[1]
TITRATION OF CYSTEINE PROTEASES
3
[1] A c t i v e Site T i t r a t i o n o f C y s t e i n e P r o t e a s e s ~ B y F. J. K~ZDY and E. T. KAISER
Cysteine proteases catalyze the hydrolysis of carboxylic acid derivatives through a double-displacement pathway involving the generaNacid general-base catalyzed formation and hydrolysis of an acyl-thiol intermediate. 2 The great similarity of this mechanism to that displayed by serine proteases assures that the active site titration methods elaborated for serine proteases ~ are also applicable without much modification to the determination of active site concentrations of sulfhydryl proteases. Indeed, such methods have been used successfully for several plant sulfhydryl proteases, with due allowance for the specificity of the individual enzymes in the choice of the titrating agent. However, the presence of an active site thiol group as the acyl acceptor confers special properties to the sulfhydryl proteases, as compared to their serine analogs. Among amino acid side chain functions the SH group of cysteine is unique by its reactivity toward electrophilic reagents, oxidizing agents, and heavy metal ions. Since in most cysteine proteases the active-site thiol is the only sulfhydryl group on the protein molecule, the design of titrating agents is greatly facilitated by this unique reactivity. 4 As an example, the use of chromophoric organomercurials could be singled out for the rapid and sensitive quantitation of sulfhydryl groups at specific sites. 5 The advantages of a single thiol group located in a catalytically favorable position at the active site are somewhat offset, however, by certain unfavorable characteristics inherent to the cysteine proteases. The high intrinsic nucleophilicity of the thiolate ion readily leads to undesired side reactions, such as disulfide exchanges within the protein molecule itself, or the reaction with trace contaminants. Also, free sulfhydryl groups are extremely sensitive toward oxidation by air, and accurate measurements should be carried out in deoxygenated solutions, under nitrogen atmosphere. Most importantly, however, these enzymes are inhibited by minute amounts of heavy metal ions present in virtually all buffers. To counteract these inactivating factors, reproducible kinetic experiments with cysteine proteases require the presence of large amounts 1Supported in part by grants GM-15951 and GM-13885 from the National Institutes of Health. 2M. L. Bender and L. J. Brubacher, J. Am. Chem. Soc. 86, 5333 (1964). F. J. K6zdy and E. T. Kaiser, this series Vol. 19 [1]. 4R. L. Heinrikson and K. J. Kramer, Prog. Bioorg. Chem. 3, 179 (1974). 5C. H. McMurray and D. R. Trenton, Biochem. J. 115, 913 (1969).
4
GENERAL ASPECTS
[ll
of protecting agents, such as 1 mM cysteine or dithiothreitol, in conjunction with comparable concentrations of chelating agents, such as EDTA or EGTA. Such conditions preclude the use of thiol-specific reagents unless the reagents incorporate active site-directed binding groups. Finally, the broad pH optimum of most of the sulfhydryl proteases results in significant autoproteolysis upon storage for even short periods. For these reasons, the enzymes are prepared and stored in an inactive form, usually as mercury or as phenylmercury complexes. They are activated then either in situ or immediately before use. Owing to the instability of these enzymes and their possible contamination by enzymatically inactive thiols, the titration of active sites by thiolspecific reagents must be accompanied by measurement of the parallel loss of enzymic activity toward a specific substrate. Optimally, a variety of reagents should be used in conjunction with the "burst titration" by a chromogenic substrate, and all methods should yield consistent results before the data are used to calibrate a convenient rate assay in terms of the molarity of the active sites. In the following discussion, papain and bromelain will be used as examples to illustrate some of the experimental approaches leading to the establishment of rate assays for cysteine proteases. The Use of an Irreversible,Covalently B o u n d Inhibitor: Titration of the Active Site of Papain with a-Bromo-4hydroxy-3-nitroacetophcnone (I)6-s
At present the titration procedure to be discussed appears to be the most convenient way of determining the operational normality of the active sites in papain solutions. The reaction that has been utilized is the rapid alkylation of the active site sulfhydryl group of papain by a-bromo4-hydroxy-3-nitroacetophenone (I). This process gives the irreversibly inactivated species shown as structure (II) in Eq. (1) below. The modification process can be followed by direct spectrophotometric titration, utilizing differences in the ultraviolet absorption spectra of the chromophoric group of the reagent (I) and the modified species (II). Alternatively, rate assays of active enzyme remaining after modification with less than stoichiometric amounts of (I) can be performed employing suitable substrates or sulfhydryl titrants. By linear extrapolation of the rate assay measurements to the point of quantitative inhibition, the equivalence point in the titration can also be established.
8R. W. Furlanetto and E. T. Kaiser, J. Am. Chem. Soc. 92, 6980 (1970). R. W. Furlanetto and E. T. Kaiser, J. Am. Chem. Soc. 95, 6786 (1973). 8R. W. Furlanetto, Ph.D. Thesis, University of Chicago, 1972.
[1]
TITRATION OF CYSTEINE PROTEASES
o
o
II
A ~
II
C-CH2Br HS--CH2
HO" T
NO~
5
+
f
A ~
~---~
Papain Ks
SO
C - CH2Br HS--CH,
t
T
Papain
NO~
(1) h2
(I)
otl
~.~C-- CHf--S--~H~ H O ~
Papain
NO2 (II)
Site o/Reaction The following evidence exists that the modification reaction shown in Eq. (1) occurs at the active site residue Cyszs. First, the scheme of Eq. (1) requires that the free sulfhydryl content of papain alkylated by reagent (I) be decreased in proportion to the concentration of the added alkylating agent. To test this, papain purified by affinity chromatography9 was treated with various concentrations of (I), and the sulfhydryl content of the resultant solutions was measured with the sulfhydryl reagent 5,5'ditihiobis(2-nitrobenzoic acid). 1° The results obtained clearly showed that the extent of alkylation was parallel to a loss in titratable sulfhydryl groups. Another type of evidence comes from a kinetic study of the alkylation of papain by (I).7 Specifically, the rate of alkylation of papain by (I) has been measured over the pH range 3 to 10.5. With the exception of the data measured for the more alkaline part of the pH range, it appears that the kinetics of the alkylation reaction can be explained in terms of the attack of the active site sulfhydryl group of papain on (I) assisted by some group in the enzyme acting as a general-base catalyst. 7 At high pH values nucleophilic attack by the thiolate form of Cys25 predominates. Thus, the same sulfhydryl group is modified at high pH as in the neutral pH region. However, while the titration of the sulfhydryl group in papain by (I) competes quite effectively in ac.idic or neutral solution with the reactions of (I) with model sulfhydryl compounds, there is no special selectivity for the sulfhydryl group of papain in very alkaline solutions. Thus, it is not recommended that titrations of the sulfhydryl group in papain be performed with (I) at pH values above pH 8. 'S. Blumberg, I. Schechter, and A. Berger, Eur. J. Biochem. 15, 97 (1970). 1°G. L. Ellman, Arch. Biochem. Biophys. 82, 70 (1959).
6
GENERAL ASPECTS
[i]
K i n e t i c Considerations
To establish firmly the theoretical basis of an active-site titration procedure, it is necessary to understand the kinetics of the reaction of the titrant with the active site of the enzyme. Some reservations have been expressed concerning the use of irreversible inhibitors as titrating agents, and arguments have been stated in favor of the use of specific substrates instead2 The major objections to the use of the irreversible inhibitors are that (1) they do not test the primary chemical reaction catalyzed by the enzyme; (2) they often react with more than one protein functional group ; (3) often the reaction does not depend on the specificity or catalytic activity of the enzyme. In the case of compound (I), because of the thorough kinetic investigation of the alkylation of papain with the reagent, only the first objection cannot be eliminated. A maior advantage in the use of specific substrates as titrants is that they do allow careful kinetic analysis of the turnover process on the enzyme, thereby providing valuable information concerning the homogeneity of the titrated species2 ,11 In the case of papain an active site titration method based on the use of specific substrate p-nitrophenyl N-benzyloxycarbonyl L-tyrosinate has been describedJ 1 However, the titration of the enzyme by this method necessitates a thorough study of the effect of substrate concentration upon the size of the burst observed, and this procedure seems to be considerably less expedient than the methods commonly used for the active site titration of chymotrypsin with substrates, for example. For this reason the direct spectrophotometric titration method described here using reagent (I) is preferred for the determination of the active-site concentrations of papain solutions. Besides the direct spectrophotometric titration procedure, it was possible to follow the course of the reaction of reagent (I) with papain, using rate assays with p-nitrophenyl N-benzyloxycarbonyl glycinate (CGN).12 These assays were carried out in 20 mM phosphate buffer, pH 6.8, 1 mM EDTA, 6.8% acetonitrile with [CGN]o >> [E]o. Under these conditions Eq. (2) describes the velocity of the enzyme-catalyzed hydrolysis and Km(a,p) ~ 1 V.M. Therefore, when [CGN]o ~ 0.1 mM, the initial rate, Vo -- kcat[E]o. Since Vo is directly proportional to [E]o, it is possible to compare relative enzyme concentrations of two solutions by comparing their initial velocities. By studying the rates of reaction of papain solutions allowed to react with various amounts of reagent (I), it is possible 1, M. L. Bender, M. L. Begue-Canton, R. L. Blakely, L. J. Brubacher, J. Feder, C. R. Gunter, F. J. K~zdy, J. V. Killheffer, Jr., T. H. Marshall, C. G. Miller, R. W. Roeske, and J. K. Stoops, J. Am. Chem. Soc. 88, 5890 (1966). 12j. F. Kirsch and M. Igelstrom, Biochemistry 5, 783 (1966).
[1]
TITRATION OF CYSTEINE PROTEASES
7
to determine the active site concentrations using assays of the remaining activity with the p-nitrophenyl ester. Once the active site concentrations of the papain solutions are determined in this way, then the true value of k~t in Eq. (2) can be calculated, permitting the use of rate assays with C G N for the measurement of absolute concentrations of active papain. v0 =
k¢~t[E]0[CGN] Km(~,,) + [CGN]
(2)
Direct Spectrophotometric Titration o] the Active Site of Papain A large change in the extinction coefficient of the o-nitrophenolate group occurs on the reaction of reagent (I) with papain. Although the reaction can be observed at either 262 nm (a drop in ~ of 6.2 X 10 ~ M -1 cm -1 at pH 3.5) and or at 323 nm (an increase in c of 6.85 }( 10 a M -1 cm -1 at pH 7.0), the longer wavelength was chosen for study because of the large protein absorption below 300 nm. It can be shown that when irreversible enzyme inhibition occurs in the presence of excess inhibitor, this is accompanied by a change in extinction coefficient at 323 nm for which the relationship of Eq. (3) holds. Here [E]o is the active site concentration, AA is the change in absorbance occurring on reaction, [I]o is the initial inhibitor concentration, and eE, c~, and cE~ are the extinction coefficients of the free enzyme, inhibitor (reagent I), and inhibited enzyme, respectively. [El0 =
AA (~
-
~i[I]o ~E
-
(3)
~i)
Typically, spectrophotometric titrations of papain solutions using the active-site titrant (I) were performed as follows, s The absorbanee of a 2-ml solution containing papain in 67 m M phosphate buffer, pH 7.0, 1 m M E D T A was measured in a 1 cm pathlength cell at 323 nm vs air (AE). A 50-~1 aliquot of a stock solution of (I) in aeetonitrile was then added with stirring. After 5 min, the absorbance of the solution was again measured at 323 nm (AE~). Finally, the absorbance of the free inhibitor was measured under exactly the same conditions of buffer, pH, wavelength and percent of acetonitrile (A~). (This reduced an error arising from uncertainty in the concentration of the stock inhibitor solution.) Care was taken to have at least a 1.2-fold excess of reagent (I) present. The final mixture contained 2.42% aeetonitrile. The active site concentration was calculated using Eq. (4) [the operational form of Eq. (3) ]. In Eq. (4), A~, A~, and A~ are defined as above, 6.85 X 103 M 1 cm-1 is the value of (c~i - - ~ - - e~) determined experi-
8
GENERAL ASPECTS
[1]
mentally and the factor 1.025 corrects for the dilution occurring during the experimental procedure. [E]0 =
(AE
AEI -- AI) X 1.025 (M) 6.85 X 103 -
-
(4)
It is clear that to have an excess of inhibitor present either the approximate enzyme concentration must be known or the titration must be repeated using at least two different concentrations of reagent (I). It should be noted t h a t the best results are obtained when enzyme concentrations range from 15 to 30 t~M. Below 15 ~M the change in absorbance on reaction is small and difficult to measure, and at concentrations above 30 uM the background absorption is large (since excess inhibitor must be used) and it becomes difficult to measure superimposed changes. The principal limitations of the method described for the titration of papain solutions is that the consumption of considerable amounts of concentrated enzyme solution is required in order to achieve reasonable accuracy. One reagent that has better spectral properties for the titration of the active site of papain, is fl-(2-hydroxy-3,5-dinitrophenyl)ethanesulfonic acid sultone. 13 This compound can be used to titrate papain at a wavelength of 400 nm, where the protein absorbance is vanishingly small and the optical change in the titration reaction is more than twice that seen with reagent (I) under optimal conditions. However, the use of the sultone is somewhat limited at the present time owing to the tedious synthetic procedure for its preparation. Syntheses Preparation o] 4-Hydroxy-3-nitroacetophenone.S, 14 Over a period of 15 rain, 10.5 g of 4-hydroxyacetophenone (Aldrich, m.p. 106°-108 °, 0.077 mole) was added to 53 ml of fuming nitric acid (dido 1.49, 79.5 g) while the temperature was maintained at --25 ° to --30 ° . The solution was stirred for an additional 60 min at --30 ° and then poured into 600 g of ice water. The yellowish precipitate was collected by filtration and recrystallized twice from ethanol, yielding 9.6 g (70%) yellow needles, m.p. 133°-134 °, lit. '4 m.p. 135 °. Preparation of a-Bromo-4-hydroxy-3-nitroacetophenone2,15 To 0.52 g of bromine (0.0053 mole) in 11.0 ml of chloroform at room temperature was added 1.00 g of 4-hydroxy-3-nitroacetophenone (0.0055 mole). The initially red solution turned clear brown, and hydrogen bromide gas 13p. Campbell and E. T. Kaiser, J. Am. Chem. Soc. 95, 3735 (1973). 14p. D. Bartlett and E. N. Trachtenberg, J. Am. Chem. Soc. 80, 5808 (1958). 15G. Sipos and R. Szabo, Acta Phys. Chem. 7, 126 (1961).
[1]
TITRATION OF CYSTEINE PROTEASES
9
evolved. The temperature was raised to 40 ° and maintained there for 15 rain; the solution was then extracted once with water and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure, and the product was recrystallized twice from carbon tetrachloride, yielding 1.02 g (75%) yellow needles, m.p. 91.5°-92.5 °, lit. ~5 m.p. 93 °. Titration of Bromelain A
The purification of the phenylmcrcury derivative of bronielain A is described in this volume [64]. For the spectrophotometric titration of the active-site thiol, the enzyme is activated by a reducing agent and then allowed to react with Ellman's reagent TM in a metal-free buffer. For the spectrophotometric burst titration the specific substrate p-nitrophenyl N"-benzyloxycarbonyl-L-lysinate (CLN) is used in the presence of 1 mM L-cysteine.
Activation of the Enzyme TM In the past, the mercury or organomercurial complexes of cysteine proteases were activated by thiocresol dissolved in toluene, followed by repeated washings with toluene in order to eliminate the excess reagent. 17 The use of immobilized thiol groups on solid supports TM simplifies considerably the activation process, eliminating at the same time the presence of a water-organic solvent interface, which invariably resulted in some denaturation of the enzyme. In the authoi's' laboratory the following process was found convenient for the activation of phenylmercury bromelain. Into a vial containing 1 g of dry Affigel 10 (Bio-Rad Laboratories) one injects with a syringe 25 ml of 0.1 M phosphate buffer, pH 7.0, saturated with L-cystine. The mixture is shaken for 24 hr at 4 °. The resulting slurry is used to prepare a 0.8 X 10 cm column. The column is first washed with 500 ml of 0.1 M phosphate buffer, pH 7.0, containing 1 M NaC1, until the eluate is free from N-hydroxysuccinimide, as monitored at 260 mm. The L-cystine is reduced by passing through the column 200 ml of 0.1 M phosphate buffer, pH 7.0 containing 1 M 2-mercaptoethanol. The column is then washed with approximately 1 liter of 0.1 M phosphate 16M. N. Reddy, unpublished observations. 17 M. Soejima and K. Shimura, J. Biochem. (Tokyo) 49, 260 (1961). 1~p. Cuatrecasas and C. B. Anfinsen, this series Vol. 22 [31].
10
GENERAL ASPECTS
[1]
buffer, pH 7.0, until the eluate is free from 2-mercaptoethanol, as measured with Ellman's reagent. For the activation of the enzyme, 1 ml of 5 X 10.4 M phenylmercury bromelain A is applied to the column at 4 ° and eluted with 0.1 M phosphate buffer pH 7.0 at a flow-rate of 15 ml/hr. Under these conditions the enzyme is more than 98% activated without any measurable loss of total protein. The column can be reactivated repeatedly by successive washings with 1 M 2-mercaptoethanol and the appropriate buffer. Sulfhydryl Group Titration ~9
The spectrophotometric titration of the thiol group of bromelain A is carried out in the presence of 60 tLM 5,5'-dithiobis(2-nitrobenzoic acid), 10 mM phosphate buffer, pH 7.04, 0.1 M KC1 at 25% Addition of a small aliquot (5-50/A) of an activated bromelain solution (10-20 mg/ml) to 3 ml of this solution yields a slow, first-order increase of the absorbance at 412 nm. In independent experiments it was shown that under the same conditions L-cysteine reacts 400 times faster with Ellman's reagent, and thus any spectral change due to small residual amounts of cysteine could be corrected for by extrapolating the slow absorbance change to the beginning of the reaction. The sulfhydryl content of the activated enzyme was determined from the total spectral change, using a A~ -- 13,600 M -1 cm -1. Experiments at several different enzyme concentrations showed that the sulfhydryl titer is linearly proportional to the protein content and the enzymic activity toward CLN. From the amount of protein added, and assuming one thiol group per enzyme molecule, an apparent MW of 36,000 was calculated, indicating that the enzyme preparation was more than 95% pure. Similar results were obtained when the loss of enzymic activity toward CLN was measured upon addition of aliquots of an aqueous solution of p-mercuribenzenesulfonate. The inhibition of the activated enzyme was proportional to the amount of inhibitor added, and total inhibition occurred at stoichiometric equivalence. Burst Titration 2°
The steady-state kinetics of the bromelain A-catalyzed hydrolysis of CLN obey Eq. (2) with kc~t ~ 7.5 sec-1 and Km= 57 ~M at pH 4.60, 25 ° and tL -- 0.1 M. The rapid initial phase of the reaction is monitored on a Durrum-Gibson stopped-flow spectrophotometer, after mixing equal 1~R. M. Silverstein and F. J. K@zdy, Arch. Biochem. Biophys. 167, 678 (1975).
R. M. Silverstein, S. H. Perlstein, and F. J. K~zdy, unpublished experiments.
[1]
TITRATION
OF CYSTEINE
PROTEASES
11
volumes of the substrate dissolved in water (1.6% acetonitrile) and the enzyme in an 0.1 M acetate buffer, pH 4.6, 1 m M L-cysteine. The progress of the reaction is measured at 340 or 370 nm. The rate of appearance of p-nitrophenol during the first 100 msec of the bromelain-catalyzed hydrolysis of CLN shows a "burst" typical of enzymic reactions involving the rapid formation of an acyl-enzyme intermediate. Analogous with the reaction of bovine trypsin with the same substrate, the reaction of CLN with bromelain is not completely deacylation rate-limiting: the steady-state turnover following the initial rapid "burst" is appreciable. By extrapolating this steady-state straight line to the initial time and by plotting the logarithm of the difference between this line and the experimental curve vs time, one can determine an apparent first-order rate constant, b, for a series of initial substrate concentrations. These constants yield a straight line when plotted as 1/b vs 1/S. From the slope and intercept of this line and the steady-state parameters of the enzymic hydrolysis at the same pH, one can calculate the mechanistically meaningful kinetic parameters defined in Eq. (5). k2
ka
E + S ~ ES --* ES' -t- P, --~ E -t- P~
(5)
Ks
E, ES, and ES' repreesnt the free enzyme, the enzyme-substrate complex, and the aeyl-enzyme, respectively, and S is the free substrate. P1 is p-nitrophenol and P._, is the product carboxylic acid. It has been shown that the constants, ks, k3, and Ks are related to the experimental constants, b, kcat, and Km, by Eqs. (6)-(8).3 1/b = 1/k: + K s ~ k 2 . 1 / S 1/kcat = 1/k2 -b 1/k3 Km = [k~/(k2 + k~)lKs
(6) (7) (8)
The values of the constants calculated from the experimental data are as follows: k2 = 140 sec -1, k3 = 7.9 sec -1, and Ks -- 1.6 mM. Since equations (6) and (8) allow one to calculate all the parameters, Eq. (7) can be used for an independent verification of the consistency of the constants. In this way, we calculated a value of k c a t = 7 -~- 2 sec -1 in good agreement with the experimentally determined value, 7.5 sec -1, based on enzymic concentrations calculated from sulfhydryl titrations. Thus, our data are fully consistent with the acyl-enzyme hypothesis as shown in Eq. (5). The extrapolated value of p-nitrophenol liberated in the presteady state (Tr) is defined for a reaction obeying Eq. (5) by Eq. (9). E0 = ~{[1 + (S//K,n)]//[]c2/(k2
~-]¢3)]} 1/2
(9)
12
GENERAL ASPECTS
[2]
With the use of this equation, the experimental value of ~r, and the true rate constants, one can calculate the concentration of molarity of active sites in the reaction mixture (Eo). At several different enzyme concentrations, the values thus obtained agreed within -----5% with those obtained by titration of the active site thiol groups, thereby demonstrating that the bromelain A preparation used in these experiments was devoid of impurities. Conclusions
Because of the high reactivity and selectivity of thiol groups toward function-specific reagents, it is tempting to use a single method such as titration with Ellman's reagent, 1° organomercurials, or alkylating agents. Although, in principle, with pure enzyme solutions the results of such titrations do yield the correct normality for enzyme active sites under appropriate reaction conditions, often competition from contaminating thiol compounds and inactive protein leads to spurious measurements. Burst titrations also have their shortcomings both experimental and theoretical. Therefore, for cysteine proteases even more than for serine proteases, it is imperative that more than one titration method be used in the determination of enzyme active site normality.
[2] S o m e S e n s i t i v e M e t h o d s f o r t h e A s s a y o f Trypsinlike Enzymes 1
By P. L. COLEMAN,H. G. LATHAM,JR., and E. N. SHAW There is a need for more sensitive assays for proteolytic enzymes as increasing numbers are discovered that are present in minute quantities and have biologically important roles. Among these, serine proteases in particular predominate, since they are essential in blood clotting, clot removal, complement action, and fertilization, to name a few areas of current interest. Most of the individual enzymes have a trypsinlike specificity. Two types of measurements are of value in the assay of proteases: the determination of the concentration of active sites, and the deter1Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Energy Research and Development Administration and with support from U.S. Public Health Service Grant 17849 from the National Institute of General Medical Sciences.
TITRATION OF CYSTEINE PROTEASES
3
[1] A c t i v e Site T i t r a t i o n o f C y s t e i n e P r o t e a s e s ~ B y F. J. K~ZDY and E. T. KAISER
Cysteine proteases catalyze the hydrolysis of carboxylic acid derivatives through a double-displacement pathway involving the generaNacid general-base catalyzed formation and hydrolysis of an acyl-thiol intermediate. 2 The great similarity of this mechanism to that displayed by serine proteases assures that the active site titration methods elaborated for serine proteases ~ are also applicable without much modification to the determination of active site concentrations of sulfhydryl proteases. Indeed, such methods have been used successfully for several plant sulfhydryl proteases, with due allowance for the specificity of the individual enzymes in the choice of the titrating agent. However, the presence of an active site thiol group as the acyl acceptor confers special properties to the sulfhydryl proteases, as compared to their serine analogs. Among amino acid side chain functions the SH group of cysteine is unique by its reactivity toward electrophilic reagents, oxidizing agents, and heavy metal ions. Since in most cysteine proteases the active-site thiol is the only sulfhydryl group on the protein molecule, the design of titrating agents is greatly facilitated by this unique reactivity. 4 As an example, the use of chromophoric organomercurials could be singled out for the rapid and sensitive quantitation of sulfhydryl groups at specific sites. 5 The advantages of a single thiol group located in a catalytically favorable position at the active site are somewhat offset, however, by certain unfavorable characteristics inherent to the cysteine proteases. The high intrinsic nucleophilicity of the thiolate ion readily leads to undesired side reactions, such as disulfide exchanges within the protein molecule itself, or the reaction with trace contaminants. Also, free sulfhydryl groups are extremely sensitive toward oxidation by air, and accurate measurements should be carried out in deoxygenated solutions, under nitrogen atmosphere. Most importantly, however, these enzymes are inhibited by minute amounts of heavy metal ions present in virtually all buffers. To counteract these inactivating factors, reproducible kinetic experiments with cysteine proteases require the presence of large amounts 1Supported in part by grants GM-15951 and GM-13885 from the National Institutes of Health. 2M. L. Bender and L. J. Brubacher, J. Am. Chem. Soc. 86, 5333 (1964). F. J. K6zdy and E. T. Kaiser, this series Vol. 19 [1]. 4R. L. Heinrikson and K. J. Kramer, Prog. Bioorg. Chem. 3, 179 (1974). 5C. H. McMurray and D. R. Trenton, Biochem. J. 115, 913 (1969).
4
GENERAL ASPECTS
[ll
of protecting agents, such as 1 mM cysteine or dithiothreitol, in conjunction with comparable concentrations of chelating agents, such as EDTA or EGTA. Such conditions preclude the use of thiol-specific reagents unless the reagents incorporate active site-directed binding groups. Finally, the broad pH optimum of most of the sulfhydryl proteases results in significant autoproteolysis upon storage for even short periods. For these reasons, the enzymes are prepared and stored in an inactive form, usually as mercury or as phenylmercury complexes. They are activated then either in situ or immediately before use. Owing to the instability of these enzymes and their possible contamination by enzymatically inactive thiols, the titration of active sites by thiolspecific reagents must be accompanied by measurement of the parallel loss of enzymic activity toward a specific substrate. Optimally, a variety of reagents should be used in conjunction with the "burst titration" by a chromogenic substrate, and all methods should yield consistent results before the data are used to calibrate a convenient rate assay in terms of the molarity of the active sites. In the following discussion, papain and bromelain will be used as examples to illustrate some of the experimental approaches leading to the establishment of rate assays for cysteine proteases. The Use of an Irreversible,Covalently B o u n d Inhibitor: Titration of the Active Site of Papain with a-Bromo-4hydroxy-3-nitroacetophcnone (I)6-s
At present the titration procedure to be discussed appears to be the most convenient way of determining the operational normality of the active sites in papain solutions. The reaction that has been utilized is the rapid alkylation of the active site sulfhydryl group of papain by a-bromo4-hydroxy-3-nitroacetophenone (I). This process gives the irreversibly inactivated species shown as structure (II) in Eq. (1) below. The modification process can be followed by direct spectrophotometric titration, utilizing differences in the ultraviolet absorption spectra of the chromophoric group of the reagent (I) and the modified species (II). Alternatively, rate assays of active enzyme remaining after modification with less than stoichiometric amounts of (I) can be performed employing suitable substrates or sulfhydryl titrants. By linear extrapolation of the rate assay measurements to the point of quantitative inhibition, the equivalence point in the titration can also be established.
8R. W. Furlanetto and E. T. Kaiser, J. Am. Chem. Soc. 92, 6980 (1970). R. W. Furlanetto and E. T. Kaiser, J. Am. Chem. Soc. 95, 6786 (1973). 8R. W. Furlanetto, Ph.D. Thesis, University of Chicago, 1972.
[1]
TITRATION OF CYSTEINE PROTEASES
o
o
II
A ~
II
C-CH2Br HS--CH2
HO" T
NO~
5
+
f
A ~
~---~
Papain Ks
SO
C - CH2Br HS--CH,
t
T
Papain
NO~
(1) h2
(I)
otl
~.~C-- CHf--S--~H~ H O ~
Papain
NO2 (II)
Site o/Reaction The following evidence exists that the modification reaction shown in Eq. (1) occurs at the active site residue Cyszs. First, the scheme of Eq. (1) requires that the free sulfhydryl content of papain alkylated by reagent (I) be decreased in proportion to the concentration of the added alkylating agent. To test this, papain purified by affinity chromatography9 was treated with various concentrations of (I), and the sulfhydryl content of the resultant solutions was measured with the sulfhydryl reagent 5,5'ditihiobis(2-nitrobenzoic acid). 1° The results obtained clearly showed that the extent of alkylation was parallel to a loss in titratable sulfhydryl groups. Another type of evidence comes from a kinetic study of the alkylation of papain by (I).7 Specifically, the rate of alkylation of papain by (I) has been measured over the pH range 3 to 10.5. With the exception of the data measured for the more alkaline part of the pH range, it appears that the kinetics of the alkylation reaction can be explained in terms of the attack of the active site sulfhydryl group of papain on (I) assisted by some group in the enzyme acting as a general-base catalyst. 7 At high pH values nucleophilic attack by the thiolate form of Cys25 predominates. Thus, the same sulfhydryl group is modified at high pH as in the neutral pH region. However, while the titration of the sulfhydryl group in papain by (I) competes quite effectively in ac.idic or neutral solution with the reactions of (I) with model sulfhydryl compounds, there is no special selectivity for the sulfhydryl group of papain in very alkaline solutions. Thus, it is not recommended that titrations of the sulfhydryl group in papain be performed with (I) at pH values above pH 8. 'S. Blumberg, I. Schechter, and A. Berger, Eur. J. Biochem. 15, 97 (1970). 1°G. L. Ellman, Arch. Biochem. Biophys. 82, 70 (1959).
6
GENERAL ASPECTS
[i]
K i n e t i c Considerations
To establish firmly the theoretical basis of an active-site titration procedure, it is necessary to understand the kinetics of the reaction of the titrant with the active site of the enzyme. Some reservations have been expressed concerning the use of irreversible inhibitors as titrating agents, and arguments have been stated in favor of the use of specific substrates instead2 The major objections to the use of the irreversible inhibitors are that (1) they do not test the primary chemical reaction catalyzed by the enzyme; (2) they often react with more than one protein functional group ; (3) often the reaction does not depend on the specificity or catalytic activity of the enzyme. In the case of compound (I), because of the thorough kinetic investigation of the alkylation of papain with the reagent, only the first objection cannot be eliminated. A maior advantage in the use of specific substrates as titrants is that they do allow careful kinetic analysis of the turnover process on the enzyme, thereby providing valuable information concerning the homogeneity of the titrated species2 ,11 In the case of papain an active site titration method based on the use of specific substrate p-nitrophenyl N-benzyloxycarbonyl L-tyrosinate has been describedJ 1 However, the titration of the enzyme by this method necessitates a thorough study of the effect of substrate concentration upon the size of the burst observed, and this procedure seems to be considerably less expedient than the methods commonly used for the active site titration of chymotrypsin with substrates, for example. For this reason the direct spectrophotometric titration method described here using reagent (I) is preferred for the determination of the active-site concentrations of papain solutions. Besides the direct spectrophotometric titration procedure, it was possible to follow the course of the reaction of reagent (I) with papain, using rate assays with p-nitrophenyl N-benzyloxycarbonyl glycinate (CGN).12 These assays were carried out in 20 mM phosphate buffer, pH 6.8, 1 mM EDTA, 6.8% acetonitrile with [CGN]o >> [E]o. Under these conditions Eq. (2) describes the velocity of the enzyme-catalyzed hydrolysis and Km(a,p) ~ 1 V.M. Therefore, when [CGN]o ~ 0.1 mM, the initial rate, Vo -- kcat[E]o. Since Vo is directly proportional to [E]o, it is possible to compare relative enzyme concentrations of two solutions by comparing their initial velocities. By studying the rates of reaction of papain solutions allowed to react with various amounts of reagent (I), it is possible 1, M. L. Bender, M. L. Begue-Canton, R. L. Blakely, L. J. Brubacher, J. Feder, C. R. Gunter, F. J. K~zdy, J. V. Killheffer, Jr., T. H. Marshall, C. G. Miller, R. W. Roeske, and J. K. Stoops, J. Am. Chem. Soc. 88, 5890 (1966). 12j. F. Kirsch and M. Igelstrom, Biochemistry 5, 783 (1966).
[1]
TITRATION OF CYSTEINE PROTEASES
7
to determine the active site concentrations using assays of the remaining activity with the p-nitrophenyl ester. Once the active site concentrations of the papain solutions are determined in this way, then the true value of k~t in Eq. (2) can be calculated, permitting the use of rate assays with C G N for the measurement of absolute concentrations of active papain. v0 =
k¢~t[E]0[CGN] Km(~,,) + [CGN]
(2)
Direct Spectrophotometric Titration o] the Active Site of Papain A large change in the extinction coefficient of the o-nitrophenolate group occurs on the reaction of reagent (I) with papain. Although the reaction can be observed at either 262 nm (a drop in ~ of 6.2 X 10 ~ M -1 cm -1 at pH 3.5) and or at 323 nm (an increase in c of 6.85 }( 10 a M -1 cm -1 at pH 7.0), the longer wavelength was chosen for study because of the large protein absorption below 300 nm. It can be shown that when irreversible enzyme inhibition occurs in the presence of excess inhibitor, this is accompanied by a change in extinction coefficient at 323 nm for which the relationship of Eq. (3) holds. Here [E]o is the active site concentration, AA is the change in absorbance occurring on reaction, [I]o is the initial inhibitor concentration, and eE, c~, and cE~ are the extinction coefficients of the free enzyme, inhibitor (reagent I), and inhibited enzyme, respectively. [El0 =
AA (~
-
~i[I]o ~E
-
(3)
~i)
Typically, spectrophotometric titrations of papain solutions using the active-site titrant (I) were performed as follows, s The absorbanee of a 2-ml solution containing papain in 67 m M phosphate buffer, pH 7.0, 1 m M E D T A was measured in a 1 cm pathlength cell at 323 nm vs air (AE). A 50-~1 aliquot of a stock solution of (I) in aeetonitrile was then added with stirring. After 5 min, the absorbance of the solution was again measured at 323 nm (AE~). Finally, the absorbance of the free inhibitor was measured under exactly the same conditions of buffer, pH, wavelength and percent of acetonitrile (A~). (This reduced an error arising from uncertainty in the concentration of the stock inhibitor solution.) Care was taken to have at least a 1.2-fold excess of reagent (I) present. The final mixture contained 2.42% aeetonitrile. The active site concentration was calculated using Eq. (4) [the operational form of Eq. (3) ]. In Eq. (4), A~, A~, and A~ are defined as above, 6.85 X 103 M 1 cm-1 is the value of (c~i - - ~ - - e~) determined experi-
8
GENERAL ASPECTS
[1]
mentally and the factor 1.025 corrects for the dilution occurring during the experimental procedure. [E]0 =
(AE
AEI -- AI) X 1.025 (M) 6.85 X 103 -
-
(4)
It is clear that to have an excess of inhibitor present either the approximate enzyme concentration must be known or the titration must be repeated using at least two different concentrations of reagent (I). It should be noted t h a t the best results are obtained when enzyme concentrations range from 15 to 30 t~M. Below 15 ~M the change in absorbance on reaction is small and difficult to measure, and at concentrations above 30 uM the background absorption is large (since excess inhibitor must be used) and it becomes difficult to measure superimposed changes. The principal limitations of the method described for the titration of papain solutions is that the consumption of considerable amounts of concentrated enzyme solution is required in order to achieve reasonable accuracy. One reagent that has better spectral properties for the titration of the active site of papain, is fl-(2-hydroxy-3,5-dinitrophenyl)ethanesulfonic acid sultone. 13 This compound can be used to titrate papain at a wavelength of 400 nm, where the protein absorbance is vanishingly small and the optical change in the titration reaction is more than twice that seen with reagent (I) under optimal conditions. However, the use of the sultone is somewhat limited at the present time owing to the tedious synthetic procedure for its preparation. Syntheses Preparation o] 4-Hydroxy-3-nitroacetophenone.S, 14 Over a period of 15 rain, 10.5 g of 4-hydroxyacetophenone (Aldrich, m.p. 106°-108 °, 0.077 mole) was added to 53 ml of fuming nitric acid (dido 1.49, 79.5 g) while the temperature was maintained at --25 ° to --30 ° . The solution was stirred for an additional 60 min at --30 ° and then poured into 600 g of ice water. The yellowish precipitate was collected by filtration and recrystallized twice from ethanol, yielding 9.6 g (70%) yellow needles, m.p. 133°-134 °, lit. '4 m.p. 135 °. Preparation of a-Bromo-4-hydroxy-3-nitroacetophenone2,15 To 0.52 g of bromine (0.0053 mole) in 11.0 ml of chloroform at room temperature was added 1.00 g of 4-hydroxy-3-nitroacetophenone (0.0055 mole). The initially red solution turned clear brown, and hydrogen bromide gas 13p. Campbell and E. T. Kaiser, J. Am. Chem. Soc. 95, 3735 (1973). 14p. D. Bartlett and E. N. Trachtenberg, J. Am. Chem. Soc. 80, 5808 (1958). 15G. Sipos and R. Szabo, Acta Phys. Chem. 7, 126 (1961).
[1]
TITRATION OF CYSTEINE PROTEASES
9
evolved. The temperature was raised to 40 ° and maintained there for 15 rain; the solution was then extracted once with water and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure, and the product was recrystallized twice from carbon tetrachloride, yielding 1.02 g (75%) yellow needles, m.p. 91.5°-92.5 °, lit. ~5 m.p. 93 °. Titration of Bromelain A
The purification of the phenylmcrcury derivative of bronielain A is described in this volume [64]. For the spectrophotometric titration of the active-site thiol, the enzyme is activated by a reducing agent and then allowed to react with Ellman's reagent TM in a metal-free buffer. For the spectrophotometric burst titration the specific substrate p-nitrophenyl N"-benzyloxycarbonyl-L-lysinate (CLN) is used in the presence of 1 mM L-cysteine.
Activation of the Enzyme TM In the past, the mercury or organomercurial complexes of cysteine proteases were activated by thiocresol dissolved in toluene, followed by repeated washings with toluene in order to eliminate the excess reagent. 17 The use of immobilized thiol groups on solid supports TM simplifies considerably the activation process, eliminating at the same time the presence of a water-organic solvent interface, which invariably resulted in some denaturation of the enzyme. In the authoi's' laboratory the following process was found convenient for the activation of phenylmercury bromelain. Into a vial containing 1 g of dry Affigel 10 (Bio-Rad Laboratories) one injects with a syringe 25 ml of 0.1 M phosphate buffer, pH 7.0, saturated with L-cystine. The mixture is shaken for 24 hr at 4 °. The resulting slurry is used to prepare a 0.8 X 10 cm column. The column is first washed with 500 ml of 0.1 M phosphate buffer, pH 7.0, containing 1 M NaC1, until the eluate is free from N-hydroxysuccinimide, as monitored at 260 mm. The L-cystine is reduced by passing through the column 200 ml of 0.1 M phosphate buffer, pH 7.0 containing 1 M 2-mercaptoethanol. The column is then washed with approximately 1 liter of 0.1 M phosphate 16M. N. Reddy, unpublished observations. 17 M. Soejima and K. Shimura, J. Biochem. (Tokyo) 49, 260 (1961). 1~p. Cuatrecasas and C. B. Anfinsen, this series Vol. 22 [31].
10
GENERAL ASPECTS
[1]
buffer, pH 7.0, until the eluate is free from 2-mercaptoethanol, as measured with Ellman's reagent. For the activation of the enzyme, 1 ml of 5 X 10.4 M phenylmercury bromelain A is applied to the column at 4 ° and eluted with 0.1 M phosphate buffer pH 7.0 at a flow-rate of 15 ml/hr. Under these conditions the enzyme is more than 98% activated without any measurable loss of total protein. The column can be reactivated repeatedly by successive washings with 1 M 2-mercaptoethanol and the appropriate buffer. Sulfhydryl Group Titration ~9
The spectrophotometric titration of the thiol group of bromelain A is carried out in the presence of 60 tLM 5,5'-dithiobis(2-nitrobenzoic acid), 10 mM phosphate buffer, pH 7.04, 0.1 M KC1 at 25% Addition of a small aliquot (5-50/A) of an activated bromelain solution (10-20 mg/ml) to 3 ml of this solution yields a slow, first-order increase of the absorbance at 412 nm. In independent experiments it was shown that under the same conditions L-cysteine reacts 400 times faster with Ellman's reagent, and thus any spectral change due to small residual amounts of cysteine could be corrected for by extrapolating the slow absorbance change to the beginning of the reaction. The sulfhydryl content of the activated enzyme was determined from the total spectral change, using a A~ -- 13,600 M -1 cm -1. Experiments at several different enzyme concentrations showed that the sulfhydryl titer is linearly proportional to the protein content and the enzymic activity toward CLN. From the amount of protein added, and assuming one thiol group per enzyme molecule, an apparent MW of 36,000 was calculated, indicating that the enzyme preparation was more than 95% pure. Similar results were obtained when the loss of enzymic activity toward CLN was measured upon addition of aliquots of an aqueous solution of p-mercuribenzenesulfonate. The inhibition of the activated enzyme was proportional to the amount of inhibitor added, and total inhibition occurred at stoichiometric equivalence. Burst Titration 2°
The steady-state kinetics of the bromelain A-catalyzed hydrolysis of CLN obey Eq. (2) with kc~t ~ 7.5 sec-1 and Km= 57 ~M at pH 4.60, 25 ° and tL -- 0.1 M. The rapid initial phase of the reaction is monitored on a Durrum-Gibson stopped-flow spectrophotometer, after mixing equal 1~R. M. Silverstein and F. J. K@zdy, Arch. Biochem. Biophys. 167, 678 (1975).
R. M. Silverstein, S. H. Perlstein, and F. J. K~zdy, unpublished experiments.
[1]
TITRATION
OF CYSTEINE
PROTEASES
11
volumes of the substrate dissolved in water (1.6% acetonitrile) and the enzyme in an 0.1 M acetate buffer, pH 4.6, 1 m M L-cysteine. The progress of the reaction is measured at 340 or 370 nm. The rate of appearance of p-nitrophenol during the first 100 msec of the bromelain-catalyzed hydrolysis of CLN shows a "burst" typical of enzymic reactions involving the rapid formation of an acyl-enzyme intermediate. Analogous with the reaction of bovine trypsin with the same substrate, the reaction of CLN with bromelain is not completely deacylation rate-limiting: the steady-state turnover following the initial rapid "burst" is appreciable. By extrapolating this steady-state straight line to the initial time and by plotting the logarithm of the difference between this line and the experimental curve vs time, one can determine an apparent first-order rate constant, b, for a series of initial substrate concentrations. These constants yield a straight line when plotted as 1/b vs 1/S. From the slope and intercept of this line and the steady-state parameters of the enzymic hydrolysis at the same pH, one can calculate the mechanistically meaningful kinetic parameters defined in Eq. (5). k2
ka
E + S ~ ES --* ES' -t- P, --~ E -t- P~
(5)
Ks
E, ES, and ES' repreesnt the free enzyme, the enzyme-substrate complex, and the aeyl-enzyme, respectively, and S is the free substrate. P1 is p-nitrophenol and P._, is the product carboxylic acid. It has been shown that the constants, ks, k3, and Ks are related to the experimental constants, b, kcat, and Km, by Eqs. (6)-(8).3 1/b = 1/k: + K s ~ k 2 . 1 / S 1/kcat = 1/k2 -b 1/k3 Km = [k~/(k2 + k~)lKs
(6) (7) (8)
The values of the constants calculated from the experimental data are as follows: k2 = 140 sec -1, k3 = 7.9 sec -1, and Ks -- 1.6 mM. Since equations (6) and (8) allow one to calculate all the parameters, Eq. (7) can be used for an independent verification of the consistency of the constants. In this way, we calculated a value of k c a t = 7 -~- 2 sec -1 in good agreement with the experimentally determined value, 7.5 sec -1, based on enzymic concentrations calculated from sulfhydryl titrations. Thus, our data are fully consistent with the acyl-enzyme hypothesis as shown in Eq. (5). The extrapolated value of p-nitrophenol liberated in the presteady state (Tr) is defined for a reaction obeying Eq. (5) by Eq. (9). E0 = ~{[1 + (S//K,n)]//[]c2/(k2
~-]¢3)]} 1/2
(9)
12
GENERAL ASPECTS
[2]
With the use of this equation, the experimental value of ~r, and the true rate constants, one can calculate the concentration of molarity of active sites in the reaction mixture (Eo). At several different enzyme concentrations, the values thus obtained agreed within -----5% with those obtained by titration of the active site thiol groups, thereby demonstrating that the bromelain A preparation used in these experiments was devoid of impurities. Conclusions
Because of the high reactivity and selectivity of thiol groups toward function-specific reagents, it is tempting to use a single method such as titration with Ellman's reagent, 1° organomercurials, or alkylating agents. Although, in principle, with pure enzyme solutions the results of such titrations do yield the correct normality for enzyme active sites under appropriate reaction conditions, often competition from contaminating thiol compounds and inactive protein leads to spurious measurements. Burst titrations also have their shortcomings both experimental and theoretical. Therefore, for cysteine proteases even more than for serine proteases, it is imperative that more than one titration method be used in the determination of enzyme active site normality.
[2] S o m e S e n s i t i v e M e t h o d s f o r t h e A s s a y o f Trypsinlike Enzymes 1
By P. L. COLEMAN,H. G. LATHAM,JR., and E. N. SHAW There is a need for more sensitive assays for proteolytic enzymes as increasing numbers are discovered that are present in minute quantities and have biologically important roles. Among these, serine proteases in particular predominate, since they are essential in blood clotting, clot removal, complement action, and fertilization, to name a few areas of current interest. Most of the individual enzymes have a trypsinlike specificity. Two types of measurements are of value in the assay of proteases: the determination of the concentration of active sites, and the deter1Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Energy Research and Development Administration and with support from U.S. Public Health Service Grant 17849 from the National Institute of General Medical Sciences.
