Biochimica et BiophysicaActa, 1076(1991)406-415 © 1991 ElsevierSciencePublishersB.V.0167-4838/91/$03.50 ADONIS 0167483891001115
4O6
BBAPRO33841
A kinetic and equilibrium study of the denaturation of aspartic proteinases from the fungi, Endothia parasitica and Mucor miehei E r i c D . B r o w n * a n d R i c k e y Y. Y a d a Departmentof FoodScience, Universityof GuelplLGuelph(Canada)
(Received18 June 1990) (Revisedmanuscriptreceived26 September1990) Key words: Asparticproteinase;Proteindenaturation: Kinetics:(E. parasitica): (M. miehei) Kinetic and equilibri,,--,n,analyses of the denaturation of Endothla parasitica and Mucor miehei aspartic proteinases were performed using enzyme activity and ultraviolet absorption as indices of denaturation. Denaturation of these pcoteinases was shown to be irreversible, suggesting that the confonnatioas of these aspartic proteinasas may be predetermined in their zymogens. Thermal and guanidine hydruchloride denaturation of these protelnases produced first-order, two-state, kinetic behaviour. Equilibrium unfolding transitions of these proteinases were highly cooperative but not entirely coincident in the two indices employed, suggesting some deviation from two-state character. Oxidation to remove 37.8% of the carbohydrate o[ M. mlehei glycoproteinase with sodium metaperiodate resulted in a substantial decrease in both kinetic and equilibrium stabilities without modification of the amino acid composition or specific activity, in addition, gel filtration subsequent to equilibrium studies indicated that partial removal of the carbohydrate from M. miehel woteilmse promoted autolysis under denaturing conditions. Intrnduetion The aspartic proteinases (EC 3.4.23) are characterized by an optimum activity at acid pH (i.e., pH < 6) and contain two catalytically essential aspartate residues. A worldwide shortage of the aspartic proteinase chymosin, traditionally used in the production of cheese, has prompted a search for adequate substitutes. Two alternatives are the aspartic proteinases from the fungi Endothia parasitica and Mucor miehei. Although M. miehei proteinase possesses high milk-clotting potential relative to its capacity for general proteolysis [1,2], high thermal stability has precluded its use as a chymosin substitute. By contrast, in spite of the low substrate specificity exhibited by E. parasitica proteinase compared to chymosin [1,2], its thermal lability has made it the substitute of choice for many cheeses [3]. It is aoteworthy that of the milk-clotting replacements studied, M. miehei proteinase exhibits the highest thermostability [4] and is the most highly glycosylated, * Present address: Gudph-WatedooCentre for Graduate Work in Chemistry and Biochemist~3', University of Guelph, Guelph, Canada. Correspondence:R.Y. Yada, Departmentof Food Science,University of Guelph, Guelph,Ontario, Canada, NIG 2WI.
possessing about 6~ carbohydrate [5]. It has been suggested that oligosaccharides help prolong the active life of a glycoprotein by stabilizing conformation and by protecting the protein from proteolytic attack [6-8]. To better understand protein stabilization, researchers have used both kinetic and thermodynamic approaches to examine protein denaturation as a twostate process. N (Native) ~ D (Denatured)
(1)
Thermodynamic studies have generally been reserved for those processes for which reversible denaturation can be demonstrated. Modifications which may discourage reversibility of denaturation include the proteolytic or autolytic processing of many proteinases and hormones. Indeed, irreversibility has been demonstrated for insulin [9] and chymotrypsin [10] after the oxidation of disulfide bonds while, under the same conditions, their respective zymogens showed reversibility. Further, the prosequence of subtilisin was recently shown to guide the refolding of Gdn-HCl-denatured subtilisin in an intermolecular process [I1]. Especially relevant m the study of aspartic proteinases is the irreversible unfolding of pepsin [12,13] even with intact disulfides and the reversible behaviour of pepsinogen, its zymogen [14]. Studies to date concerning the stability of aspartic proteinases other than pepsin, have focused on the
407 thermal inactivation of microbial rennets and have relied on enzyme activity as a measure of integrity [3]. These studies were kinetic in nature and reported values for the energy of activation of denaturation. In the work presented here, the stabilities of the proteinases from E. parasitica and M. miehei as well as a carbohydratereduced form of the latter, were assessed using both specific activity and ultraviolet absorption as indices of denaturation. As well as kinetic experiments, equilibrium studies were undertaken, despite apparent irreversibility of inactivation, and the practicality of such an approach examined. In addition, the importance of the glycan of the glycoprotein M. miehei in stabilizing the enzyme against both autolysis and gross conformational changes was investigated. Ma~d~sandMethods Proteinases were purified to homogeneity from commercial microbial rennets as described previously [15]. Enzyme concentrations were determined using extinction coefficients of 11.8 and 11.5 for 1 mg/ml solutions of M. miehei proteinase [16] and E. parasitica [17], respectively. Guanidine hydrochloride (Gdn-HCI) (reagent grade, Fisher Scientific, Fair Lawn, N J) was twice recrystallized according to Nozaki [18]. Precise concentrations of Gdn-HCI were routinely determined using a refractive index technique [18]. One unit of enzyme activity was the change in absorbance at 280 nm per win of a trichloroacetic acid superuatant after incubation of the afiquot with a 2.5~ (w/v) solution of acid denatured bemoglobin (pH 1.8) for 15 rain at 37°C [19]. Gel-exclusion-purified enzymes, native and oxidized were assayed in quadruplicate for total carbohydrate using glucose as a standard [20]. Amino acid analyses were as described previously [15]. Periodate oxidation. Removal of oxidizable carbohydrate from M. miehei proteinase was as described by McBride-Warren and Rickert [21]. 25 mg of lyophilized, ion-exchange-purified (partially purified) M. miehei proteinase [1'3] was oxidized in 0.1 M sodium metaperiodate ~:t 0 ° C for 4.5 h and quenched with 50~ (v/v) ethylene glycol. The oxidized protein was recovered by application of the mixwre to a 1 × 25 cm column of Sephadex G-25 (Pharmacia) equilibrated at 4 ° C with 10 mM IVies buffer (pH 5.4) and lyophilized. Tni,, preparation was reconstituted in a minimum amount of distilled water and further purified, as was the control enzyme (i.e., not oxidized), using a Superose 12 (Pharmacia) gel-exclusion column [15!, equilibrated in 0.05 M sodium acetate buffer (pH 4.0) for kinetic experiments and 0.025 M (2-(N-morpholino)ethanesulfonic acid) (IVies) buffer (pH 5.4) for equilibrium experiments. The chromatogram demonstrated, in addition to one major peak, a higher molecular weight
shoulder, presumably containing less oxidized species. Only the material of the major lower-molecular-weight peak was collected. Enzyme activities of the control and oxidized enzymes x~e~c determined in duplicate at five different enzyme concentrations. Specific activities were then derived from a plot of absorbance at 280 nm of the trichloroacetic acid supernatant vs. enzyme concentration as suggested by McBride-Warren and Rickert [21]. Kinetic experiments. Absorbance measurements and difference spectra were made on a Shimadzu UV-260 Recording Spectrophotometer (TekScience, Mississauga, ON). Ultraviolet difference spectroscopy was used to identify critical wavelengths for following chemical or thermal denaturation through scan subtraction, of the native from the perturbed spectrum of protein. Difference spectra were principal in identifying wavelengths of maximum difference for both kinetic and equilibrium studies. A time-course study of the denaturation of M. miehei and E. parasitica proteinases in 4.0 and 2.9 M guanidine hydrochloride, respectively, involved rapid mixing through inversion of a Teflon stoppered cuvette and subsequent monitoring of the absorbance at 285.7 nm. Data analysis was performed to confirm that the reaction was first order and involved the construction of semilogarithmic plots according to the first-order integrated rate equation [N] (A,-Af) n [--~Tol= m ~ = - kt .
(2)
where [N]/[N0] represents ,'he fraction native at time t and where At, A 0 and A t were the absorbances of denatured enzyme, native enzyme and enzyme incubated in Gdn-HCi for time t, respectively. Kinetic temperature jump studies were performed using E. parasitica, M. miehei and degiycosylated M. miehei proteinases. A Haake C water bath (Fisher Scientific, Fair Lawn, N J) controlled with a Haake F3 Digital Immersion Circulator was used to maintain the temperature of jacketed sample and reference cells to within 0.01°C. At each temperature, 1.8 ml of 0.05 M sodium acetate buffer (pH 4.0) was preheated to the target temperature in a Teflon stoppered jacketed sample cuvette, A blank of the same composition was preheated in a stoppered reference cell. After equilibration, the sample cell was unstoppered and a 0.250 ml aliquot of protein solution, approximate concentration 2.5 mg/ml, was defivered, followed by 10 s of mixing using a mechanical cuvette stirrer consisting of a Rotary Whip and Precision Controller (Instech Laboratories, Horsham, PA). Subsequent to the initial mixing a time zero reading at 285.7 nm was recorded, an additional 30 s of mechanical mixing followed, and the Teflon stopper on the sample cell was replaced. Data analysis was first order and involved the construction of semilogarithmic
408 plots as described above (Eqn. 2). The activation energy (E~) of denaturation was derived according to the Arrhenius equation
was used to generate fN, the fraction native, which was given by fN = ( R - Rf )/( Ro- Rf )
k = A e -E./RT
(3)
by measuring the rate constant, k, over a range of denaturing temperatures. The kinetics of enzyme inactivation was investigated for E. parasitica, M. miehei and deglycosylated M. meihei proteinases. This involved the delivery of aliquots of 40/LI of protein (concentration 0.2 mg/ml) into the bottom of 1.5 ml disposable microcentrifuge tubes (Fisher Scientific, Fair Lawn, N J) which were then placed in the H aake water bath, previously equilibrated at the target temperature. Tubes were removed at successive intervals and immersed in a room temperature bath for 30 s. Subsequently, the top 3 cm of the tube was clipped with pliers and discarded such that the remaining abbreviated tube was small enough to be added directly to the bemoglobin assay mixture 60 s after removal from the high temperature water bath. Incubation temperatures of the stopped enzyme assay were 17" C for the enzyme from E. parasitica and 37 ° C for that from M. miehei, owing to differences in proteolytie activity. First-order kinetic analyses of inactivation were as described for ultraviolet experiments and involved the construction of semilogarithmic plots of residual activity vs. time. Temperature influence was again investigated as in spectral studies using the Arrhenius relationship (Eqn. 3) to derive the activation energy of denaturation. Equilibrium studies. To evaluate the reversibility of denaturation in Gdn-HCI, M. miehei and E. parasitiea proteinases were equilibrated (3.5 h) in 4 M Gdn-HCI, a denaturing concentration for each, and exhaustively dialysed against 0.05 M citrate phosphate buffer of pH values 2.7, 3.6, 4.7, 5.7 and 6.6. Specific activities were subsequently assayed. Equilibrium unfolding studies involved equilibration (3.5 h) of proteinases in incremental concentrations of Gdn-HCI buffered with 0.025 M Mes (pH 5.4) at protein concentrations of approx. 0.25 m g / m l for spectroscopic studies and 0.15 m g / m l for inactivation experiments. The absorbance of these equilibrated solutions was measured at 257 and 285.7 nm (maximum and minimum wavelengths identified through difference spectroscopy) or assayed for activity. While equilibrium studies with E. parasitica and M. miehei proteinases employed both spectral and inactivation experiments, only inactivation was used as an index of denaturation for the carbohydrate-reduced M. miehei proteinase. For spectroscopic experiments, the ratio R = A2s~/A2ss.7
(4)
(5)
where Rf, R o and R were the ratios for the denatured enzyme, native enzyme and the enzyme incubated in Gdn-HCI, respectively. Values of Rf and R o in the transition region were obtained from extrapolating from the pre- and post-transition regions using least-squares regression analysis as suggested by Pace et al. [22[. Using enzyme activity as an index of denaturation, 0.100 and 0.050 ml aliquots of M. miehei and E. parasitica proteinases were assayed at 37 and 27 ° C, respectively, following equilibration. In this case fraction native, fN was fN = ( U-Uf )/( Uo-Uf )
(6)
where Uf, Uo and U were the relative enzyme activities of the denatured enzyme, native enzyme and enzyme equilibrated in Gdn-HCI, respectively. In the enzyme activity experiments, finear extrapolation of the pos~transition region was employed to generate values of Uf. Since the activation of the enzyme acti~ty in the pretransition region was judged to be non.linear and seemed to reach a threshold value prior to the transition, a baseline of slope equal to zero at the threshold valt~e was used to approximate Uo. For all equilibrium experiments, the equilibrium constant K D and free energy of denaturation (AGD) were calculated for the transition. O-fN) gc,= ~ A G D = -- R T In
O) KD
~S)
As suggested by Tanford [23] only those K D values within the range 0.1 to 10 were used in the analysis of the transition. The limiting values of the transition were determined through regression analysis of the line given by dG D =dG ° - re[D]
(9)
where [D] is the concentration of denaturant and m is the slope, to obtain an estimate of the free energy of denaturation under standard conditions (AGO). Parameters from the regressed line (i.e., AG ° and m) were used to generate the model curve frw the data (i.e., fN at given values of [D]) given by ~,G ° - m i D ] = - R T I n [ ( l - f N ) / f N ]
(lO)
~l]te model curve was then plotted with the raw data to examine the fit of the estimate. lt, vestigation of autolysis. Subsequent to the 3.5 h incubation period, samples were frozen in liquid nitro-
409 gen and stored at - 2 0 ° C until chromatographed. Three samples, representative of the denaturation curve, having Gdn-HCI concentrations corresponding approximately to the top crest, the midpoint and the bottom of the transition curve, were chromatographed. Samples were warmed to room temperature immediately prior to chromatography, which was performed using FPLC equipped with a Superose 12 (Pharmacia) gel-exclusion column with detection at 280 nm as described previously [15]. Chromatograms were corrected for dution of Gdn-tiCI at the column volume of approx. 18 mi by using a 'blank' having an equal concentration of GdnHCI.
in this experiment due to the perJodate treatment of M. miehei proteinase during the 4.5 h incubation (data not shown). Moreover, no differences were demonstrated in the respective amino acid compositions (data not shown). Under the same conditions as those u~sedin this study, McBride-Warren and Rickert [21] reported that the amino acid composition of oxidized M. miehei proteinase, compared with the control (unmodified), was unchanged. Difference ultraviolet spectroscopy. Time-course difference spectral scans for the Gdn-HCI induced denaturation of M. miehei proteinase (Fig. la) showed a shift to lower wavelength accompanied by a decrease in molar extinction coefficient. The difference spectrum of E. parasitica proteinase was highly similar and demonstrated the same points of isosbecity and inflection (data not shown). Such a shift is typical of the exposure of chromophores, largely tyrosine and tryptophan, originating in the hydrophobic interior of a protein, consequent on the disordering of protein structure [24]. Significant in both difference spectra was the presence of distinct points of isosbecity and the persistence of a relatively fiat baseline in the region from 350 to 310 nm, which suggested that no aggregation, chemical reactions or other forms of non-ideality occurred as a result of the unfolding reaction. Time-course difference spectral scans for the thermal denaturation of M. michel and E. partzsitica protinases demonstrated the same basic lea-
Results and Discussion
Deglycosylation of M. michel proteinase. Oxidation of M. michel proteinase with periodate followed by gel-exclusion to remove some residual unmodified species was successful in reducing the carbohydrate content of the enzyme by 37.9~g (carbohydrate contents of the native and oxidized enzymes were respectively, 6.9 4- 0.4% and 4.3 ± 0.3%). McBride-Warren and Rickert [21] reported 39% reduction under the same conditions of oxidation and demonstrated that exposure of M. miehei proteinase to 0.1 M periodate for more than 5 h resulted in little additional loss of carbohydrate, but caused a large decrease in activity. No activity loss could be detected
0 27
oo: .o 2. -o3. -o4,
0 26.
.o5. -o6, .o7. -o6.
0.25,
O.
.og,
c
.11.
e B
~ o24
-13, -~4,
o ....
,3
6
~ o.23 ,,
o ' ~ " ~ '~"
;6"2b'~'4'
T,me (m,n~
o
---7 D 0.22=
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•
~45
,
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,
,
=
200 Wavelength (rim)
,
,
,
,
0
350
D U O D
n
O
21
0 T i m e (rnin)
Fig. L Repeated spectral scan (a) during the denaturation of M. raiehei proteinas¢ in 3,8 M Gdn-HCL Enzyme concez~tration was 0,,LSOm g / m l in 0.025 M Mes buffer (pH 5.4). Scanning times wc.~e I, 2.3, 5.2, 7.S, 11.5, 18.5 and 45.2 rain after mixin~ for curves I, 2, 3, 4, S, 6 and 7, respectively. Decrease in absorbance Co) at 285.7 nm for the proteinase of M. michel in 4 M Gdn-HCI. Enzyme concentration was 0.250 mg,/nd in 0.025 M Mes buffer (pH 5.4). Inset shows the f'wsborder transformation of time-course data and the regressed line.
410 tures as those for Gdn-HCI denaturation (data not shown). In particular, the minimum at 285.7 nm was also demonstrated in temperture jump experiments. Kinetics of denaturation. The time-course of Gdn-HCI induced denaturation, evidenced by a decrease in absorption at 285.7 nm for the enzymes from M. michel (Fig. lb) and E. parasitica (data not shown), was found to follow first-order kinetics. This suggested that denaturation was best approximated by a two-state model where-only native and denatured molecules exist in significant concentrations. These findings are in agreement with studies of the two-state kinetics of urea and Gdn-HCi denaturation of pepsin [25,26] and of pepsinogen [14]. Rate constants found through regression of the linear transformation (Fig. l b inset) were 0.058 and 0.028 rain-t for M. mtehei and E. parasitica proteinases (data not shown), respectively. Fig. 2a depicts typical first-order semilogarithmie plots of the isothermal denaturation of E. parasitica proteinase as evidenced by a decrease in absorbance at 285.7 nm. This plot and those for M. miehei and the carbohydrate-reduced M. miehei proteinases (data not shown) demonstrated the first-order, two-state behaviour already documented for thermal inactivation of these proteinases [4,27] and confirmed the persistence of this behaviour in the modified M. miehei proteinase. While the Arrhenius plots for E. parasitica proteinase and the unmodified M. miehei enzyme (Fig. 2b) appeared linear in character, that for the deglycosylated form of M. miehei was somewhat curvilinear (Fig. 2b). This suggested that a heterogeneous population existed which may have consisted of differentially deglyco-
.o,.
,
TABLE 1
Activation energies a in kJ/mol for the denaturation of M. miehei, E. parasitica and deglycosylated M. miehei proteinases followed by ultraviolet spectroscopy and enzyme activity Method
Proteinase
M. michel E. par~itica Ultraviolet spectroscopy 437± 12 Enzyme activity 458 ± 41
250± l0 225 ± 11
M. michel (deglycosylated) 370± 19 339 ± 30
a Activation energy determined from simple linear regression of the Arrhenius plot (error term is the standard error for the determination of slope or Ea ).
sylated species. In experiments employing enzyme activity as a measure of structural integrity, first-order plots of residual activity (data not shown) confirmed the first-order nature of the reaction and were used to derive the energy of activation from Arrhenius plots. Again, the Arrhenins plot for the modified enzyme from M. miehei (data not shown) was somewhat curvilinear. Activation energies for the denaturation of M. michel and E. parasitica proteinases were respectively, in ultraviolet experiments 437 and 250 l d / m o l and in inactivation studies 458 and 225 kJ/mol (Table I). That the values were not grossly different using the two methods, suggested that irreversible enzyme inactivation was coincident with a gross conformational change and provided further support for the two-state approximation. Using inactivation as the index for denaturation Hyslop et al. [4] reported a somewhat higher value (552 Ll/mol) -0 Bi O -o.91o -111
b ~ ~
-11L -12~-
~..
-13[-
g_o~
\
-1.4!.
÷
-'5~
+~
-o;
-'~B[ -tg| -2[
-08 -09 -1
.... o '
-21~ ~ '~-'
6
Time ( rnlnJ
. . . .8. . .
:o
,.
-22 I , t , , • k , 290292 294 296 298 300 302 304 306 30B lIT
(x10"3 K)
Fig. 2. Semilogarithmic plots (a) of Iog(At-Af)/(A o - Af), where absorbanee, abbreviated A, was monitored at 285.7 nm vs. time for the isothermal denaturation of E parasitica proteinase at temperatures 52.5°C (ll), 55.5°C (
4O6
BBAPRO33841
A kinetic and equilibrium study of the denaturation of aspartic proteinases from the fungi, Endothia parasitica and Mucor miehei E r i c D . B r o w n * a n d R i c k e y Y. Y a d a Departmentof FoodScience, Universityof GuelplLGuelph(Canada)
(Received18 June 1990) (Revisedmanuscriptreceived26 September1990) Key words: Asparticproteinase;Proteindenaturation: Kinetics:(E. parasitica): (M. miehei) Kinetic and equilibri,,--,n,analyses of the denaturation of Endothla parasitica and Mucor miehei aspartic proteinases were performed using enzyme activity and ultraviolet absorption as indices of denaturation. Denaturation of these pcoteinases was shown to be irreversible, suggesting that the confonnatioas of these aspartic proteinasas may be predetermined in their zymogens. Thermal and guanidine hydruchloride denaturation of these protelnases produced first-order, two-state, kinetic behaviour. Equilibrium unfolding transitions of these proteinases were highly cooperative but not entirely coincident in the two indices employed, suggesting some deviation from two-state character. Oxidation to remove 37.8% of the carbohydrate o[ M. mlehei glycoproteinase with sodium metaperiodate resulted in a substantial decrease in both kinetic and equilibrium stabilities without modification of the amino acid composition or specific activity, in addition, gel filtration subsequent to equilibrium studies indicated that partial removal of the carbohydrate from M. miehel woteilmse promoted autolysis under denaturing conditions. Intrnduetion The aspartic proteinases (EC 3.4.23) are characterized by an optimum activity at acid pH (i.e., pH < 6) and contain two catalytically essential aspartate residues. A worldwide shortage of the aspartic proteinase chymosin, traditionally used in the production of cheese, has prompted a search for adequate substitutes. Two alternatives are the aspartic proteinases from the fungi Endothia parasitica and Mucor miehei. Although M. miehei proteinase possesses high milk-clotting potential relative to its capacity for general proteolysis [1,2], high thermal stability has precluded its use as a chymosin substitute. By contrast, in spite of the low substrate specificity exhibited by E. parasitica proteinase compared to chymosin [1,2], its thermal lability has made it the substitute of choice for many cheeses [3]. It is aoteworthy that of the milk-clotting replacements studied, M. miehei proteinase exhibits the highest thermostability [4] and is the most highly glycosylated, * Present address: Gudph-WatedooCentre for Graduate Work in Chemistry and Biochemist~3', University of Guelph, Guelph, Canada. Correspondence:R.Y. Yada, Departmentof Food Science,University of Guelph, Guelph,Ontario, Canada, NIG 2WI.
possessing about 6~ carbohydrate [5]. It has been suggested that oligosaccharides help prolong the active life of a glycoprotein by stabilizing conformation and by protecting the protein from proteolytic attack [6-8]. To better understand protein stabilization, researchers have used both kinetic and thermodynamic approaches to examine protein denaturation as a twostate process. N (Native) ~ D (Denatured)
(1)
Thermodynamic studies have generally been reserved for those processes for which reversible denaturation can be demonstrated. Modifications which may discourage reversibility of denaturation include the proteolytic or autolytic processing of many proteinases and hormones. Indeed, irreversibility has been demonstrated for insulin [9] and chymotrypsin [10] after the oxidation of disulfide bonds while, under the same conditions, their respective zymogens showed reversibility. Further, the prosequence of subtilisin was recently shown to guide the refolding of Gdn-HCl-denatured subtilisin in an intermolecular process [I1]. Especially relevant m the study of aspartic proteinases is the irreversible unfolding of pepsin [12,13] even with intact disulfides and the reversible behaviour of pepsinogen, its zymogen [14]. Studies to date concerning the stability of aspartic proteinases other than pepsin, have focused on the
407 thermal inactivation of microbial rennets and have relied on enzyme activity as a measure of integrity [3]. These studies were kinetic in nature and reported values for the energy of activation of denaturation. In the work presented here, the stabilities of the proteinases from E. parasitica and M. miehei as well as a carbohydratereduced form of the latter, were assessed using both specific activity and ultraviolet absorption as indices of denaturation. As well as kinetic experiments, equilibrium studies were undertaken, despite apparent irreversibility of inactivation, and the practicality of such an approach examined. In addition, the importance of the glycan of the glycoprotein M. miehei in stabilizing the enzyme against both autolysis and gross conformational changes was investigated. Ma~d~sandMethods Proteinases were purified to homogeneity from commercial microbial rennets as described previously [15]. Enzyme concentrations were determined using extinction coefficients of 11.8 and 11.5 for 1 mg/ml solutions of M. miehei proteinase [16] and E. parasitica [17], respectively. Guanidine hydrochloride (Gdn-HCI) (reagent grade, Fisher Scientific, Fair Lawn, N J) was twice recrystallized according to Nozaki [18]. Precise concentrations of Gdn-HCI were routinely determined using a refractive index technique [18]. One unit of enzyme activity was the change in absorbance at 280 nm per win of a trichloroacetic acid superuatant after incubation of the afiquot with a 2.5~ (w/v) solution of acid denatured bemoglobin (pH 1.8) for 15 rain at 37°C [19]. Gel-exclusion-purified enzymes, native and oxidized were assayed in quadruplicate for total carbohydrate using glucose as a standard [20]. Amino acid analyses were as described previously [15]. Periodate oxidation. Removal of oxidizable carbohydrate from M. miehei proteinase was as described by McBride-Warren and Rickert [21]. 25 mg of lyophilized, ion-exchange-purified (partially purified) M. miehei proteinase [1'3] was oxidized in 0.1 M sodium metaperiodate ~:t 0 ° C for 4.5 h and quenched with 50~ (v/v) ethylene glycol. The oxidized protein was recovered by application of the mixwre to a 1 × 25 cm column of Sephadex G-25 (Pharmacia) equilibrated at 4 ° C with 10 mM IVies buffer (pH 5.4) and lyophilized. Tni,, preparation was reconstituted in a minimum amount of distilled water and further purified, as was the control enzyme (i.e., not oxidized), using a Superose 12 (Pharmacia) gel-exclusion column [15!, equilibrated in 0.05 M sodium acetate buffer (pH 4.0) for kinetic experiments and 0.025 M (2-(N-morpholino)ethanesulfonic acid) (IVies) buffer (pH 5.4) for equilibrium experiments. The chromatogram demonstrated, in addition to one major peak, a higher molecular weight
shoulder, presumably containing less oxidized species. Only the material of the major lower-molecular-weight peak was collected. Enzyme activities of the control and oxidized enzymes x~e~c determined in duplicate at five different enzyme concentrations. Specific activities were then derived from a plot of absorbance at 280 nm of the trichloroacetic acid supernatant vs. enzyme concentration as suggested by McBride-Warren and Rickert [21]. Kinetic experiments. Absorbance measurements and difference spectra were made on a Shimadzu UV-260 Recording Spectrophotometer (TekScience, Mississauga, ON). Ultraviolet difference spectroscopy was used to identify critical wavelengths for following chemical or thermal denaturation through scan subtraction, of the native from the perturbed spectrum of protein. Difference spectra were principal in identifying wavelengths of maximum difference for both kinetic and equilibrium studies. A time-course study of the denaturation of M. miehei and E. parasitica proteinases in 4.0 and 2.9 M guanidine hydrochloride, respectively, involved rapid mixing through inversion of a Teflon stoppered cuvette and subsequent monitoring of the absorbance at 285.7 nm. Data analysis was performed to confirm that the reaction was first order and involved the construction of semilogarithmic plots according to the first-order integrated rate equation [N] (A,-Af) n [--~Tol= m ~ = - kt .
(2)
where [N]/[N0] represents ,'he fraction native at time t and where At, A 0 and A t were the absorbances of denatured enzyme, native enzyme and enzyme incubated in Gdn-HCi for time t, respectively. Kinetic temperature jump studies were performed using E. parasitica, M. miehei and degiycosylated M. miehei proteinases. A Haake C water bath (Fisher Scientific, Fair Lawn, N J) controlled with a Haake F3 Digital Immersion Circulator was used to maintain the temperature of jacketed sample and reference cells to within 0.01°C. At each temperature, 1.8 ml of 0.05 M sodium acetate buffer (pH 4.0) was preheated to the target temperature in a Teflon stoppered jacketed sample cuvette, A blank of the same composition was preheated in a stoppered reference cell. After equilibration, the sample cell was unstoppered and a 0.250 ml aliquot of protein solution, approximate concentration 2.5 mg/ml, was defivered, followed by 10 s of mixing using a mechanical cuvette stirrer consisting of a Rotary Whip and Precision Controller (Instech Laboratories, Horsham, PA). Subsequent to the initial mixing a time zero reading at 285.7 nm was recorded, an additional 30 s of mechanical mixing followed, and the Teflon stopper on the sample cell was replaced. Data analysis was first order and involved the construction of semilogarithmic
408 plots as described above (Eqn. 2). The activation energy (E~) of denaturation was derived according to the Arrhenius equation
was used to generate fN, the fraction native, which was given by fN = ( R - Rf )/( Ro- Rf )
k = A e -E./RT
(3)
by measuring the rate constant, k, over a range of denaturing temperatures. The kinetics of enzyme inactivation was investigated for E. parasitica, M. miehei and deglycosylated M. meihei proteinases. This involved the delivery of aliquots of 40/LI of protein (concentration 0.2 mg/ml) into the bottom of 1.5 ml disposable microcentrifuge tubes (Fisher Scientific, Fair Lawn, N J) which were then placed in the H aake water bath, previously equilibrated at the target temperature. Tubes were removed at successive intervals and immersed in a room temperature bath for 30 s. Subsequently, the top 3 cm of the tube was clipped with pliers and discarded such that the remaining abbreviated tube was small enough to be added directly to the bemoglobin assay mixture 60 s after removal from the high temperature water bath. Incubation temperatures of the stopped enzyme assay were 17" C for the enzyme from E. parasitica and 37 ° C for that from M. miehei, owing to differences in proteolytie activity. First-order kinetic analyses of inactivation were as described for ultraviolet experiments and involved the construction of semilogarithmic plots of residual activity vs. time. Temperature influence was again investigated as in spectral studies using the Arrhenius relationship (Eqn. 3) to derive the activation energy of denaturation. Equilibrium studies. To evaluate the reversibility of denaturation in Gdn-HCI, M. miehei and E. parasitiea proteinases were equilibrated (3.5 h) in 4 M Gdn-HCI, a denaturing concentration for each, and exhaustively dialysed against 0.05 M citrate phosphate buffer of pH values 2.7, 3.6, 4.7, 5.7 and 6.6. Specific activities were subsequently assayed. Equilibrium unfolding studies involved equilibration (3.5 h) of proteinases in incremental concentrations of Gdn-HCI buffered with 0.025 M Mes (pH 5.4) at protein concentrations of approx. 0.25 m g / m l for spectroscopic studies and 0.15 m g / m l for inactivation experiments. The absorbance of these equilibrated solutions was measured at 257 and 285.7 nm (maximum and minimum wavelengths identified through difference spectroscopy) or assayed for activity. While equilibrium studies with E. parasitica and M. miehei proteinases employed both spectral and inactivation experiments, only inactivation was used as an index of denaturation for the carbohydrate-reduced M. miehei proteinase. For spectroscopic experiments, the ratio R = A2s~/A2ss.7
(4)
(5)
where Rf, R o and R were the ratios for the denatured enzyme, native enzyme and the enzyme incubated in Gdn-HCI, respectively. Values of Rf and R o in the transition region were obtained from extrapolating from the pre- and post-transition regions using least-squares regression analysis as suggested by Pace et al. [22[. Using enzyme activity as an index of denaturation, 0.100 and 0.050 ml aliquots of M. miehei and E. parasitica proteinases were assayed at 37 and 27 ° C, respectively, following equilibration. In this case fraction native, fN was fN = ( U-Uf )/( Uo-Uf )
(6)
where Uf, Uo and U were the relative enzyme activities of the denatured enzyme, native enzyme and enzyme equilibrated in Gdn-HCI, respectively. In the enzyme activity experiments, finear extrapolation of the pos~transition region was employed to generate values of Uf. Since the activation of the enzyme acti~ty in the pretransition region was judged to be non.linear and seemed to reach a threshold value prior to the transition, a baseline of slope equal to zero at the threshold valt~e was used to approximate Uo. For all equilibrium experiments, the equilibrium constant K D and free energy of denaturation (AGD) were calculated for the transition. O-fN) gc,= ~ A G D = -- R T In
O) KD
~S)
As suggested by Tanford [23] only those K D values within the range 0.1 to 10 were used in the analysis of the transition. The limiting values of the transition were determined through regression analysis of the line given by dG D =dG ° - re[D]
(9)
where [D] is the concentration of denaturant and m is the slope, to obtain an estimate of the free energy of denaturation under standard conditions (AGO). Parameters from the regressed line (i.e., AG ° and m) were used to generate the model curve frw the data (i.e., fN at given values of [D]) given by ~,G ° - m i D ] = - R T I n [ ( l - f N ) / f N ]
(lO)
~l]te model curve was then plotted with the raw data to examine the fit of the estimate. lt, vestigation of autolysis. Subsequent to the 3.5 h incubation period, samples were frozen in liquid nitro-
409 gen and stored at - 2 0 ° C until chromatographed. Three samples, representative of the denaturation curve, having Gdn-HCI concentrations corresponding approximately to the top crest, the midpoint and the bottom of the transition curve, were chromatographed. Samples were warmed to room temperature immediately prior to chromatography, which was performed using FPLC equipped with a Superose 12 (Pharmacia) gel-exclusion column with detection at 280 nm as described previously [15]. Chromatograms were corrected for dution of Gdn-tiCI at the column volume of approx. 18 mi by using a 'blank' having an equal concentration of GdnHCI.
in this experiment due to the perJodate treatment of M. miehei proteinase during the 4.5 h incubation (data not shown). Moreover, no differences were demonstrated in the respective amino acid compositions (data not shown). Under the same conditions as those u~sedin this study, McBride-Warren and Rickert [21] reported that the amino acid composition of oxidized M. miehei proteinase, compared with the control (unmodified), was unchanged. Difference ultraviolet spectroscopy. Time-course difference spectral scans for the Gdn-HCI induced denaturation of M. miehei proteinase (Fig. la) showed a shift to lower wavelength accompanied by a decrease in molar extinction coefficient. The difference spectrum of E. parasitica proteinase was highly similar and demonstrated the same points of isosbecity and inflection (data not shown). Such a shift is typical of the exposure of chromophores, largely tyrosine and tryptophan, originating in the hydrophobic interior of a protein, consequent on the disordering of protein structure [24]. Significant in both difference spectra was the presence of distinct points of isosbecity and the persistence of a relatively fiat baseline in the region from 350 to 310 nm, which suggested that no aggregation, chemical reactions or other forms of non-ideality occurred as a result of the unfolding reaction. Time-course difference spectral scans for the thermal denaturation of M. michel and E. partzsitica protinases demonstrated the same basic lea-
Results and Discussion
Deglycosylation of M. michel proteinase. Oxidation of M. michel proteinase with periodate followed by gel-exclusion to remove some residual unmodified species was successful in reducing the carbohydrate content of the enzyme by 37.9~g (carbohydrate contents of the native and oxidized enzymes were respectively, 6.9 4- 0.4% and 4.3 ± 0.3%). McBride-Warren and Rickert [21] reported 39% reduction under the same conditions of oxidation and demonstrated that exposure of M. miehei proteinase to 0.1 M periodate for more than 5 h resulted in little additional loss of carbohydrate, but caused a large decrease in activity. No activity loss could be detected
0 27
oo: .o 2. -o3. -o4,
0 26.
.o5. -o6, .o7. -o6.
0.25,
O.
.og,
c
.11.
e B
~ o24
-13, -~4,
o ....
,3
6
~ o.23 ,,
o ' ~ " ~ '~"
;6"2b'~'4'
T,me (m,n~
o
---7 D 0.22=
-0.~
•
~45
,
•
,
,
=
200 Wavelength (rim)
,
,
,
,
0
350
D U O D
n
O
21
0 T i m e (rnin)
Fig. L Repeated spectral scan (a) during the denaturation of M. raiehei proteinas¢ in 3,8 M Gdn-HCL Enzyme concez~tration was 0,,LSOm g / m l in 0.025 M Mes buffer (pH 5.4). Scanning times wc.~e I, 2.3, 5.2, 7.S, 11.5, 18.5 and 45.2 rain after mixin~ for curves I, 2, 3, 4, S, 6 and 7, respectively. Decrease in absorbance Co) at 285.7 nm for the proteinase of M. michel in 4 M Gdn-HCI. Enzyme concentration was 0.250 mg,/nd in 0.025 M Mes buffer (pH 5.4). Inset shows the f'wsborder transformation of time-course data and the regressed line.
410 tures as those for Gdn-HCI denaturation (data not shown). In particular, the minimum at 285.7 nm was also demonstrated in temperture jump experiments. Kinetics of denaturation. The time-course of Gdn-HCI induced denaturation, evidenced by a decrease in absorption at 285.7 nm for the enzymes from M. michel (Fig. lb) and E. parasitica (data not shown), was found to follow first-order kinetics. This suggested that denaturation was best approximated by a two-state model where-only native and denatured molecules exist in significant concentrations. These findings are in agreement with studies of the two-state kinetics of urea and Gdn-HCi denaturation of pepsin [25,26] and of pepsinogen [14]. Rate constants found through regression of the linear transformation (Fig. l b inset) were 0.058 and 0.028 rain-t for M. mtehei and E. parasitica proteinases (data not shown), respectively. Fig. 2a depicts typical first-order semilogarithmie plots of the isothermal denaturation of E. parasitica proteinase as evidenced by a decrease in absorbance at 285.7 nm. This plot and those for M. miehei and the carbohydrate-reduced M. miehei proteinases (data not shown) demonstrated the first-order, two-state behaviour already documented for thermal inactivation of these proteinases [4,27] and confirmed the persistence of this behaviour in the modified M. miehei proteinase. While the Arrhenius plots for E. parasitica proteinase and the unmodified M. miehei enzyme (Fig. 2b) appeared linear in character, that for the deglycosylated form of M. miehei was somewhat curvilinear (Fig. 2b). This suggested that a heterogeneous population existed which may have consisted of differentially deglyco-
.o,.
,
TABLE 1
Activation energies a in kJ/mol for the denaturation of M. miehei, E. parasitica and deglycosylated M. miehei proteinases followed by ultraviolet spectroscopy and enzyme activity Method
Proteinase
M. michel E. par~itica Ultraviolet spectroscopy 437± 12 Enzyme activity 458 ± 41
250± l0 225 ± 11
M. michel (deglycosylated) 370± 19 339 ± 30
a Activation energy determined from simple linear regression of the Arrhenius plot (error term is the standard error for the determination of slope or Ea ).
sylated species. In experiments employing enzyme activity as a measure of structural integrity, first-order plots of residual activity (data not shown) confirmed the first-order nature of the reaction and were used to derive the energy of activation from Arrhenius plots. Again, the Arrhenins plot for the modified enzyme from M. miehei (data not shown) was somewhat curvilinear. Activation energies for the denaturation of M. michel and E. parasitica proteinases were respectively, in ultraviolet experiments 437 and 250 l d / m o l and in inactivation studies 458 and 225 kJ/mol (Table I). That the values were not grossly different using the two methods, suggested that irreversible enzyme inactivation was coincident with a gross conformational change and provided further support for the two-state approximation. Using inactivation as the index for denaturation Hyslop et al. [4] reported a somewhat higher value (552 Ll/mol) -0 Bi O -o.91o -111
b ~ ~
-11L -12~-
~..
-13[-
g_o~
\
-1.4!.
÷
-'5~
+~
-o;
-'~B[ -tg| -2[
-08 -09 -1
.... o '
-21~ ~ '~-'
6
Time ( rnlnJ
. . . .8. . .
:o
,.
-22 I , t , , • k , 290292 294 296 298 300 302 304 306 30B lIT
(x10"3 K)
Fig. 2. Semilogarithmic plots (a) of Iog(At-Af)/(A o - Af), where absorbanee, abbreviated A, was monitored at 285.7 nm vs. time for the isothermal denaturation of E parasitica proteinase at temperatures 52.5°C (ll), 55.5°C (
