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Separation and characterization of venom components in Loxosceles reclusa—II. Protease enzyme activity
0041-0IOIJ79/1101-0329SOZ.00/0
Torkww, Vol . 17, pp . 329-037 . O PerQamon Pana Ltd . 1979. Printed in Great Britain.
SEPARATION AND CHARACTERIZATION OF VENOM COMPONENTS IN LOXOSCELES RECLUSA-II. PROTEASE ENZYME ACTTVITY* YAw-SHONG JONG,t
B. R. Nolt>siErrr$ and JAi~s R. HE1TZfi
Departments of Biochemistryj' and Entomology$, Mississippi Agricultural and Forestry Experiment Station, Mississippi Stato University, Mississippi State, MS 39762, U.S .A. (Acceptedjor publication 29 January 1979) Ynw-Sxotvo JONG, B. R. NoxMenrr and Jn~rns R. HErrz. Separation and characterization of venom components in Loxosceles reclusa-II . Protease enzyme activity . Toxicon 17, 529-537. 1979 .-A protease from brown recluse spider venom hydrolyzes the amide linkage of amino acids containing aliphatic, aromatic, or basic side chains. The mol. wt of tho enzyme was 29,600 by SDS electrophoresis. The Km for L-leucyl-ß-naphthylamide is 375 x 10 -' M and the activation energy was 49,000 cal/mole. The pH profile indicates that the enzymo has a narrow active region centered at pH 7~2. Spider venom protease is inhibited irreversibly by ethyknediamine tetraacetic acid, N-ethylmaleimide and 8uorescein mercuric acetate. INTRODUCTION
Txs sltowx recluse spider, Loxosceles reclusa, has attracted much attention in the past 10 yr as the gravity of the problems caused by the bite of this spider have become known. The clinical manifestation of brown recluse venom on man is well established. However, basic information on the venom and venom components is still lacking. NAZHAT (M.S . Thesis, Oklahoma State Univ ., 1968) reported that there was no protease or phospholipase C or D activity, although some lipase activity was observed in the venom. HALL (M .S . Thesis, Oklahoma State Univ ., 1970) reported that the venom contained no phospholipase A activity, however, some hyaluronidase activity was found . In other studies esterase and hyaluronidase have been reported in L. reclusa venom; but alkaline phosphatase, collagenase, phospholipase A, dipeptidase, acetylcholinesterase, ribonuclease A and deoxyribonuclease were not found (WRIGHT et al., 1973 ; W1ucxT, 1973). WRIGHT and co-workers also characterized hyaluronidase which was considered to be a "spreading factor". Subsequently, alkaline phosphatase activity was demonstrated and characterized by HeaTZ and NoxltmlvT (1974) . Although some enzymes have been found in L. reclusa venom, the toxins which were separated by G»ri et al. (197 were not directly associated with the enzymatic activities of hyaluronidase, alkaline phosphatase and 5'-ribonucleotidase . EsxAFt and NOx i~T (197 histochemically demonstrated venom protease, esterase, lipase and alkaline phosphatase in L. reclusa envenomated larvae of Heliothis virescens and Musca domestica . The minute quantity of spider venom obtained per specimen prohibits many of the more routine enzyme studies. Purification of large amounts of an individual protein component is not feasible through ordinary biochemical methods. Thus, only the most sensitive methods can be utilized. Recently, JoNG et al. (1979) separated L. reclusa venom proteins +Publication No. 4033 of the Mississippi Agricultural and Forestry Experiment Station. This research was supported by Grant No. SROlES00651 from the National Institute of Health. 529
53 0
YAW-SHONG JONG, B. R. NORMENT and JAMFS R. HEI'TZ
utilizing preparative-disc electrophoresis in a one-step purification . Ten fractions were resolved with high reproducibility ; fraction 1 has been identified as a nucleotide. This report presents a further study of the enzyme components present in these spider venom fractions ; in this case, the purification and characterization of a proteolytic enzyme . MATERIALS AND METHODS Venom protease (Fig. 1) was assayed with slight modification of the procedure of Rorer (1965) . All substrates were prepared fresh before each experiment as 1~0 x 10 -' M in methanol stock solutions except L-cystina~di-ß-naphthylamide which was dissolved in dimethylformamide. Enzyme catalyzed reactions were calculated as the increase in fluorescence intensity per hr (~F/hr) against control reactions containing no enzyme . There was evidence of fluorescence intensity caused by the spontaneous decomposition of substrate, however, the linearity of the true enzyme activity with respect to enzyme concentration showed that the assay was reliable. H HN
~NHZ R- ~\ H C-N 0 ~-Ar~inoacyl-ß-rwphthylamide
~-Nophthylamine protease
(highty fluorescent product)
Hz 0
t
l ran-fluorescent substrate) R-CH
/NH2
~COOH
HYDROLYTIC CLEAVAGE
-Amino add
FIG. 1. OF L-AMINOACYL-ß-NAPFITHYLAMTDE DERIVATIVE4 IN TFIE PRESENCE OF PROTEASE. Different L-aminoacyl derivatives are variably substituted at R. The ß-naphthylamine is a highly fluorescent product whose excitation wavelength and emission wavelength are 335 and 410 nm, respectively .
Venom extraction, separation and collection procedures have boon previously described (JONG et al., 1979). The position of the spider venom protease was then determined by measuring activity in 0~2 ml aliquots from every second test tube fraction of each preparative-disc electrophoretic separation using L-alanyl-ß-naphthylamide as a substrate. The protease position was also determined using L-leucyl-ßnaphthylamide as a substrate. Protein concentration could not be determined in each fraction due to the small volumes. Venom protein was mixed with inhibitor solutions : 4~0 x 10 -' M ethylenediaminetetra-acetic acid (EDTA), 4~0 x 10-' M N-ethylmaleimide (NEM), or 2~0 x 10 -' M fluorescein mercuric acetate (FMA), in phosphate buffer for various times of pre-incubation before L-leucyl-ß-naphthylamide was added to initiate the enzyme catalyzed reaction. Enzyme activity was recorded at zero time and after 1 hr of incubation at 32°C and theAF/hr of each reaction was calculated . Inhibition was determined by comparing this activity with that observed in control reactions to which no inhibitorhad been added. L-leucyl-ß-naphthylamide was prepared in concentrations from 2~0 x 10 - ' M to 2~0 x 10 - ' M in phosphate buffer. Loxosceles reclusa crude venom protein initiated the enzyme reaction at 35 °C in the absence of MgCI, in sodium phosphate buffer, pH 7~2. Activities (~F/hr) were recorded at different substrate concentrations to calculate the K value according to the method of LINEWEAVER and BURR (1934) . The Km values at seven different temperatures were obtained in the same manner except the temperature of the waterbath was adjusted between 2945 and308 K. The theoretical basis of the temperature effect on spider venom protease activity is referred to in DDCON and W®H (1964) and SEGEL (1975) . A plot of In Ko vs 1/T gave the activation energy (E,) of the reaction . The E, value was derived from the slope of the line, -E,/R. The pool of fraction 4 from the preparative-disc eloctrophoretic separation was added to dialysis casing (Sigma Chemical Co . 250-9u) and was dialyzed against distilled water at 4°C for 6 hr . The protein component was rinsed three times with distilled water into a VirTis bottle after dialysis and lyophilized with a VirTis Lyophilizer (Model No. 10-145 MR BA). The dried sample was dissolved by adding 80111 of 0~1 M phosphate buffer, pH 7~0. SDS-polyacrylamide gel electrophoresis was conducted according to the procedar; of WEHER and OsaoRN (1969) . For the preparation of the sample solution, 40111 of venom protease
Separation and Characterization of Venom Components in Loxosceks reclusa--II
531
solution was added to 20 lIl of 2 M sucrose and 20 iIl of 005 ~ bromophenol blue . The sample was diluted to 0~1 ml and incubated overnight at room temperature . The standard proteins were made up in 001 M sodium phosphate at 5 mg/ml and were treated as described for the spider venom protease solution. The standard proteins bovine serum albumen, alcohol dehydrogenase (yeast), alcohol dehydrogenase (horse liver), hemoglobin and cytochrome C, were obtained from the Sigma Chemical Company, as was the denaturant, SDS. Electrophoresis was performed at a constant current of 3 mA per gel with the positive electrode in the lower chamber. Under these conditions the marker dye reached 1 cm from the bottom of the gel in approximately 5 hr . After electrophoresis, the gels were removed from the tubes and stained overnight at room temperature with 025 ~ Coomassie Blue in SO ~ methanol containing 10 ~ acetic acid . The gels were dostained for 15 min in 7~ acetic acid with a Canalco 1801 Quick Gel Destainer . The L-ar~oacyl-ß-naphthylamide derivatives, listed in Table 1, were obtained from ICN Pharmaceuticals, Inc. including: tyrosino-, tryptophan-, phenylalanino-, lysine-, arginine-, proline-, hydroxyproline-, isoleucino-, leucine-, valino-, glycine- and serino-ß-naphthylamide. L-Alanyl-ß-naphthylamido was obtained from the Sigma Chemical Company. IrLeucyl-ß.naphthylamide in Figs . 2-7 and Table 2 and Iralanyl-ß-ttaphthylamide in Fig. 7 were also obtained from the Sigma Chemical Company. RESULTS AND DISCUSSION
1.-Leucyl-ß-naphthylamide was the best protease substrate (+~--}-), whereas the aliphatic and basic aminoacyl derivatives, valine-, alanine-, arginine-, and lysine-, were hydrolyzed less rapidly (~---~) (Table 1). The venom protease hydrolyzed the aromatic TAaI~
I. ACTION OF L. rtC1USa VENOM
PROTEASE
ON
VARIOUS IrAMINOACYL-ß-NAPHYTiYLAA~E DERIVATIVES
$Ub6trateS
Bufer System
t-Aminoacyl-ß-naphthylamide derivatives Tyrosine Tryptophan l~Ienylahuvne Lysine Arginine Proline Hydroxyproline Isoleucine Leucine Valine Alanine Glycine Serine Cystine-di-ß-naphthylamide
CO, 1/2 H,O free base HBr salt HBr salt HBr salt HCl salt HBr salt HBr salt free base di-HBr salt
Glycine-HCl iPH = 2'64) -
Tris-HCI ~PH = ~'~) + + +-f++ 1-++ ++ ++ _
Glycine-NaOH ~PH = 9.40)
Couches containing 100 pg crude venom protein in 0~1 ml Tris~lycine buffer at pH 8~3, SO lIl 0~1 M MgCI, solution in 235 ml buffer solution. Reactions were initiated by adding SO pl 1~0 x 10 -' M L-aminoacyl-ß-naphthylamide solution . Fluorescence intensity was recorded at time zero and 1 hr later.
aminoacyl derivatives tryptophan and phenylalanine slowly (-~-), and L-isoleucyl-ßnaphthyhunide was not hydrolyzed at all (-). Similarly, there was no specific enryme in L. reclusa venom which cleaved the disulfide bond. There was also no protease activity recorded for the proline-, serine-, hydroxyproline- and glycine-derivatives. The brown recluse venom protease acted on substrate in neutral solution only ; no recordable activity was found in the acidic or basic pH ranges. This protease apparently has a high reactivity for R groups which are hydrophobic or contain a positive charge . L. reclusa venom protease possessed a narrow pH range of optimal activity centering at approximately 7"2 (Fig. 2). The enzyme assays recorded in Table 1 were in Tris-buffer, pH 7"29, which is close to the optimum pH value. Previously, s-amino-n-caproic acid had been utilized as a competitive inhibitor for protease, because there is both an amino group and an acid group on the molecule (LORAND
53 2
YAW-SHON(} JONG, H. R NORMENT and JAMES R. HEITZ
PH
FYo. 2. L. IECIYBR VFNOM PROTEASE ACITVITY AS A FUNCTION OF pH . Reaction mixtures contained 6 x 10'' M MgCI, and 100 ~g crude venom solution in 2"S ml phosphate buffer ( ") or glycine-NaOH buffer (O). After pre-incubation for 5 min at 33 "5°C, SO pl of 1" 0 x 10- ' M Uleucyl-ß-naphthylamide were added. Fluorescence intensity was recorded at time zero and 1 hr later.
and Coxnrr, 196 . However, results indicated that e-amino-n-caproic acid had no effect on L. rechrsa venom protease activity (Table 2). Similarly, the lack of inhibition by a serinetype inhibitor, phenyhnethylsulfonyl fluoride, indicated that serine was not a functional amino acid in the active site of L. reclusa venom protease . TA$LE
Enzyme Venom -F Venom Venom Venom -F Venom Venom
-
8 8 8 8
x x x x
2.
10'' M 10'' M 10'' M 10 - ' M
IxmetTCOx oF
L. reclusa vENOrt PROTEASE ~crmrY
Addition None None Ethylenediaminetetra-acetic acid Amino-n~aproic acid N-ethylmaleimide Phenylmethylsulfonyl fluoride
Activity ( 0 100 18 100 53 97
n
Enzyme activities were measured in cuvettes containing 2"0 x 10'' M Uleucyl-~-naphtl~ylamide, 8 x 10 -' M inhibitors and 60 ~g of venom protein in 2~5 ml total volume of 0"1 M phosphate buffer at pH 7"2. Fluorescence intensity was recorded at time zero and after 1 hr incubation .
Both EDTA and N-ethylmaleimide (NEM) inhibited the protease activity (Table 2). When the chelating agent, EDTA, was employed at 4"0 x 10 - ' M, the time of pre-incubation of the chelating agent with the spider venom altered the degree of inhibition observed (Fig. 3). The inactivation reaction was first order with a half life of approximately 12 min. The inhibition caused by EDTA suggested that the presence of a metal ion in the protein is critical to enzyme activity. The reaction rate of venom protease was higher in sodium phosphate buffer than in Tris-buffer at the same pH without any activators . Comparatively, in controls containing no activators, the enzymatic activity increased to 150 ~ by adding 0~1 M sodium chloride in Tris-buffer. Thus, the sodium ion or increased ionic strength may play a role as an activator for venom protease . The inactivation of venom protease by NEM suggested that a sulfhydryl group was necessary for the enzyme action (Table 2, Fig. 4). The results of inactivation studies using a more potent sulfhydryl inhibitor, fluores-
Tlms,
min
Fm. 3. Fiasr ORDER RATE OF INACTIVATION OF L. rCCIu30 VENOM PROTEASE HY EDTA . Incubation mixtures were prepared containing 4~0 x 10~' M EDTA and 60 pg crude venom protein in 2~3 ml total volume of 0~1 M phosphate buffer at pH 7~2. After various limos of incubation, the reactions wero initiated by addition of 50 lIl of 1'0 x 10'' M L-leucyl-ßnaphthylamide. ao
do
Fra. 4.
Tlms,
min
INI~IIION of L . recluses vENOM PROTEASE HY N-Ei'HYL~7ALEII~DE (NEM) AND FLUORESCEIN MERCURIC ACETATE (FMA). Inwbation mixtures were prepared containing 4~0 x 10 -' M NEM ( ") of 2'0 x 10 - ' M FMA (x) and 601Ig crude venom protein in a total volume of 2~5 ml of 0~1 M phosphate buffer at pH 7~2. After various times of incubation ; 50 ul of 1~0 x 10 -' M crleucyl-ß-napht1~ylamido wero added to the reaction mixtures . Enzyme activities were measured at zero time and after 1 hr of incubation at 32°C.
534
YAW-SHONG JONG, B. R. NORMENT and JAMES R HE1TZ
coin mercuric acetate (FMA), indicated rapid inactivation (Fig. 4). FMA has been reported to inhibit sulfhydryl enzymes so rapidly that conventional kinetic measurements were impossible (KARUSH et al., 1964; HEITZ and AxDERSOx, 1968 ; HErrz, 1973) . There may be a sulfhydryl group necessary for the binding of substrate or catalysis at the active site of spider venom protease . The ability of FMA to approach and react with this sulfhydryl group supports the concept that the active site ofthis enzyme is large and bulk tolerant. The Xm value of 357 x 10 - ' M suggested a high affinity between venom protease and substrate with a low reaction rate (Fig. ~. It further suggested that there was a high energy barrier (activation energy) which reduced the reaction rate .
-2 .S
0
2.S
v~-b,kyi-ß-rwar+ttria~,~as, Fta. S. LnvEwEAVeR-BURx Pear of
5.0
M-' x io'
vEr~ PRarEASE ACr1VITY oN t-~sUCn-ßNAPHl'HYLAI~E. Gtivettea contained from 2~0 x 10 -' M to 2~0 x 10 - ' M, L-leucyl-ß_naphthylamide in 2~5 nil total volume of 0~1 M phosphate buffer at pH 72. Reactions were initiated by addition of 60 hg of crude venom protein . The onzyme reactions were incubated for 1 hr at 35°C. L . reelusa
The effect of temperature on the kinetic properties of the enzyme showed that the enzyme catalyzed reaction follows the Arrhenius equation, i.e . a linear relationship between the logarithm of the velocity and the reciprocal of the absolute temperature (Fig. 6) WiLSOx (1971) determined the activation energy (EJ from the slope ofa plot ofthe natural logarithm of the rate of the enzyme catalyzed reaction vs the reciprocal of the absolute temperature. PAULE (1971) calculated an enthalpy of activation (0H') using a plot of the natural logarithm of the equilibrium constant of the reaction (K~) vs the reciprocal of the absolute temperature . In some cases, enzymes do not conform to this equation as has been discussed by DucoN and WEBS (1964) . However, for a simple, one step chemical reaction : REACTANT
k, a=+ k_
1
PRODUCT.
The equilibrium constant (K~) is equal to kl/k_, and is used in the Arrhenius equation : In K~ _ -E /R x 1/T -I- log A. The value of E, is determined experimentally by plotting In Ka, vs 1/T. The Michaelis-
Separation and Charactarizatlon of Venom Components in Loxoseeles recluse-Il
533
-e
_9
32.3
33.0
33.3
34 .0
I/T (dsgres absolutd~z 104) Tea~ewxv~ ~$LT otv L. recluse v~to~ rROre~se wcnvrnr. FYa. 6. All conditions were identical to those of Fig. 5 except that the substrate concentration waa S~0 x 10"' M and the temperature was varkd between 215°C and 35°C. Menten constant (K~ is defined as : 1~ _ (k~ -~- (k_~/k,, according to the basic kinetic equation : k,
k,
E-~S .~E-S---" E+P. k_,
For some enzyme reactions, E, values cannot be obtained by plotting In ~ vs 1/T since the ~value is not necessarily directly related to the K~ value. However, in some enzymatic reactions k_ 1 is very large compared with ka, in which case the rate constant k~ becomes negligibly small and Km simplifies to the expression Km . kllkl where Km is approximately equal to the reciprocal of ~a . Experimentally, this situation appears to be valid for a variety of hydrolytic enzymes (WI~TB et al., 1973) . Spider venom protease hydrolyzed r.-aminoacyl-ß-naphthylamide and followed a pseudo first order reaction . Thus, it was assumed that 1~ - 1/K,q and log Km was plotted vs 1/T to calculate E, . The E, value was calculated to be 49,000 cal/mole from 215° to 35°C . Figure 6 shows the straight line determined by a linear regression analysis using a Canola 167 P program . Excellent agreement between the experimental points and the linear relationship is shown by the correlation coefiîcient of 099. Some precautions in the use of this method deserve discussion . The thermal denaturation of the enzyme over the temperature range should be considered. Additionally, the possibility exists that the pH of the medium may change with temperature since spider venom protease acted on substrates in a narrow pH range (7~2) of optimal activity. However, in the temperature range of 21~~-35°C, the variant factors described above were negligible. In order to identify the protease fraction of spider venom, protease assays were performed immediately after preparative-disc electrophoresic separation. Fraction 4 (preparative electrophoresis) was the only component acting on both L-leucyl-ß-naphthylamide and r,-alanyl-ß-naphthylamide (Fig. 7~. Furthermore, the enzyme activity in fraction 4 was negligible within 48 hr after the separation which thus suggested that the protease is subject
536
YAW-SHONG JONß, B. R. NORMENT and JAMES R. HETTZ
o .oi
0 N
IQD
T~st tub~ No.
Î. L.
rCCINSa VENOM PROTEASE ACrIVITIF3 SHOWN IN FRACTION 4 OF THE PREPARATIVE-DISC ELECTROPHORESIC SEPARATION . lncubation mixtures were prepared containing 0~2 ml aliquots of the various fractions in a total volume of 2~5 ml of 0~1 M phosphate buffer at pH 7~2. The reactions wero initiated by addition of 50 lIl of 1~0 x 10 - ' M L-leucyl-ß-naphthylamide ( ") or L-alanyl-~naphthylFIG .
amide (O) .
either to denaturation in a highly dilute solution or to autolysis. Autolysis would be less likely in . a dilute solution which implies that denaturation is taking place. The mol. wt estimation of spider venom protease is shown in Fig. 8. SDS gel electro-
ô x
ti+onwty FIG . S. THE MOLECULAR WEIGHT OF PURIFIED SPII)ER VENOM PROTEASE, FRACTION 4 OF TFiß PR~AAATiVE-DISC BLECIROPHORBTIC ~PARATION, ESTIMATED HY SDS-POLYACRYLAMIDE OEL ELECTROPHORESIS. The standard proteins used were : (~), bovine serum albumin ; (p), horse liver alcohol dehydrogenase ; ( "), yeast alcohol dehydrogenase ; (p), hemoglobin ; ( " ), cytochrome C. Spider venom protease is represented by Q.
Separation and Characterization of Venom Components in Loxosceles reclusa-II
537
phoresis of fraction 4 of the preparative-disc electrophoretic separation showed that the protease exhibited a mol. wt of 29,600 . The protease activity in brown recluse spider venom appears to have a broad substrate specificity and a narrow pH optimum. Optimum activity depends on a free sulfhydryl group and a metal cofactor. The enryme is of moderate size, mol. wt, 29,600. REFERENCES Dtxox, M. and WIiHIi, E. C. (1964) Enzymes, Znd edn., p. 145. London : Longmans . Esw~Ft, F. M. tmd Nottt~rrr, B. R. (1976) Physiological action of Loxosceks reclusa (GBtM) venom on insect larvae . Toxicon 14, 7. Gettruv, C. R., Ct~rt, T. K., Howra.t., D. E. and Onet.r., G. V. (1976) Isolation and characterization of toxins from brown recluse spider venom (Loxosceles reclusa). Archs Blochem. Biophys. 174, 90 . HEtrz, J. R and AtvntatsoN, B. M. (1968) Selective binding of fluorescein mercuric acetate to yeast alcohol dehydrogenase. Archs Blochem. Biophys. 127, 637. Hrarz, J. R. (1973) The reaction of fluorescein mercuric acetate with sorbitol dehydrogenase. J. biol. Chem . 248, 5790 . HErrz, J. R. and Notv~r, B. R. (1974) Characteristics of an alkaline phosphatase activity in brown recluse venom. Toxicon 12, 181 . Joxa, Y. S., Notu~tvr, B. R. and Hertz, J. R. (1979) Separation and characterization of venomcomponents in Loxosceles reclusa-I. Preparative-disc electrophoresis. Toxicon 17, 307. KARUSA, F., Kt uv~x, N. R. and Mains, R. (1964) An assay method for disulßde groups by fluorescence quenching. Analyt. Blochem. 9, 100. Ltrrswsnviat, H. and Bvatc, D. (1934) The determination of enzyme dissociation constants. J. Am. them . Soc. 56, 658. Lottexn, L. and Comer, V. (1965) Ester hydrolysis by umkinase. Blochem. 14, 265. P~ULa, M. R. (1971) The effect of temperature on the kinetics of adenosine diphosphogluoose pyrophosphorylase from Rhodospirlllum rubum. Blochen. J. 10, 4509. Rorer, M. (1965) Enzymes in Clinical Chemistry, p. 10. Amsterdam: ELsevier. S6OfiL, I. H. (1975) Enzyme Kinetics, p. 930. New York : Wiley-Intertcience . Wea>:x, K. and Oseottx, M. (1969) The reliability of molecular weight determination by dodecyl sulfatepolyacrylamide gel electrophoresis. J. blol. Chem . 244, 4406 . Witze, A., H~xnL~t, P, and SMtrx, E. L. (1973) Principles of Biochemistry, 5th edn., p. 23. New York : McGraw-Hill. WnsoN, J. E. (1971) An expeditious method for determ ination of activation energies of enzymatic reactions. Archs Blochem. Biophys. 147, 471 . Watat~r, R. P., EweRr, K. D., CAMPBELL, B. J, and BARRETT, J. T. (1973) Hyaluronidase and esterase activities of the venom of the poisonous brown recluse spider. Archs Blochem. Biophys. 159, 415 . WRIOI~I', R. P. (1973) Enzymic characterization of brown recluse spider venom. Bull. Mo. Aced. Sci. Supplement 2, 94 pp.
Torkww, Vol . 17, pp . 329-037 . O PerQamon Pana Ltd . 1979. Printed in Great Britain.
SEPARATION AND CHARACTERIZATION OF VENOM COMPONENTS IN LOXOSCELES RECLUSA-II. PROTEASE ENZYME ACTTVITY* YAw-SHONG JONG,t
B. R. Nolt>siErrr$ and JAi~s R. HE1TZfi
Departments of Biochemistryj' and Entomology$, Mississippi Agricultural and Forestry Experiment Station, Mississippi Stato University, Mississippi State, MS 39762, U.S .A. (Acceptedjor publication 29 January 1979) Ynw-Sxotvo JONG, B. R. NoxMenrr and Jn~rns R. HErrz. Separation and characterization of venom components in Loxosceles reclusa-II . Protease enzyme activity . Toxicon 17, 529-537. 1979 .-A protease from brown recluse spider venom hydrolyzes the amide linkage of amino acids containing aliphatic, aromatic, or basic side chains. The mol. wt of tho enzyme was 29,600 by SDS electrophoresis. The Km for L-leucyl-ß-naphthylamide is 375 x 10 -' M and the activation energy was 49,000 cal/mole. The pH profile indicates that the enzymo has a narrow active region centered at pH 7~2. Spider venom protease is inhibited irreversibly by ethyknediamine tetraacetic acid, N-ethylmaleimide and 8uorescein mercuric acetate. INTRODUCTION
Txs sltowx recluse spider, Loxosceles reclusa, has attracted much attention in the past 10 yr as the gravity of the problems caused by the bite of this spider have become known. The clinical manifestation of brown recluse venom on man is well established. However, basic information on the venom and venom components is still lacking. NAZHAT (M.S . Thesis, Oklahoma State Univ ., 1968) reported that there was no protease or phospholipase C or D activity, although some lipase activity was observed in the venom. HALL (M .S . Thesis, Oklahoma State Univ ., 1970) reported that the venom contained no phospholipase A activity, however, some hyaluronidase activity was found . In other studies esterase and hyaluronidase have been reported in L. reclusa venom; but alkaline phosphatase, collagenase, phospholipase A, dipeptidase, acetylcholinesterase, ribonuclease A and deoxyribonuclease were not found (WRIGHT et al., 1973 ; W1ucxT, 1973). WRIGHT and co-workers also characterized hyaluronidase which was considered to be a "spreading factor". Subsequently, alkaline phosphatase activity was demonstrated and characterized by HeaTZ and NoxltmlvT (1974) . Although some enzymes have been found in L. reclusa venom, the toxins which were separated by G»ri et al. (197 were not directly associated with the enzymatic activities of hyaluronidase, alkaline phosphatase and 5'-ribonucleotidase . EsxAFt and NOx i~T (197 histochemically demonstrated venom protease, esterase, lipase and alkaline phosphatase in L. reclusa envenomated larvae of Heliothis virescens and Musca domestica . The minute quantity of spider venom obtained per specimen prohibits many of the more routine enzyme studies. Purification of large amounts of an individual protein component is not feasible through ordinary biochemical methods. Thus, only the most sensitive methods can be utilized. Recently, JoNG et al. (1979) separated L. reclusa venom proteins +Publication No. 4033 of the Mississippi Agricultural and Forestry Experiment Station. This research was supported by Grant No. SROlES00651 from the National Institute of Health. 529
53 0
YAW-SHONG JONG, B. R. NORMENT and JAMFS R. HEI'TZ
utilizing preparative-disc electrophoresis in a one-step purification . Ten fractions were resolved with high reproducibility ; fraction 1 has been identified as a nucleotide. This report presents a further study of the enzyme components present in these spider venom fractions ; in this case, the purification and characterization of a proteolytic enzyme . MATERIALS AND METHODS Venom protease (Fig. 1) was assayed with slight modification of the procedure of Rorer (1965) . All substrates were prepared fresh before each experiment as 1~0 x 10 -' M in methanol stock solutions except L-cystina~di-ß-naphthylamide which was dissolved in dimethylformamide. Enzyme catalyzed reactions were calculated as the increase in fluorescence intensity per hr (~F/hr) against control reactions containing no enzyme . There was evidence of fluorescence intensity caused by the spontaneous decomposition of substrate, however, the linearity of the true enzyme activity with respect to enzyme concentration showed that the assay was reliable. H HN
~NHZ R- ~\ H C-N 0 ~-Ar~inoacyl-ß-rwphthylamide
~-Nophthylamine protease
(highty fluorescent product)
Hz 0
t
l ran-fluorescent substrate) R-CH
/NH2
~COOH
HYDROLYTIC CLEAVAGE
-Amino add
FIG. 1. OF L-AMINOACYL-ß-NAPFITHYLAMTDE DERIVATIVE4 IN TFIE PRESENCE OF PROTEASE. Different L-aminoacyl derivatives are variably substituted at R. The ß-naphthylamine is a highly fluorescent product whose excitation wavelength and emission wavelength are 335 and 410 nm, respectively .
Venom extraction, separation and collection procedures have boon previously described (JONG et al., 1979). The position of the spider venom protease was then determined by measuring activity in 0~2 ml aliquots from every second test tube fraction of each preparative-disc electrophoretic separation using L-alanyl-ß-naphthylamide as a substrate. The protease position was also determined using L-leucyl-ßnaphthylamide as a substrate. Protein concentration could not be determined in each fraction due to the small volumes. Venom protein was mixed with inhibitor solutions : 4~0 x 10 -' M ethylenediaminetetra-acetic acid (EDTA), 4~0 x 10-' M N-ethylmaleimide (NEM), or 2~0 x 10 -' M fluorescein mercuric acetate (FMA), in phosphate buffer for various times of pre-incubation before L-leucyl-ß-naphthylamide was added to initiate the enzyme catalyzed reaction. Enzyme activity was recorded at zero time and after 1 hr of incubation at 32°C and theAF/hr of each reaction was calculated . Inhibition was determined by comparing this activity with that observed in control reactions to which no inhibitorhad been added. L-leucyl-ß-naphthylamide was prepared in concentrations from 2~0 x 10 - ' M to 2~0 x 10 - ' M in phosphate buffer. Loxosceles reclusa crude venom protein initiated the enzyme reaction at 35 °C in the absence of MgCI, in sodium phosphate buffer, pH 7~2. Activities (~F/hr) were recorded at different substrate concentrations to calculate the K value according to the method of LINEWEAVER and BURR (1934) . The Km values at seven different temperatures were obtained in the same manner except the temperature of the waterbath was adjusted between 2945 and308 K. The theoretical basis of the temperature effect on spider venom protease activity is referred to in DDCON and W®H (1964) and SEGEL (1975) . A plot of In Ko vs 1/T gave the activation energy (E,) of the reaction . The E, value was derived from the slope of the line, -E,/R. The pool of fraction 4 from the preparative-disc eloctrophoretic separation was added to dialysis casing (Sigma Chemical Co . 250-9u) and was dialyzed against distilled water at 4°C for 6 hr . The protein component was rinsed three times with distilled water into a VirTis bottle after dialysis and lyophilized with a VirTis Lyophilizer (Model No. 10-145 MR BA). The dried sample was dissolved by adding 80111 of 0~1 M phosphate buffer, pH 7~0. SDS-polyacrylamide gel electrophoresis was conducted according to the procedar; of WEHER and OsaoRN (1969) . For the preparation of the sample solution, 40111 of venom protease
Separation and Characterization of Venom Components in Loxosceks reclusa--II
531
solution was added to 20 lIl of 2 M sucrose and 20 iIl of 005 ~ bromophenol blue . The sample was diluted to 0~1 ml and incubated overnight at room temperature . The standard proteins were made up in 001 M sodium phosphate at 5 mg/ml and were treated as described for the spider venom protease solution. The standard proteins bovine serum albumen, alcohol dehydrogenase (yeast), alcohol dehydrogenase (horse liver), hemoglobin and cytochrome C, were obtained from the Sigma Chemical Company, as was the denaturant, SDS. Electrophoresis was performed at a constant current of 3 mA per gel with the positive electrode in the lower chamber. Under these conditions the marker dye reached 1 cm from the bottom of the gel in approximately 5 hr . After electrophoresis, the gels were removed from the tubes and stained overnight at room temperature with 025 ~ Coomassie Blue in SO ~ methanol containing 10 ~ acetic acid . The gels were dostained for 15 min in 7~ acetic acid with a Canalco 1801 Quick Gel Destainer . The L-ar~oacyl-ß-naphthylamide derivatives, listed in Table 1, were obtained from ICN Pharmaceuticals, Inc. including: tyrosino-, tryptophan-, phenylalanino-, lysine-, arginine-, proline-, hydroxyproline-, isoleucino-, leucine-, valino-, glycine- and serino-ß-naphthylamide. L-Alanyl-ß-naphthylamido was obtained from the Sigma Chemical Company. IrLeucyl-ß.naphthylamide in Figs . 2-7 and Table 2 and Iralanyl-ß-ttaphthylamide in Fig. 7 were also obtained from the Sigma Chemical Company. RESULTS AND DISCUSSION
1.-Leucyl-ß-naphthylamide was the best protease substrate (+~--}-), whereas the aliphatic and basic aminoacyl derivatives, valine-, alanine-, arginine-, and lysine-, were hydrolyzed less rapidly (~---~) (Table 1). The venom protease hydrolyzed the aromatic TAaI~
I. ACTION OF L. rtC1USa VENOM
PROTEASE
ON
VARIOUS IrAMINOACYL-ß-NAPHYTiYLAA~E DERIVATIVES
$Ub6trateS
Bufer System
t-Aminoacyl-ß-naphthylamide derivatives Tyrosine Tryptophan l~Ienylahuvne Lysine Arginine Proline Hydroxyproline Isoleucine Leucine Valine Alanine Glycine Serine Cystine-di-ß-naphthylamide
CO, 1/2 H,O free base HBr salt HBr salt HBr salt HCl salt HBr salt HBr salt free base di-HBr salt
Glycine-HCl iPH = 2'64) -
Tris-HCI ~PH = ~'~) + + +-f++ 1-++ ++ ++ _
Glycine-NaOH ~PH = 9.40)
Couches containing 100 pg crude venom protein in 0~1 ml Tris~lycine buffer at pH 8~3, SO lIl 0~1 M MgCI, solution in 235 ml buffer solution. Reactions were initiated by adding SO pl 1~0 x 10 -' M L-aminoacyl-ß-naphthylamide solution . Fluorescence intensity was recorded at time zero and 1 hr later.
aminoacyl derivatives tryptophan and phenylalanine slowly (-~-), and L-isoleucyl-ßnaphthyhunide was not hydrolyzed at all (-). Similarly, there was no specific enryme in L. reclusa venom which cleaved the disulfide bond. There was also no protease activity recorded for the proline-, serine-, hydroxyproline- and glycine-derivatives. The brown recluse venom protease acted on substrate in neutral solution only ; no recordable activity was found in the acidic or basic pH ranges. This protease apparently has a high reactivity for R groups which are hydrophobic or contain a positive charge . L. reclusa venom protease possessed a narrow pH range of optimal activity centering at approximately 7"2 (Fig. 2). The enzyme assays recorded in Table 1 were in Tris-buffer, pH 7"29, which is close to the optimum pH value. Previously, s-amino-n-caproic acid had been utilized as a competitive inhibitor for protease, because there is both an amino group and an acid group on the molecule (LORAND
53 2
YAW-SHON(} JONG, H. R NORMENT and JAMES R. HEITZ
PH
FYo. 2. L. IECIYBR VFNOM PROTEASE ACITVITY AS A FUNCTION OF pH . Reaction mixtures contained 6 x 10'' M MgCI, and 100 ~g crude venom solution in 2"S ml phosphate buffer ( ") or glycine-NaOH buffer (O). After pre-incubation for 5 min at 33 "5°C, SO pl of 1" 0 x 10- ' M Uleucyl-ß-naphthylamide were added. Fluorescence intensity was recorded at time zero and 1 hr later.
and Coxnrr, 196 . However, results indicated that e-amino-n-caproic acid had no effect on L. rechrsa venom protease activity (Table 2). Similarly, the lack of inhibition by a serinetype inhibitor, phenyhnethylsulfonyl fluoride, indicated that serine was not a functional amino acid in the active site of L. reclusa venom protease . TA$LE
Enzyme Venom -F Venom Venom Venom -F Venom Venom
-
8 8 8 8
x x x x
2.
10'' M 10'' M 10'' M 10 - ' M
IxmetTCOx oF
L. reclusa vENOrt PROTEASE ~crmrY
Addition None None Ethylenediaminetetra-acetic acid Amino-n~aproic acid N-ethylmaleimide Phenylmethylsulfonyl fluoride
Activity ( 0 100 18 100 53 97
n
Enzyme activities were measured in cuvettes containing 2"0 x 10'' M Uleucyl-~-naphtl~ylamide, 8 x 10 -' M inhibitors and 60 ~g of venom protein in 2~5 ml total volume of 0"1 M phosphate buffer at pH 7"2. Fluorescence intensity was recorded at time zero and after 1 hr incubation .
Both EDTA and N-ethylmaleimide (NEM) inhibited the protease activity (Table 2). When the chelating agent, EDTA, was employed at 4"0 x 10 - ' M, the time of pre-incubation of the chelating agent with the spider venom altered the degree of inhibition observed (Fig. 3). The inactivation reaction was first order with a half life of approximately 12 min. The inhibition caused by EDTA suggested that the presence of a metal ion in the protein is critical to enzyme activity. The reaction rate of venom protease was higher in sodium phosphate buffer than in Tris-buffer at the same pH without any activators . Comparatively, in controls containing no activators, the enzymatic activity increased to 150 ~ by adding 0~1 M sodium chloride in Tris-buffer. Thus, the sodium ion or increased ionic strength may play a role as an activator for venom protease . The inactivation of venom protease by NEM suggested that a sulfhydryl group was necessary for the enzyme action (Table 2, Fig. 4). The results of inactivation studies using a more potent sulfhydryl inhibitor, fluores-
Tlms,
min
Fm. 3. Fiasr ORDER RATE OF INACTIVATION OF L. rCCIu30 VENOM PROTEASE HY EDTA . Incubation mixtures were prepared containing 4~0 x 10~' M EDTA and 60 pg crude venom protein in 2~3 ml total volume of 0~1 M phosphate buffer at pH 7~2. After various limos of incubation, the reactions wero initiated by addition of 50 lIl of 1'0 x 10'' M L-leucyl-ßnaphthylamide. ao
do
Fra. 4.
Tlms,
min
INI~IIION of L . recluses vENOM PROTEASE HY N-Ei'HYL~7ALEII~DE (NEM) AND FLUORESCEIN MERCURIC ACETATE (FMA). Inwbation mixtures were prepared containing 4~0 x 10 -' M NEM ( ") of 2'0 x 10 - ' M FMA (x) and 601Ig crude venom protein in a total volume of 2~5 ml of 0~1 M phosphate buffer at pH 7~2. After various times of incubation ; 50 ul of 1~0 x 10 -' M crleucyl-ß-napht1~ylamido wero added to the reaction mixtures . Enzyme activities were measured at zero time and after 1 hr of incubation at 32°C.
534
YAW-SHONG JONG, B. R. NORMENT and JAMES R HE1TZ
coin mercuric acetate (FMA), indicated rapid inactivation (Fig. 4). FMA has been reported to inhibit sulfhydryl enzymes so rapidly that conventional kinetic measurements were impossible (KARUSH et al., 1964; HEITZ and AxDERSOx, 1968 ; HErrz, 1973) . There may be a sulfhydryl group necessary for the binding of substrate or catalysis at the active site of spider venom protease . The ability of FMA to approach and react with this sulfhydryl group supports the concept that the active site ofthis enzyme is large and bulk tolerant. The Xm value of 357 x 10 - ' M suggested a high affinity between venom protease and substrate with a low reaction rate (Fig. ~. It further suggested that there was a high energy barrier (activation energy) which reduced the reaction rate .
-2 .S
0
2.S
v~-b,kyi-ß-rwar+ttria~,~as, Fta. S. LnvEwEAVeR-BURx Pear of
5.0
M-' x io'
vEr~ PRarEASE ACr1VITY oN t-~sUCn-ßNAPHl'HYLAI~E. Gtivettea contained from 2~0 x 10 -' M to 2~0 x 10 - ' M, L-leucyl-ß_naphthylamide in 2~5 nil total volume of 0~1 M phosphate buffer at pH 72. Reactions were initiated by addition of 60 hg of crude venom protein . The onzyme reactions were incubated for 1 hr at 35°C. L . reelusa
The effect of temperature on the kinetic properties of the enzyme showed that the enzyme catalyzed reaction follows the Arrhenius equation, i.e . a linear relationship between the logarithm of the velocity and the reciprocal of the absolute temperature (Fig. 6) WiLSOx (1971) determined the activation energy (EJ from the slope ofa plot ofthe natural logarithm of the rate of the enzyme catalyzed reaction vs the reciprocal of the absolute temperature. PAULE (1971) calculated an enthalpy of activation (0H') using a plot of the natural logarithm of the equilibrium constant of the reaction (K~) vs the reciprocal of the absolute temperature . In some cases, enzymes do not conform to this equation as has been discussed by DucoN and WEBS (1964) . However, for a simple, one step chemical reaction : REACTANT
k, a=+ k_
1
PRODUCT.
The equilibrium constant (K~) is equal to kl/k_, and is used in the Arrhenius equation : In K~ _ -E /R x 1/T -I- log A. The value of E, is determined experimentally by plotting In Ka, vs 1/T. The Michaelis-
Separation and Charactarizatlon of Venom Components in Loxoseeles recluse-Il
533
-e
_9
32.3
33.0
33.3
34 .0
I/T (dsgres absolutd~z 104) Tea~ewxv~ ~$LT otv L. recluse v~to~ rROre~se wcnvrnr. FYa. 6. All conditions were identical to those of Fig. 5 except that the substrate concentration waa S~0 x 10"' M and the temperature was varkd between 215°C and 35°C. Menten constant (K~ is defined as : 1~ _ (k~ -~- (k_~/k,, according to the basic kinetic equation : k,
k,
E-~S .~E-S---" E+P. k_,
For some enzyme reactions, E, values cannot be obtained by plotting In ~ vs 1/T since the ~value is not necessarily directly related to the K~ value. However, in some enzymatic reactions k_ 1 is very large compared with ka, in which case the rate constant k~ becomes negligibly small and Km simplifies to the expression Km . kllkl where Km is approximately equal to the reciprocal of ~a . Experimentally, this situation appears to be valid for a variety of hydrolytic enzymes (WI~TB et al., 1973) . Spider venom protease hydrolyzed r.-aminoacyl-ß-naphthylamide and followed a pseudo first order reaction . Thus, it was assumed that 1~ - 1/K,q and log Km was plotted vs 1/T to calculate E, . The E, value was calculated to be 49,000 cal/mole from 215° to 35°C . Figure 6 shows the straight line determined by a linear regression analysis using a Canola 167 P program . Excellent agreement between the experimental points and the linear relationship is shown by the correlation coefiîcient of 099. Some precautions in the use of this method deserve discussion . The thermal denaturation of the enzyme over the temperature range should be considered. Additionally, the possibility exists that the pH of the medium may change with temperature since spider venom protease acted on substrates in a narrow pH range (7~2) of optimal activity. However, in the temperature range of 21~~-35°C, the variant factors described above were negligible. In order to identify the protease fraction of spider venom, protease assays were performed immediately after preparative-disc electrophoresic separation. Fraction 4 (preparative electrophoresis) was the only component acting on both L-leucyl-ß-naphthylamide and r,-alanyl-ß-naphthylamide (Fig. 7~. Furthermore, the enzyme activity in fraction 4 was negligible within 48 hr after the separation which thus suggested that the protease is subject
536
YAW-SHONG JONß, B. R. NORMENT and JAMES R. HETTZ
o .oi
0 N
IQD
T~st tub~ No.
Î. L.
rCCINSa VENOM PROTEASE ACrIVITIF3 SHOWN IN FRACTION 4 OF THE PREPARATIVE-DISC ELECTROPHORESIC SEPARATION . lncubation mixtures were prepared containing 0~2 ml aliquots of the various fractions in a total volume of 2~5 ml of 0~1 M phosphate buffer at pH 7~2. The reactions wero initiated by addition of 50 lIl of 1~0 x 10 - ' M L-leucyl-ß-naphthylamide ( ") or L-alanyl-~naphthylFIG .
amide (O) .
either to denaturation in a highly dilute solution or to autolysis. Autolysis would be less likely in . a dilute solution which implies that denaturation is taking place. The mol. wt estimation of spider venom protease is shown in Fig. 8. SDS gel electro-
ô x
ti+onwty FIG . S. THE MOLECULAR WEIGHT OF PURIFIED SPII)ER VENOM PROTEASE, FRACTION 4 OF TFiß PR~AAATiVE-DISC BLECIROPHORBTIC ~PARATION, ESTIMATED HY SDS-POLYACRYLAMIDE OEL ELECTROPHORESIS. The standard proteins used were : (~), bovine serum albumin ; (p), horse liver alcohol dehydrogenase ; ( "), yeast alcohol dehydrogenase ; (p), hemoglobin ; ( " ), cytochrome C. Spider venom protease is represented by Q.
Separation and Characterization of Venom Components in Loxosceles reclusa-II
537
phoresis of fraction 4 of the preparative-disc electrophoretic separation showed that the protease exhibited a mol. wt of 29,600 . The protease activity in brown recluse spider venom appears to have a broad substrate specificity and a narrow pH optimum. Optimum activity depends on a free sulfhydryl group and a metal cofactor. The enryme is of moderate size, mol. wt, 29,600. REFERENCES Dtxox, M. and WIiHIi, E. C. (1964) Enzymes, Znd edn., p. 145. London : Longmans . Esw~Ft, F. M. tmd Nottt~rrr, B. R. (1976) Physiological action of Loxosceks reclusa (GBtM) venom on insect larvae . Toxicon 14, 7. Gettruv, C. R., Ct~rt, T. K., Howra.t., D. E. and Onet.r., G. V. (1976) Isolation and characterization of toxins from brown recluse spider venom (Loxosceles reclusa). Archs Blochem. Biophys. 174, 90 . HEtrz, J. R and AtvntatsoN, B. M. (1968) Selective binding of fluorescein mercuric acetate to yeast alcohol dehydrogenase. Archs Blochem. Biophys. 127, 637. Hrarz, J. R. (1973) The reaction of fluorescein mercuric acetate with sorbitol dehydrogenase. J. biol. Chem . 248, 5790 . HErrz, J. R. and Notv~r, B. R. (1974) Characteristics of an alkaline phosphatase activity in brown recluse venom. Toxicon 12, 181 . Joxa, Y. S., Notu~tvr, B. R. and Hertz, J. R. (1979) Separation and characterization of venomcomponents in Loxosceles reclusa-I. Preparative-disc electrophoresis. Toxicon 17, 307. KARUSA, F., Kt uv~x, N. R. and Mains, R. (1964) An assay method for disulßde groups by fluorescence quenching. Analyt. Blochem. 9, 100. Ltrrswsnviat, H. and Bvatc, D. (1934) The determination of enzyme dissociation constants. J. Am. them . Soc. 56, 658. Lottexn, L. and Comer, V. (1965) Ester hydrolysis by umkinase. Blochem. 14, 265. P~ULa, M. R. (1971) The effect of temperature on the kinetics of adenosine diphosphogluoose pyrophosphorylase from Rhodospirlllum rubum. Blochen. J. 10, 4509. Rorer, M. (1965) Enzymes in Clinical Chemistry, p. 10. Amsterdam: ELsevier. S6OfiL, I. H. (1975) Enzyme Kinetics, p. 930. New York : Wiley-Intertcience . Wea>:x, K. and Oseottx, M. (1969) The reliability of molecular weight determination by dodecyl sulfatepolyacrylamide gel electrophoresis. J. blol. Chem . 244, 4406 . Witze, A., H~xnL~t, P, and SMtrx, E. L. (1973) Principles of Biochemistry, 5th edn., p. 23. New York : McGraw-Hill. WnsoN, J. E. (1971) An expeditious method for determ ination of activation energies of enzymatic reactions. Archs Blochem. Biophys. 147, 471 . Watat~r, R. P., EweRr, K. D., CAMPBELL, B. J, and BARRETT, J. T. (1973) Hyaluronidase and esterase activities of the venom of the poisonous brown recluse spider. Archs Blochem. Biophys. 159, 415 . WRIOI~I', R. P. (1973) Enzymic characterization of brown recluse spider venom. Bull. Mo. Aced. Sci. Supplement 2, 94 pp.