis, G. T.; Pocker, Y. J. Chem. Educ. 1994,71, 715–718.
temperature, especially in the presence of alcohols.
14. Bevington, P. R. Data Reduction and Error Analysis for thePhysical Sciences; McGraw-Hill: New York, 1969. Suggestions for Further Experiments
15. Chase, T.; Shaw, E. Method. Enzymol.1974,19, 20–27. 16. Jaenicke, R. Prog. Biophys. Mol. Biol.1999,71, 155–241. One. Students may learn how to determine the real con-
17. Santoro, M. M. Unfolding of β-Trypsin at pH 3.0; Presented
centration of trypsin by active-site titration with nitrophenyl
at International Symposium on Calorimetry and Chemical
Thermodynamics; Campinas, S. P., Brazil, 1998. Two. The effect of organic solvents may be studied in
18. Khmelnitsky, Y. L.; Mozhaev, V. V.; Belova, A. B.; Sergeeva, M. V.;
different ways. For example, the effect of incubating the en-
Martinek, K. Eur. J. Biochem. 1991,198, 31–41.
zyme in organic media on its stability can be evaluated. Also,
19. Simon, L. M.; László, K.; Vértesi, A.; Bagi, K.; Szajáni, B. Vmax and Km values can be determined in the presence of
J. Mol. Catal. B 1998, 4, 41–45.
20. Dias, C. L.; Rogana, E. Braz. J. Med. Biol. Res. 1986,19, 11–18.
15th International Conference on Behçet’s Disease POSTER PROGRAM （ Friday, July 13 ） Poster Session 1 Immunology and Genetics P1-1-001 Behçet's Disease and HLA A26 Zemfira S Alekberova, M Krylov, Regina Goloeva, J Guseva, Evgenii L Nasonov Research Institute of Rheumatology, Moscow, Russia P1-1-002 Correlation between plasma homocysteine level and HLA-B51 in patients