A comparative study was performed on the conformational stabilities of trypsin and a-chymotrypsin. At 45ºC, trypsin was most stable at pH 3, while the highest stability of a -chymotrypsin was observed at pH 5. With both ester and amide substrates, trypsin displayed activation at pH 3. In the case of a-chymotrypsin, activation was detected at pH 5 only with the amide substrate. The time curves of heat inactivation were complex. For both enzymes, autolysis proceeded with the highest velocity at pH 8. The results obtained on a-chymotrypsin suggested consecutive reactions: the first step, heat denaturation of the protein, is followed by digestion of the damaged molecules

Lehoczkiné Simon, Mária

English

Lehoczkiné Simon Mária

University of Szeged

Volume 45(1-4):43-49, 2001Acta Biologica Szegediensishttp://www.sci.u-szeged.hu/ABSARTICLEDepartment of Biochemistry, University of Szeged, Szeged, HungaryA comparative study of the conformational stabilities oftrypsin and -chymotrypsinMária L. Simon*, Kinga László, Márta Kotormán, Béla SzajániABSTRACT                        A comparative study was performed on the conformational stabilities of trypsinand α-chymotrypsin. At 45ºC, trypsin was most stable at pH 3, while the highest stability of α-chymotrypsin was observed at pH 5. With both ester and amide substrates, trypsin displayedactivation at pH 3. In the case of α-chymotrypsin, activation was detected at pH 5 only withthe amide substrate. The time curves of heat inactivation were complex. For both enzymes,autolysis proceeded with the highest velocity at pH 8. The results obtained on α-chymotrypsinsuggested consecutive reactions: the first step, heat denaturation of the protein, is followedby digestion of the damaged molecules. Acta Biol Szeged 45(1-4):43-49 (2001)KEY WORDStrypsinα-chymotrypsinconformational stabilityautolysispH effectAccepted November 28, 2000*Corresponding author. E-mail: simon@biocom.bio.u-szeged.hu43too. The present paper reports results on the heat inactivationof trypsin and α-chymotrypsin.Materials and MethodsMaterialsBovine pancreas trypsin (EC 3.4.21.4), α-chymotrypsin (EC3.4.21.1), N-benzoyl-L-arginine ethyl ester (BAEE), N-acetyl-L-tyrosine ethyl ester (ATEE), N-benzoyl-DL-ar-ginine-p-nitroanilide (BAPNA) and N-carbobenzoxy-L-phenylalanine-p-nitroanilide (CPPNA) were purchased fromSigma-Aldrich Company (Budapest, Hungary). The specificactivities were 40-60 units/mg for α-chymotrypsin and10,000 units/mg for trypsin. All other chemicals were reagentgrade products (Reanal, Budapest, Hungary).Assays of enzyme activitiesThe activity of trypsin was measured by following theincrease in absorbance at 253 nm (Geiger and Fritz 1984) ina reaction mixture (3 ml) containing 46.7 mM Tris/HClbuffer (pH 8.0), 19 mM CaCl2 and 0.9 mM BAEE, thereaction being initiated by the addition of 5 units of enzyme.One unit of enzyme activity was defined as the amount ofenzyme that hydrolyses 1 µM of BAEE per min at pH 8.0 andat 25ºC. The activity measurements with BAPNA werecarried out as follows: the reaction mixture contained 150mM triethanolamine/HCl (pH 8.0), 15 mM CaCl2, 0.8 mMBAPNA and 5-10 units of trypsin (Erlanger et al. 1961). Theamount of p-nitroanilide released was monitored via theincrease in absorbance at 410 nm. For the measurement ofα-chymotrypsin activity, ATEE was used and the changes inabsorbance at 237 nm were followed in a reaction mixture(3 ml) containing 40 mM Tris/HCl (pH 8.0), 50 mM CaCl2Trypsin and α-chymotrypsin are well-known serine proteases(Desnuelle 1971; Keil 1971; Cohen et al. 1981; Journak andMcPherson 1987). The serine proteases exhibit structural andchemical similarities, but their specificities are different(Polgár 1989).According to early observations, trypsin is stable at pH3 at low temperatures for weeks. It can be reversibly heatdenaturated (Lazdunski and Delaage 1965). Lazdunski andDelaage (1967) investigated the effect of pH on the tempera-ture-induced reversible denaturation of bovine trypsin.D’Albis (1970) conducted a thermodynamic study on thereversible thermodenaturation of trypsin in the pH range 1.0-3.4. The conformation of trypsin is well ordered between pH7 and 8, but is considerably less ordered at more acidic oralkaline pH values. Both enzymes are susceptible to autoly-sis. Chymotrypsin A is most stable at pH 3, but even at thispH autolysis proceeds, although very slowly. At pHs lowerthan 3 or higher than 10, the enzyme undergoes conforma-tional changes (Walsh and Wilcox 1970).The conversion of trypsin to chymotrypsin and vice versaby site-directed mutagenesis is a model for protein engineers(Gráf et al. 1987; Heldstrom et al. 1992). Site-directedmutagenesis could modify the conformational stabilities ofthe enzyme derivatives. For an appraisal of the stabilitychanges induced, a kinetic re-evaluation of the conforma-tional stabilities of the parent enzymes appeared reasonable.Heat denaturation experiments comprise a simple andinexpensive method of investigation of the conformationalstabilities of proteins, and are useful for comparative studies,44Simon et al.and 0.5 mM ATEE (Schwert and Takenaka 1955). Thereaction was initiated by the addition of 4.5 units of enzyme.One unit of enzyme activity was defined as the amount ofenzyme that catalyses the hydrolysis of 1 µM of ATEE permin at pH 8.0 and at 25ºC. For the activity determination withCPPNA as substrate, the reaction mixture (3.0 ml) contained50 mM Tris/HCl (pH 8.0), 0.1 mM CPPNA in DMF and 5units of α-chymotrypsin (Delmar et al. 1979). The amountof p-nitroanilide released was monitored via the increase inabsorbance at 410 nm.Stability testsThe pH dependencies of the conformational stabilities oftrypsin and α-chymotrypsin were studied in the pH range 3-7 by using 0.1 M glycine/HCl buffer (pH 3), 0.1 M aceticacid/NaOH buffer (pH 4-5), 0.1 M citric acid/NaOH buffer(pH 6) and 0.1 M triethanolamine/HCl buffer (pH 7), respec-tively. Enzyme solutions of 0.1 and 1.0 mg/ml were preparedwith the different buffers and incubated for 5 h at varioustemperatures. Aliquots of 100-200 µl were withdrawn and theresidual activities were determined.Measurements of ninhydrin-positive speciesThe appearance of ninhidrin-positive substances during heattreatment was followed quantitatively according to theprocedure of Moore and Stein (1948).ResultsEffects of pH on stabilities of trypsin and a-chymotrypsinThe pH dependences of the stabilities of trypsin and α-chymotrypsin were studied at 45ºC, both ester and amidesubstrates being used for the determination of residualactivities. The protein concentration of the enzyme solutionwas 1 mg/ml.For trypsin, similar results were obtained with eitherBAEE or BAPNA as substrate (Fig. 1), but the loss inamidase activity was somewhat faster, especially in the acidicmedia. At pH 3, activation was observed with both substrates(18-20% and 16-17%, respectively). Trypsin exhibited thehighest stability at this pH. The inactivation was faster in thesolutions with pH > 6 than that in the media with lower pHs.The results obtained with α-chymotrypsin are depicted inFig. 2. Significant differences were found in the stabilities ofesterase (ATEE substrate) and amidase (CPPNA substrate)activities, especially at pH < 6. The highest stability of α-chymotrypsin was observed at pH 5. At this pH, the esteraseactivity was preserved for at least 4 h, while with the amidesubstrate activation of at most 24% was measured. Above pH6 the inactivation was more rapid than that in the media withlower pHs. At pH 9, the enzyme was practically inactivatedduring the first 20 min of incubation.Effects of temperature on stabilities of trypsinand α-chymotrypsinThe temperature dependence of the stability of α-chymo-trypsin was studied at pH 4 in citrate buffer and at pH 7 inphosphate buffer, with ATEE as substrate. The proteinconcentration was 1 mg/ml. The results are presented in Fig.3. At pH 4, the enzyme retained about 20% of its startingactivity after incubation for 5 h at 50ºC, while at pH 7 theenzyme practically lost all of its activity. At 55ºC, theinactivation was complete during the first 20 min of incuba-tion. In a 1 mg/ml solution, in phosphate buffer (pH 8) at55ºC, trypsin lost more than 90% of its initial esterase andamidase activities during a 5-min incubation.Effects of protein concentration on stabilitiesof trypsin and α-chymotrypsinThe effects of 0.1 and 1 mg/ml protein concentrations on thestability of trypsin were studied in Tris/HCl buffer (pH 8) at55ºC, with both ester (BAEE) and amide (BAPNA) assubstrates. After incubation for 2.5 min in the 0.1 mg/mlsolution, trypsin had lost 55.6% of its original esteraseactivity, while in the 1 mg/ml solution only 11.5% of thestarting activity was preserved. As regards the amidaseactivity, after incubation for 5 min in 0.1 mg/ml solution theactivity loss was 61.6%, while in 1 mg/ml solution it was93.2%. In the case of α-chymotrypsin, the effects of theprotein concentration on the stability were studied at 50ºC atpH 4 (sodium citrate) and pH 7 (potassium phosphate) in 0.1and 1 mg/ml solutions, with ester (ATEE) and amide(CPPNA) as substrates. The inactivation in the 0.1 mg/mlsolution was faster for both types of substrates (Fig. 4).Autolysis of trypsin and α-chymotrypsinSamples from the heat inactivation experiments were submit-ted to the ninhydrin test. The time curves of liberation ofninhydrin-positive species from trypsin are shown in Fig. 5.In 1 mg/ml solutions at pH 3 and 4, ninhydrin-positivesubstances could not be demonstrated, but at pH > 5 theautolysis proceeded rapidly. The highest rate was experi-enced at pH 8 in Tris/HCl buffer. The process involved atleast two phases, a fast and a slower one. At that pH in the0.1 mg/ml solution, the liberation of ninhydrin-positivesubstances was not detected at 45ºC and 55ºC.In 0.1 mg/ml α-chymotrypsin solutions, heat-treated at45ºC and 50ºC and at pH 4 and 7, respectively, ninhydrin-positive species were not liberated. In 1 mg/ml solutions at45ºC and in the pH range 3-4.5, ninhydrin-positive substanc-es could likewise not be detected. At pH 6, a lag period wasfollowed by the accelerated formation of autolysis products.At pH > 7, the time curves did not exhibit any lag period andthe process proceeded rapidly. The maximum velocity wasmeasured at pH 8 in potassium phosphate or Tris/HCl buffer45Stabilities of trypsin and α-chymotrypsin(Fig. 6). The autolysis involved at least two phases, similarlyas for trypsin. At higher temperature (50ºC), the lag periodwas observed only at pH 4 in the first 25 min of incubation(Fig. 7).DiscussionTrypsin and α-chymotrypsin display close structural similar-ities. The backbone structure of the two proteases are highlyhomologous and the homology also extends to the catalytictriad and substrate-binding pocket regions (Steitz et al. 1969;Birktoft and Blow 1972; Polgár 1989). In spite of the struc-tural similarities, however, there are significant differencesin their conformational stabilities. In earlier work (Simon etal. 1998), we established that, in miscible polar solvents suchas acetonitrile, ethanol and 1,4-dioxane, α-chymotrypsin hasquite different behaviour from that of trypsin. The differencesin the conformational stability are confirmed by the heattreatment experiments. At 45ºC, trypsin is most stable at pH3, while the highest stability of α-chymotrypsin was ob-served at pH 5. With both ester and amide substrates, trypsinshows activation at pH 3. In the case of α-chymotrypsin,activation was detected at pH 5 only with the amide substrate.The time curves of heat inactivation are complex for bothenzymes, in consequence of the existence of differentFigure 1. Effects of pH on inactivation of trypsin at 45ºC. Protein concentration: 1 mg/ml. Substrates: BAEE (A, B) and BAPNA (C, D). Buffers(0.1 M): citrate (◊) pH 3, (♦) pH 4, (Ο) pH 5, (l) pH 6, phosphate (x) pH 6, (∆) pH 7, (o) pH 8; borate (n) pH 9. For details, see text.46Simon et al.molecular forms. The stabilities of the different molecularforms of trypsin are temperature- and pH-dependent (Laz-dunski and Delaage 1967). At 20ºC, the acidification oftrypsin from pH 8 to pH 0.5 results in the appearance of 3reversible equilibria. The most important structural changein the alkaline range involves the unmasking of the abnormaltyrosines. This process is reversible, but is followed by anirreversible denaturation. α-chymotrypsin can exist in twomajor conformational states, only one of which is active.Stoesz and Lumry (1978) examined the pH and ionic strengthdependence of the transition between the active and inactiveforms. At low pH (pH 2.0-6.0), the equilibrium is verydependent on the salt concentration; high salt concentrationseffectively stabilize the active conformation. This apparentstabilization is an artifact due to the dimerization of the activeform of α-chymotrypsin. At pH 6.0-8.0, the dimerizationdoes not occur. At pH > 6, the pH dependence can be de-scribed by a two-ionization mechanism at all ionic strengths.The self-association of α-chymotrypsin was studied byPandit and Rao (1974). We suspect that the transient activa-Figure 2. Effects of pH on inactivation of α-chymotrypsin at 45ºC. Protein concentration: 1 mg/ml. Substrates: ATEE (A,B) and CPPNA (C, D).Buffers (0.1 M): citrate (◊) pH 3, (♦) pH 4, (Ο) pH 5, (l) pH 6, phosphate (x) pH 6, (∆) pH 7, (o) pH 8; borate (n) pH 9. For details, see text.47Stabilities of trypsin and α-chymotrypsintions during the heat treatment stem from the rise of amolecular subform with a higher catalytic activity, but alower stability.The autolysis proceeds with the highest velocity at pH 8for both enzymes. At pH 3 and 4, the liberation of theninhydrin-positive substances from α-chymotrypsin mol-ecules cannot be detected. A similar phenomenon wasobserved in 0.1 mg/ml solutions (in spite of the fast heatdenaturation) at pH 4 and 7 at 45ºC and 50ºC for α-chymo-trypsin and at pH 8 at 45ºC and 50ºC for trypsin. The detailedinvestigation by Kumar and Hein (1970) suggested that themechanism of autolysis of α-chymotrypsin can be explainedby an apparent second-order inactivation process. Autodi-gestion is chemically distinguishable from the process ofdenaturation. Our experimental results support these find-ings.Figure 3. Effects of temperature on inactivation of α-chymotrypsin at pH 4 in citrate buffer (A) and at pH 7 in phosphate buffer (B) with ATEEas substrate. Protein concentration: 1 mg/ml. Temperatures: (*) 45ºC, (s) 50ºC, (+) 55ºC. For details, see text.Figure 4. Effects of protein concentration on inactivation of α-chymotrypsin at 50ºC at pH 4 in citrate buffer and at pH 7 in phosphate buffer,with ATEE (A) and CPPNA (B) as substrates. Protein concentrations and pHs: (O) 0.1 mg/ml and pH 4, (l) 1 mg/ml and pH 4, (∆) 0.1 mg/ml andpH 7, (s) 1 mg/ml and pH 7. For details, see text.48Simon et al.The results obtained on the heat denaturation of α-chymotrypsin at pH 6 and at 45ºC point to consecutivereactions: the first step, heat denaturation, is followed by thedigestion of the damaged molecules. Similar kinetics couldnot be observed for trypsin. We presume a higher sensitivityof trypsin for autodigestion, resulting in a very short, unde-tectable lag period.ReferencesBirktoft JJ, Blow DM (1972) Structure of chrystalline α-chymotrypsin. JMol Biol 68:187- 240.Cohen GH, Silverton EW, Davie DR (1981) Refined chrystal structure ofγ-chymotrypsin in 1.9 A resolution. J Mol Biol 148:449-479.D’Albis A (1970) Étude thermodinamique de la denaturation termiqueréversible de la trypsine entre pH 1.0 et 3.4. Biochim Biophys Acta200:34-39.Figure 5. Effects of pH on autolysis of trypsin at 45ºC. Protein concentration: 1 mg/ml. Buffers (0.1 M): citrate (O) pH 5, (l) pH 6; phosphate(∆) pH 7, (o) pH 8; Tris/HCl (–) pH 8; borate (n) pH 9, (x) pH 10. For details, see text.Figure 6. Effects of pH on autolysis of α-chymotrypsin at 45ºC. Protein concentration: 1 mg/ml. Buffers (0.1 M): citrate (O) pH 5, (l) pH 6;phosphate (X) pH 6, (∆) pH 7, (+) pH 7.5, (o) pH 8; Tris/HCl (–) pH 8; borate (n) pH 9, (x) pH 10. For details, see text.49Stabilities of trypsin and α-chymotrypsinDelmar EG, Largman C, Brodrick JW, Geokas MC (1979) A sensitive newsubstrate for chymotrypsin. Anal Biochem 99:316-320.Desnuelle P (1971) The structure of chymotrypsin. In Boyer PD, ed., TheEnzymes, Academic Press, New York, 8:185-193.Erlanger BF, Kokowsky M, Cohen W (1961) The preparation and propertiesof two new chromogenic substrates for trypsin. Arch Biochem Biophys95:271-278.Geiger R, Fritz H (1984) Trypsin, In Bergmeyer HU, ed., Methods ofEnzymatic Analysis, Verlag Chemie, Weinheim, 5:119-123.Gráf L, Craik ChS, Patthy A, Rozniak S, Fletterick RJ, Rutter WJ (1987)Selective alteration of substrate specificity by replacement asparticacid-189 with lysine in the binding pocket of trypsin. Biochemistry26:2616-2623.Heldstrom L, Szilágyi L, Rutter WJ (1992) Converting trypsin to chymo-trypsin: the role of surface loops. Science 225:1249-1253.Jurnak FA, McPherson A, eds., (1987) Catalytic properties of trypsin,Biological macromolecules and assemblies: active site of enzymes,Wiley, New York, 377-385.Keil B (1971) Trypsin. In Boyer PD, ed., The Enzymes, Academic Press,New York, 8:248- 275.Kumar S, Hein GE (1970) Concerning the mechanism of autolysis of α-chymotrypsin. Biochemistry 9:291-297.Lazdunski M, Delaage M (1965) Sur la morphologie des trypsines de porcet de bceuf étude des denaturations reversibles. Biochim Biophys Acta105:541-561.Lazdunski M, Delaage M (1967) Étude structurale du trypsinogéne et dela trypsine. Les diagrammes d’ état. Biochim Biophys Acta 140:417-434.Moor S, Stein WH (1948) Photometric ninhydrin method for use in thechromatography of amino acids. J Biol Chem 176:367-388.Polgár L (1989) Mechanism of protease action. CRC Press, Boca RatonSchwert GW, Takenaka Y (1955) A spectrophotometric determination oftrypsin and chymotrypsin. Biochim Biophys Acta 16:570-575.Simon LM, László K, Vértesi A, Bagi K, Szajáni B (1998) Stability ofhydrolytic enzymes in water-organic solvent systems. J Mol Catal B:Enz 4:41-45.Steitz TA, Henderson R.D, Blow M (1969) Structure of christalline α-chymotrypsin. J Mol Biol 46:337-348.Stoesz JD, Lumry RW (1978) Refolding transition of α-chymotrypsin: pHand salt dependence. Biochemistry 17:3693-3699.Pandit MW, Narasinga Rao MS, (1974) Studies on self-association ofproteins. The self- association of α-chymotrypsin at pH 8.3 and ionicstrength 0.05. Biochemistry 13:1048- 1053.Walsh KA, Wilcox PE (1970) Serine proteases, In Perlmann GE, LorandL, eds., Methods in Enzymology, Academic Press, New York, 19:31-42.Figure 7. Effects of temperature on autolysis of α-chymotrypsinEnzyme concentration: 1 mg/ml. Buffers (0.1 M) and temperatures:citrate (O) pH 4 and 4 ºC, (l) pH 45 and 50ºC, phosphate (∆) pH 7and 45ºC, (s) pH 7 and 50 ºC. For details, see text.

A comparative study of the conformational stabilities of trypsin and α-chymotrypsin

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A comparative study of the conformational stabilities of trypsin and α-chymotrypsin

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