The Development of Autocatalytic Structural Materials for ...
Autocatalytic activation of trypsinogen by trypsin
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Autocatalytic Activation of Trypsinogen by Trypsin Salvatore F. Russo and Erin T. Wahl Western Washington University, Bellingham. WA 98225 This 3-hour experiment provides an example of the bio- logically important process of zymogen activation. It utilizes an active site titrant for trypsin based on the acyl enzyme mechanism for this enzyme. The activation of an inactive zymogen to a functioning enzyme is important in digestion, blood coagulation, fibrino- lysis, complement activation, hormone production, meta- morphosis, fertilization,,and supramolecular assembly (I). The activation process provides a prompt and irreversible response in these systems. The zvmoeens secreted hv the nancreas durine dieestion have been w i l characterized: ~ he~b~siolo~ical a&&on of trvosinoeen is initiated bv entero~entidase. This enzvme is se"&etedfrom the brush border ofthe small intestine where- as trvosinogen orkinates in the acinar cells of the Dancreas. ~hui,.the Gte of generation of active trypsin is r e s h . e d to the confluence of these two secretory streams. Active trypsin can then act as the catalyst in the limited hydroly& of chymotrypsinogen and procarhoxypeptidase. Trypsin is specific for the hydrolysis of peptide bonds when the carhon- yl is contributed by either a lysine or an arginine residue (2). In this experiment, the activation of trypsinogen is cata- lyzed by added trypsin H20 + ~rypsinogen- Trypsin + Hexapeptide In an autocatalytic process, the reaction is catalyzed by one of the products. In this case, trypsin is produced in the reaction as well as initiating the process (3). Activity is moni- tored as a function of time using the ester p-nitrophenyl-pr- guanidinohenzoate (NPGB). This synthetic substrate is bound and hydrolyzed by trypsin due to its guanidino group which is also found in an arginine residue of a natural sub- strate. NPGB is hydrolyzed according to the acyl enzyme mechanism (4) where E = enzyme, S = substrate (NPGB), ES = Michaelis- Menten complex, ES' = acyl enzyme, PI = p-nitrophenolate, and P, = p-guanidinobenzoate. The burst of p-nitropheno- late can he monitored spectrophotometrically (5). This yields a direct measure of the stoichiometric, active enzyme concentration provided the rate constant for deacylation is much smaller than that for acylation and also that KM(.~~ .... ~) << [S]bbi (4). The absolute standard in an active site titration is the small organic titrant (NPGB) rather than the enzyme which must be the absolute standard in a rate assay procedure (6). Experlrnenial SoMions Needed Trypsmugrrt. 1)issolve 75.0 mi: trypsinogen (H'orthington Bio- chemical Cwp) in 25.0 ml. of 0.001 Al llCl netermrne the roncen- tratim hased on s specific nhsrrptivity of 1.39 cm-' ml. me (71 at 280 nm. Trypsin: Dissolve 10.0 mg trypsin (Worthington Biochemical Corp) in 10.0 mL of 0.001 M HCI. Determine the concentration hased on a specific absorptivity of 1.44 cm-' mL/mg (7) at 280 nm. Concentrated buffer for actiuation: 0.20 M tris(hydroxymethyl)- aminomethane and 0.04 MCaClt at pH 8.3. ~. Dilure Rullrrfw Asoy 6.10 .U tristh~droxymethyllaminometh- ane and 0.02 A!CaCl? at pH 8.3. Subrrrore ni,nolw 6.8 mg p-n~tmphenyl-p'-~uanidinobenzc#ate hydrorhlwide INurritional Hiochemicals Corp.) in '1 0 ml. S,N-di- methylformamide. Soluent for Reference Cuvette: 0.001MHC1-0.02 MCaClz Activation Mix Add 10.0 mL of the trypsinogen solution to 11.0 mL concentrated buffer. The inclusion of Ca2+in the buffer improves the yield of trypsin (8). A timer should be started concurrent with the addition of 1.0 mL trypsin solution. Enzyme Assay The following procedure is a modification of that proposed by Chase and Shaw (5): Pipet 1.000mL from the activation mix into the sample cuvette followed by 1.000 mL dilute buffer. Mix by covering the cuvette with Parafilm and inverting the covered cuvette. Pipet 1.000mLofO.OOlMHC1-0.02MCaClzand 1.000 mLof dilute buffer into the reference cuvette. Mix again by inverting the covered cu- vette. Balance the double beam spectrophotometer (Cary 14)at Am = 0. Then add 25 aL of substrate salution to the reference cuvette with mixing. The reaction is started by adding 25 aL of substrate solution to the sample cuvette witb mixing. Record the initial Am and time elapsed since the addition of trypsin to trypsinogen. The use of a double beam spectrophotometer witb sample and reference euvettes automatically corrects for nonenzymatic hydrolysis. In classical experiments, the activation mix is kept at 0' and assay is performed at 25'. However, we have chosen to do both at 25' in order to maintain a simpler experimental protocol. Results The figure shows A410 as a function of time in the antocat- alytic activation of trypsinogen and trypsin. The point a t zero time was obtained in a control experiment in which the 0.001 M HC1 was substituted for the trvpsinopen in the . . - activation mix. The stock trypsinogen solution was 3.00 mg/mL (by weight) but only 2.58 mg/mL based on absorbance at 280 nm (9). Similarly, the trypsin solution was 1.0 mgImL (by weight) but only 0.9 mg/mL based on absorbance at 280 nm. If all the trypsinogen were converted to trypsin, we would expect 26.7 mg of active trypsin or TIME (MIN) Autocatalytic activation of trypsinogen. A,lo due to the bmt of pnitropheno- late is measured as a function of activation time. Volume 64 Number 1 January 1987 83
Transcript of Autocatalytic activation of trypsinogen by trypsin
Autocatalytic Activation of Trypsinogen by Trypsin Salvatore F. Russo and Erin T. Wahl Western Washington University, Bellingham. WA 98225
This 3-hour experiment provides an example of the bio- logically important process of zymogen activation. It utilizes an active site titrant for trypsin based on the acyl enzyme mechanism for this enzyme.
The activation of an inactive zymogen to a functioning enzyme is important in digestion, blood coagulation, fibrino- lysis, complement activation, hormone production, meta- morphosis, fertilization,,and supramolecular assembly ( I ) . The activation process provides a prompt and irreversible response in these systems.
The zvmoeens secreted hv the nancreas durine dieestion have been w i l characterized: ~ h e ~ b ~ s i o l o ~ i c a l a&&on of trvosinoeen is initiated bv entero~entidase. This enzvme is se"&etedfrom the brush border ofthe small intestine where- as trvosinogen orkinates in the acinar cells of the Dancreas. ~hui , . the Gte of generation of active trypsin is r e s h . e d to the confluence of these two secretory streams. Active trypsin can then act as the catalyst in the limited hydroly& of chymotrypsinogen and procarhoxypeptidase. Trypsin is specific for the hydrolysis of peptide bonds when the carhon- yl is contributed by either a lysine or an arginine residue (2).
In this experiment, the activation of trypsinogen is cata- lyzed by added trypsin
H20 + ~rypsinogen- Trypsin + Hexapeptide
In an autocatalytic process, the reaction is catalyzed by one of the products. In this case, trypsin is produced in the reaction as well as initiating the process (3). Activity is moni- tored as a function of time using the ester p-nitrophenyl-pr- guanidinohenzoate (NPGB). This synthetic substrate is bound and hydrolyzed by trypsin due to its guanidino group which is also found in an arginine residue of a natural sub- strate. NPGB is hydrolyzed according to the acyl enzyme mechanism (4)
where E = enzyme, S = substrate (NPGB), ES = Michaelis- Menten complex, ES' = acyl enzyme, PI = p-nitrophenolate, and P, = p-guanidinobenzoate. The burst of p-nitropheno- late can he monitored spectrophotometrically (5). This yields a direct measure of the stoichiometric, active enzyme concentration provided the rate constant for deacylation is much smaller than that for acylation and also that
KM(.~~.. . .~) << [S]bbi (4). The absolute standard in an active site titration is the small organic titrant (NPGB) rather than the enzyme which must be the absolute standard in a rate assay procedure (6).
Experlrnenial
SoMions Needed Trypsmugrrt. 1)issolve 75.0 mi: trypsinogen (H'orthington Bio-
chemical Cwp) in 25.0 ml. of 0.001 Al llCl netermrne the roncen- tratim hased on s specific nhsrrptivity of 1.39 cm-' ml. me ( 7 1 at 280 nm.
Trypsin: Dissolve 10.0 mg trypsin (Worthington Biochemical Corp) in 10.0 mL of 0.001 M HCI. Determine the concentration hased on a specific absorptivity of 1.44 cm-' mL/mg (7) at 280 nm.
Concentrated buffer for actiuation: 0.20 M tris(hydroxymethyl)- aminomethane and 0.04 MCaClt at pH 8.3. ~.
Dilure R u l l r r f w A s o y 6.10 .U tristh~droxymethyllaminometh- ane and 0.02 A!CaCl? at pH 8.3.
Subrrrore ni,nolw 6.8 mg p-n~tmphenyl-p'-~uanidinobenzc#ate hydrorhlwide INurritional Hiochemicals Corp.) in '1 0 ml. S,N-di- methylformamide.
Soluent for Reference Cuvette: 0.001 MHC1-0.02 MCaClz
Activation Mix Add 10.0 mL of the trypsinogen solution to 11.0 mL concentrated
buffer. The inclusion of Ca2+ in the buffer improves the yield of trypsin (8). A timer should be started concurrent with the addition of 1.0 mL trypsin solution.
Enzyme Assay The following procedure is a modification of that proposed by
Chase and Shaw (5): Pipet 1.000mL from the activation mix into the sample cuvette followed by 1.000 mL dilute buffer. Mix by covering the cuvette with Parafilm and inverting the covered cuvette. Pipet 1.000mLofO.OOlMHC1-0.02MCaClzand 1.000 mLof dilute buffer into the reference cuvette. Mix again by inverting the covered cu- vette. Balance the double beam spectrophotometer (Cary 14) at A m = 0. Then add 25 aL of substrate salution to the reference cuvette with mixing. The reaction is started by adding 25 aL of substrate solution to the sample cuvette witb mixing. Record the initial A m and time elapsed since the addition of trypsin to trypsinogen. The use of a double beam spectrophotometer witb sample and reference euvettes automatically corrects for nonenzymatic hydrolysis. In classical experiments, the activation mix is kept at 0' and assay is performed at 25'. However, we have chosen to do both at 25' in order to maintain a simpler experimental protocol.
Results The figure shows A410 as a function of time in the antocat-
alytic activation of trypsinogen and trypsin. The point a t zero time was obtained in a control experiment in which the 0.001 M HC1 was substituted for the trvpsinopen in the . . - activation mix.
The stock trypsinogen solution was 3.00 mg/mL (by weight) but only 2.58 mg/mL based on absorbance a t 280 nm (9). Similarly, the trypsin solution was 1.0 mgImL (by weight) but only 0.9 mg/mL based on absorbance a t 280 nm. If all the trypsinogen were converted to trypsin, we would expect 26.7 mg of active trypsin or
TIME (MIN)
Autocatalytic activation of trypsinogen. A,lo due to the b m t of pnitropheno- late is measured as a function of activation time.
Volume 64 Number 1 January 1987 83
maximum trypsin concentration at the conclusion of activa- 0.30 - X 100 = 71%
tion. The maximum possible absorbance a t 410 nm would be 0.42
of the trypsinogen was activated. 1.000 mL
Max A,,n = (16,600 M-' em-') (1 cm) - 2.025 mL
(5.10 x M) Literature Cited (1) Neursth, H.: Walsh, K. A.Pmr. Norl. Acod. Sci. USA 1976.73.38'25. (2) Neursth, H.: Schwert G. W. Chem. Re". 1950,46,69. (3) Pechere J.-F.; Neursth. H. J. B i d Chem. 1957,229,389.
where 1.000 mLl2.025 mL represents the dilution factor and ce ~ ~ ~ d ~ ~ , M. L.; K O Z ~ Y . F. J.: wedler, F. C. J them. E ~ U C . 1967.44, M. 16,600 M-1 cm-l is the molar absorptivity of pnitropheno- (5) Chase. T.. Jr.; Shaw. E. Biochom. Biophy. Re- Commun. 1967,29,508.
(6) Bender, M. L.; Begue-Cantbn. M. L.; Blakeley, R. L., Brubscher. L. J.: Feder, J.: late (5). Therefore, maximum A d l o = 0.42. The control ex- G~~~~~.C.R.;K~~~~,F.J.:K~II~~~~~~.J.V.,J~.;M~~~~II,T.H.;M~II.~,C.G.:R~~, periment takes into account the amount of trypsin originally R. w . : s toop , J. K. J. ~ m e r . chem. sot. 1 9 6 6 , ~ . 5890.
added to the activation mix. Hence, from the figure A A m = (7) Davie, E. W.; Neurath, H. J. B i d . Chem. 1955,212,515. (8) McDonald, M. R.; Kunitz, M. J. Gan. Phyaiol. 1941.25.53.
0.32 - 0.02 = 0.30. Finally, we calculate that 19) R W ~ O , S. F.; nohrnan, T. J. ckm. E ~ U C . 1 ~ 7 , 5 4 , 6 0 .
84 Journal of Chemical Education
