In the Laboratory
W
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 nucleophilic catalysis with acid–base assistance and depends on the presence of a catalytic triad (3). Since the enzyme is also relatively inexpensive, it can be used as a tool for teaching kinetic and specificity properties of enzymes in undergraduate experimental courses. It is known that organic solvents affect the catalytic activity of enzymes (4–6 ), including proteases (7, 8). On the other hand, the use of organic solvents may be advantageous to improve the solubility of hydrophobic substrates or to change 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 performed in aqueous–organic media. A number of protocols describing enzyme kinetic experiments for undergraduate students have been published. Here, we suggest a different approach that includes the investigation of effects of a series of alcohols on enzyme catalysis. We also investigated other parameters such as enzyme and substrate concentration, pH, and temperature. Johnson studied catalase activity, employing an ingenious method for measuring enzyme kinetics (11). Cornely et al. proposed an experiment for investigating the hydrolysis of Nα-benzoyl-arginine-p-nitroanilide (BApNA) using papain (12). Other kinetic experiments with trypsin have also been proposed (13). With our experimental protocol, the students are challenged with questions regarding basic concepts of enzyme kinetics and spectrophotometric analyses. They will also be able to discuss the effect of nonaqueous media on enzyme catalysis, which has technological implications. As a project-oriented approach, the protocol provides minimal tutoring and students are encouraged to find the solution for each problem for themselves. The project takes about four months with four hours per week spent in the laboratory. Materials and Methods
Reagents Nα-benzoyl-DL-arginine-p-nitroanilide and p-nitroaniline were purchased from Sigma and Merck. Tris-HCl, glycine hydrochloride, and sodium acetate were analytical grade salts. Milli-Q water was employed. Pancreatic porcine trypsin (E.C. 3.4.21.4) was a Sigma product (13,700 units/mg protein for Nα-benzoyl-arginine ethyl ester hydrolysis). The organic solvents were of analytical grade.
Spectrophotometric Assay Kinetic assays were performed through continuous measurement of p-nitroaniline release from the hydrolysis of BApNA in a Shimadzu UV160A spectrophotometer with controlled cell temperature. The initial velocities were obtained from the slopes of the absorbance versus time plot. Enzymatic reaction rates (mmol min᎑1 L᎑1) were obtained after dividing the absorbance slopes by the product molar extinction coefficient. Parameter Effects on Substrate Hydrolysis Rate BApNA Concentration A stock BApNA solution (4.05 × 10᎑3 mol L᎑1) was prepared 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 buffer and 15 µL of a trypsin solution (1.0 mg mL᎑1 in HCl 1.0 × 10 ᎑3 mol L᎑1) were added to the spectrophotometer cuvette. After temperature equilibration (5 min, 37 °C), different volumes of the BApNA solution [(400 – x) µL)] and DMSO solution (25%, x µL) were added to keep co-solvent concentration constant. Absorbance increments at 410 nm were recorded for 10 min. Reaction rates were obtained as described previously. Data were plotted according to the equation of Michaelis–Menten, as the double reciprocal plot (Lineweaver–Burk plot), and as the Hanes plot ([S]/v vs [S]). Temperature After the addition of buffer (1.0 mL of 40 mmol L᎑1 TrisHCl, pH 7.4, containing 20 mmol L᎑1 CaCl2), and enzyme solution (15 µL of 1.0 mg mL᎑1 in HCl 1.0 × 10᎑3 mol L᎑1) the temperature was equilibrated (15 to 70 °C). Then 200 µL of the 4.05 × 10᎑3 mol L᎑1 BApNA solution (in the buffer containing 25% DMSO) was added and absorbance values were recorded at 410 nm up to 15 min. Reaction rates were obtained as described previously. pH 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, 3.0 to 9.0. The pH values were adjusted with HCl or NaOH. After addition of buffer and enzyme solution (15 µL of 1.0 mg mL᎑1 in HCl 1.0 × 10᎑3 mol L᎑1) and after temperature equilibration, we added 200 µL of the 4.05 × 10᎑3 mol L᎑1 BApNA solution (prepared in the respective buffers containing 25% DMSO). Absorbance values at 410 nm were recorded up to 15 min.
JChemEd.chem.wisc.edu • Vol. 78 No. 11 November 2001 • Journal of Chemical Education
1535
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 mmol L᎑1 CaCl2 and 4% DMSO, or in 35% aqueous alcohol (containing 4% DMSO). Aliquots of this solution (10–200 µL) were added to the spectrophotometer cell and the volume made up to 1.4 mL with the same buffer or alcohol solution. Absorbance values were recorded at 410 nm to furnish the ε ([1/mol L᎑1] cm᎑1) values. Each experiment was repeated at least three times.
0.4
0.3
0.2
0.1
0.0 0.0
In the first experiment we determined the enzyme concentration to be used in the following investigations. The value of 1.2 × 10᎑2 mg mL᎑1 was chosen because it resulted in a linear relationship of the reaction rate with time for 5 min even for low BApNA concentration. To simplify the procedure, active-site titration was not performed (15). The dependence of reaction rate on substrate concentration 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 ± 0.44 mmol L᎑1 and 1.12 ± 0.35 mmol L᎑1 min᎑1 for Km and Vmax, respectively. 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. For instance, the distribution of errors is more uniform in the Hanes plot than in the Lineweaver–Burk plot.
1536
0.4
0.6
0.8
1.0
1.2
6
B 5
4
3
2
1
0 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
(1/[N-benzoyl-arginine-p-nitroanilide]) / (L mmol ᎑1) 3.0
C 2.5
([S]/V) / (103 min)
Results and Discussion
0.2
[N-benzoyl-arginine-p-nitroanilide] / (mmol L᎑1)
Hazards 1-Propanol is mildly irritating to eyes and mucous membranes. Ingestion or inhalation of large quantities of 2-propanol may cause flushing, headache, dizziness, mental depression, nausea, vomiting, narcosis, anaesthesia, and coma. Methanol is very toxic from ingestion and can lead to visual impairment or complete blindness. Dimethyl sulfoxide is irritating through skin contact.
A
0.5
(1/Reaction Rate) / (min L µmol ᎑1)
Organic Co-solvent Solutions of methanol, ethanol, 1-propanol, and 2propanol at 35% in 40 mmol L᎑1 Tris-HCl buffer, pH 7.4 (with 20 mmol L᎑1 CaCl2 and 4% DMSO) were prepared. 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 by addition 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 respective molar extinction coefficient (Table 1). Error propagation calculations were performed (14).
Reaction Rate / (µmol min᎑1 L᎑1)
In the Laboratory
2.0
1.5
1.0
0.5
0.0 0.2
0.4
0.6
0.8
1.0
1.2
(1/[N-benzoyl-arginine-p-nitroanilide]) / (L mmol ᎑1)
Figure 1. A: Michaelis–Menten, B: Lineweaver–Burk, and C: Hanes plots of hydrolysis of BApNA catalyzed by trypsin.
Table 1. Molar Extinction Coefficients Values and Reaction Rates for Tr ypsin-Catalyzed Hydrolysis of BApNA ε/M᎑1 cm᎑1
Reaction Rate/ µmol min᎑1 L᎑1
Tris-HCl buffer
7,680 ± 350
0.253 ± 0.023
Methanol
7,810 ± 700
0.200 ± 0.037
Ethanol
8,760 ± 840
0.122 ± 0.017
1-Propanol
8,950 ± 580
0.161 ± 0.043
2-Propanol
7,570 ± 990
0.164 ± 0.026
Solvent
Journal of Chemical Education • Vol. 78 No. 11 November 2001 • JChemEd.chem.wisc.edu
In the Laboratory
The optimum pH value determined was 8.0, which corresponds to the expected value (1, 2). The optimum temperature was approximately 40 °C. As pointed out by Jaenicke (16 ), the optimum temperature for enzyme catalysis depends on the thermodynamic stability of the enzyme and not 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 active at 40 °C and pH 7.4. 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 may cause enzyme inactivation (18). The enzyme maintained its catalytic activity in the presence of all four alcohols (Table 1). Nevertheless, the reaction rate was reduced in the presence of methanol and further reduced by ethanol, 1-propanol, and 2-propanol. Since polar organic solvents are usually harmful to protein structure (4–6 ), it was surprising to find that methanol had the least effect on enzyme activity. The denaturing effect of alcohols on trypsin has been reported by others in the following order: methanol < ethanol < 2-propanol < 1-propanol (18 ). According to these studies, at 35% alcohol volume, only 1-propanol causes denaturation of trypsin. Simon showed that trypsin activity is not significantly affected by the presence of organic co-solvents in volume percentages up to 80% (19). Thus, the students will learn that enzyme studies in organic media are not straightforward and that controversial data have been reported. To obtain correct rate values, the molar extinction coefficients were calculated in the presence of each alcohol as shown in Table 1. The students may be asked which parameters may affect the molar extinction coefficient. The ε values seem to be higher in the presence of linear-chain alkyl alcohols than in buffer. Values were recorded after temperature equilibration because the ε value of p-nitroaniline varied with temperature, especially in the presence of alcohols. Suggestions for Further Experiments One. Students may learn how to determine the real concentration of trypsin by active-site titration with nitrophenyl p-guanidinobenzoate (15). Two. The effect of organic solvents may be studied in different ways. For example, the effect of incubating the enzyme in organic media on its stability can be evaluated. Also, Vmax and Km values can be determined in the presence of organic solvents.
Three. Commercial trypsin is a mixture of enzyme molecules. β-Trypsin, for instance, can be purified in one step by ion-exchange chromatography (20) and then kinetic data can be obtained using a molecularly defined catalyst. WSupplemental
Material
Some further experimental observations and data are available in this issue of JCE Online. Literature Cited 1. Beynon, R. J.; Bond, J. S. Proteolytic Enzymes: a Practical Approach; Oxford University Press: Oxford, 1989. 2. Johnson, A.; Gautham, N.; Pattabhi, V. Biochim. Biophys. Acta 1999, 1435, 7–21. 3. Dodson, G.; Wlodawer, A. Trends Biochem. Sci. 1998, 23, 347–352. 4. Halling, P. J. Enzyme Microb. Technol. 1994, 16, 178–206. 5. Klibanov, A. M. Trends Biotechnol. 1997, 15, 97–101. 6. Carrea, G.; Riva, S. Angew. Chem., Int. Ed. Engl. 2000, 22, 2226–2254. 7. Bemquerer, M. P.; Adlercreutz, P.; Tominaga, M. Int. J. Pept. Prot. Res. 1994, 44, 448–456. 8. Wangikar, P. P.; Rich, J. O.; Clark, D. S.; Dordick, J. S. J. Am. Chem. Soc. 1995, 34, 12302–12310. 9. Partridge, J.; Moore, B. D.; Halling, P. J. J. Mol. Catal. B 1999, 6, 11–20. 10. Gill, I.; López-Fandiño, R.; Jorba, X.; Vulfson, E. N. Enzyme Microb. Technol. 1996, 18, 162–183. 11. Johnson, A. K. A. J. Chem. Educ. 2000, 77, 1451–1452. 12. Cornely, K.; Crespo, E.; Earley, M.; Kloter, R.; Levesque, A.; Pickering, M. J. Chem. Educ. 1999, 76, 644–646. 13. Anderson, J.; Byrne, T.; Woelfel, K. J.; Meany, J. E.; Spyridis, G. T.; Pocker, Y. J. Chem. Educ. 1994, 71, 715–718. 14. Bevington, P. R. Data Reduction and Error Analysis for the Physical Sciences; McGraw-Hill: New York, 1969. 15. Chase, T.; Shaw, E. Method. Enzymol. 1974, 19, 20–27. 16. Jaenicke, R. Prog. Biophys. Mol. Biol. 1999, 71, 155–241. 17. Santoro, M. M. Unfolding of β-Trypsin at pH 3.0; Presented at International Symposium on Calorimetry and Chemical Thermodynamics; Campinas, S. P., Brazil, 1998. 18. Khmelnitsky, Y. L.; Mozhaev, V. V.; Belova, A. B.; Sergeeva, M. V.; Martinek, K. Eur. J. Biochem. 1991, 198, 31–41. 19. Simon, L. M.; László, K.; Vértesi, A.; Bagi, K.; Szajáni, B. J. Mol. Catal. B 1998, 4, 41–45. 20. Dias, C. L.; Rogana, E. Braz. J. Med. Biol. Res. 1986, 19, 11–18.
JChemEd.chem.wisc.edu • Vol. 78 No. 11 November 2001 • Journal of Chemical Education
1537
