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P1535

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; *mpbemque@icb.ufmg.br
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 3.4.21.4) 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% Literature Cited
(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 1999, 1435, 7–21.
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 the Physical 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 p-guanidinobenzoate (15).
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.

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