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[32] Leucostoma peptidase A
Leucostoma PEPTIDASE A
[32l
397
[32] L e u c o s t o m a P e p t i d a s e A
By JOH~ M. PRESCOTTand FREDW. WAG~ER Leucostoma peptidase A is the most abundant protease in the venom of the western cottonmouth moccasin (Agkistrodon piscivorus leucostoma) from which it can be isolated in good yield and a high degree of homogeneity. 1,2 This endopeptidase possesses some characteristics similar to those of bacterial neutral proteases, inasmuch as it is inhibited by metal complexing reagents, is a metalloprotein that contains calcium and zinc, and has a substrate specificity dictated primarily by the identity of the residue contributing the amino group to the susceptible peptide bond.
Assay Procedures Leucostoma peptidase A readily digests casein and denatured hemoglobin, either of which can be used as a substrate to assay the proteolytic activity of fractions obtained during enzyme isolation. Details for the assay with hemoglobin substrate are given below. In addition, it is also advisable to assay for esterase and arylamidase activities at each stage of the procedure in order to monitor the elimination of enzymes that accompany Ieucostoma peptidase A through part of the purification procedure. Hemoglobin Assay for Proteolytic Activity. The procedure is essentially the same as that of Anson, 3 and involves determining the increase in trichloroacetic acid-soluble products that result from digestion of urea-denatured hemoglobin. The substrate is prepared by suspending 5 g of lyophilized, dialyzed hemoglobin (Worthington) in 80 ml of water with 80 g of urea. After the mixture has been incubated at 37 ° for 1 hr, 125 ml of 0.133 M phosphate buffer (pH 8.5) and 10 g of urea are added. The mixture is permitted to stand overnight and the pH is checked to ensure that it is 8.5. Assays are run by adding 1 ml of suitably diluted enzyme solution to 5 ml of the substrate and incubating the mixture at 37 ° for 10 min. The addition of 10 ml of 5% trichloroacetic acid serves to stop the reaction and to precipitate undigested protein, which is removed by filtration through Whatman No. 3 paper. The absorbance of 1F. W. Wagner, A. M. Spiekerman, and J. M. Prescott, J. Biol. Chem. 243, 4486 (1968). "A. M. Spiekerman, K. K. Fredericks, :F. W. Wagner, and J. M. Prescott, Biochim. Biophys. Acta 293, 464 (1973). 3M. L. Anson, Y. Gen. Physiol. 22, 79 (1938).
398
ENDOPEPTIDASES
[32]
the filtrate at 280 nm is then determined against a blank made by adding trichloroaeetic acid to the substrate prior to addition of the enzyme. One unit is defined as that quantity of enzyme that produces an increase of 1.0 in al)sorbance in 10 rain. Arylamidase Assay. Although the venom of A. p. leucostoma does not contain true aminopeptidase activity, it catalyzes the hydrolysis of L-leucyl-fl-naphthylamide' and thus contains one or more enzymes that can be classified for operational convenience as arylamidases, the activities of which are assayed by the aminopeptidase assay procedure of Goldbarg and Rutenburg. '~ The reagents required are a-Leucyl-fl-naphthylamide-HC1, (Leu-NA) 0.2 mg/ml in 0.1 M citrate buffer, pH 6.7 Trichloroacetic acid, 40% (w/w) NaNO,, 0.1% Ammonium sulfamate, 0.5% 1-Naphthylethylenediamine dihydrochloride (NEDD), 0.05% in 95% ethanol Assays are run by incuhating 0.5 ml of enzyme solution with 0.5 ml of Leu-NA substrate for 30 min, and the reaction is terminated by the addition of 0.5 ml of 40% trichloroacetic acid. To the mixture is added 1 ml of the NaNO., solution and the reaction is allowed to proceed for 3 min to convert the enzymically liberated B-naphthylamine to its diazonium salt. Excess nitrous acid is neutralized by the addition of 1 ml of 0.5% ammonium sulfamate, and after 2 rain the diazonium salt is coupled to NEDD by the addition of 2 ml of the NEDD reagent. Color development is complete in 20 rain, after which absorbance is measured at 560 nm. One unit of arylamidase activity is defined as the amount of enzyme required to liberate, in 30 min, sufficient fl-naphthylamine to yield a diazotized product having an absorbance of 1.0 at 560 nm. Esterase Assay. The speetrophotometric assay of Schwert and Takenaka 6 is used to measure the increase in absorbance at 253 nm when N-benzoyl-L-arginine ethyl ester (BAEE) is hydrolyzed. The substrate consists of 0.1 mM BAEE in 5 mM Tris-HC1 buffer, pH 7.6, equilibrated at 25 °. Cuvettes containing 2.9 ml of substrate are placed in a recording spectrophotometer, and the reaction is started by thoroughly mixing 0.1 ml of enzyme with the substrate. The increase in absorbance is recorded for 2-3 min while the temperature in the cell compartment: is maintained 4 F. W. Wagner and J. M. Prescott, Comp. Biochem. Physiol. 17, 191 (1966). J. A. Goldbarg and A. M. Rutenburg, Ca~cer 11,283 (1958). 6 G. W. Schwert and Y. Takenaka, Biochim. Biophys. Acta 16, 570 (1955).
[32]
Leucostoma PEPTIDASE A
399
at 25 °. The initial velocity of the reaction is determined from the recorder tracing, and 1 unit of esterase activity is defined as the amount of enzyme that. causes an increase of 1.0 in absorbance in 1 min at 25 °.
Enzyme Isolation All chromatographic procedures are carried out at 40-5 ° in order to minimize loss of enzymic activity, and it is convenient, but by no means necessary, to have continuous monitoring of the column effluents at 280 nm. Step 1. Preparation o/ Venom /or Chromatography. Lyophilized venom of A. p. leucostoma is purchased from Miami Serpentarium, Miami, Florida, and stored at --10 ° until used. Normally, 1-5 g of the dried venom are used in an isolation experiment; the volumes suggested below are suitable for a sample of about 2 g. The venom is dissolved in 20-50 ml of 5 m M Tris-HC1 buffer, pH 8.5, and dialyzed against this buffer overnight. The somewhat turbid solution is clarified by centrifugation at 5400 g for 5-10 rain and the clear supernatant fluid is stored at 4 ° until used. Step 2. Chromatography on DEAE-Sephadex A-50. DEAE-Sephadex A-50 is suspended in distilled water and allowed to stand for several hours, after which the fine particles are decanted. The ion-exchange material is suspended in 0.5 N HC1, filtered immediately, resuspended in distilled water, then washed several times in distilled water. The DEAE-Sephadex is next suspended in 0.5 N NaOH, allowed to stand for 15-20 min then filtered and again suspended in 0.5 N N a O H for a few minutes. After filtration, the resin is washed in distilled water until the washings are neutral to pH paper. The hydroxide form of D E A E Sephadex thus prepared is suspended in 5 m M Tris-HC1 buffer, pH 8.5, and allowed to equilibrate before being poured in a 2.5 X 50 cm column which is throughly washed with the buffer at 5 °. The sample from step 1 is applied and the column is washed with the same buffer at a rate of 60 ml/hr until the unadsorbed material emerges. The chromatogram is then developed by the application of a linear gradient from 0 to 0.1 M NaC1, with the effluent being collected in 10 ml fractions at a flow rate of 50-60 ml/hr. Typically, the first cylinder of the gradient-producing apparatus contains 1 liter of the 5 m M Tris-HCl buffer, pH 8.5, and the second contains an equal amount of the same buffer that is 0.1 M in NaC1. For experiments in which less than 1 g of venom is used as the starting material, the total volume of the gradient is reduced to 600 ml and for samples over 5 g it should be increased correspondingly. In some experiments we have completed the ehromatogram by washing the column
400
ENDOPEPTIDASES
[321
sequentially with 0.2 M and with 1.0 M NaC1 as shown in Fig. 1 to ensure elution of all proteolytic activity, but this step is not essential inasmuch as leucostoma peptidase A is eluted at about 0.05 M NaC1. This fraction always contains the greatest amount of activity toward hemoglobin substrate, although a small amount of proteolytic activity emerges in the breakthrough fraction, and some lots of venom contain a third chromatographically distinct proteolytic peak. Step 3. Rechromatography on DEAE-Sephadex A-50. The protease from step 2 (fraction I in Fig. 1) is dialyzed against 5 mM borateNaOH buffer, pH 9.2, and applied to a 2.5 X 50 cm column of DEAESephadex A-50 that has been equilibrated with this buffer. The chromatogram is developed with approximately 700 ml of a linear gradient of increasing NaC1 concentration as in step 2 (0-0.1 M NaC1), with a flow rate of 60 ml/hr. Fractions of 10 ml are collected in tubes containing 1 ml of 0.1 M Na2HPO~, pH 8.0, to lower the pH of the eluate. Each tube is read at 280 nm, and every fifth fraction is assayed for the three types of enzymic activity. The proteolytic activity emerges in a prominent peak that is only partially separated from smaller protein fractions that contain esterase and arylamidase activities.
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145 150
190 215
FIG. 1. Chromatography of c~.ude, dialyzed Agkistrodon piscivorus leucostoma venom on DEAE-Sephadex A-50 in 5 m M Tris-HC1, pH 8.5. A 2-g sample of venom was dialyzed, then applied to a 2.5 X 50 cm column and eluted at approximately 50 ml/hr into fractions containing 12 ml. - - , absorbance at 280 nm; Q, proteolytic activity toward denatured hemoglobin; A, esterase activity toward N-benzoyl-Larginine ethyl ester; [-1, arylamidase activity toward Leu-NA; - - - , NaC1 gradient. Fraction I (tubes 95-145) was selected for further purification. From F. W. Wagner, A. M. Spiekerman and J. M. Prescott, J. Biol. Chem. 243, 4486 (1968).
[32]
Leucostoma PEPTIDASE A
401
Step 4. Gel Filtration. The active material from step 3 is concentrated to approximately 5-10 ml by ultrafiltration with an Amicon UM-05 membrane under a nitrogen atmosphere of 50 psi. The concentrated sample is applied to a 2.5 X 50 cm column of Sephadex G-75 previously equilibrated with 5 mM Tris-HC1 buffer, pH 8.5, that is 0.1 M in NaC1, and eluted at a flow rate of 15-20 ml/hr until a total volume of 150-250 ml has been collected in fractions of 3-5 ml. The exact volume at which the ehromatogram is considered to be fully developed should be ascerrained by monitoring the column effluent at 280 nm, either manually or by a flow analyzer. The emergence of the proteolytie activity frequently is preceded by two relatively small components possessing esterase, but no protease, activity. The third and largest fraction is highly proteolytic but also contains some BAEE activity. In some experiments, this fraction may also be contaminated by arylamidase, and, if so, rechromatography under the same conditions is advisable. Step 5. Rechromatography on DEAE-Sephadex. The protease fraction from step 4 is dialyzed for 18 hr against 5 mM borate-NaOH buffer, pH 9.6, and applied to a 2.5 X 50 cm column of DEAE-Sephadex A-50, which has been equilibrated with the same buffer. The protease is eluted with 800-850 ml of a linear gradient of NaC1 (20 mM to 0.5 M) in the same buffer, with fractions of 6 ml collected at a rate of 15 ml/hr in tubes containing 1 ml of 0.1 M Tris-HC1 buffer, pH 7.8. Three components are separated by this procedure; the second and most prominent of these is protease, uncontaminated by esterase; the latter enzyme emerges in the third peak. Those fractions that show protease activity but no esterase are combined to form the final preparation. A summary of a purification experiment is shown in the table.
Properties Purity. Preparations of leucostoma peptidase A, purified as described above, have been found to be homogeneous by moving boundary electrophoresis at several pH values and by polyacrylamide gel electrophoresis at pH 9.5. Moreover, enzymic assays for activity toward BAEE and Leu-NA show the absence of detectable amounts of. esterase and arylamidase, respectively. The absence of esterase activity is an important criterion for purity because of the confusion generated by early workers who used unfractionated venoms as a source of enzyme in characterizing proteolytic activity. They found that not only were hemoglobin and casein rapidly hydrolyzed, but a number of venoms also cleaved such trypsin and chymotrypsin substrates as BAEE and N-acetyl-Ltyrosine ethyl ester, respectively. By analogy with the well known animal
402
ENDOPEPTIDASES
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Leucostoma PEPTIDASE A
403
enzymes, but without experimental evidence, some of these workers assumed that the proteolytic and esterolytic activities resulted from a single enzyme; this assumption led to the view that snake venoms contain "trypsin-like" and "chymotrypsin-like" proteases. Subsequently, experiments with carefully isolated proteases, including leucostoma peptidase A, showed the enzymes that are responsible for most of the proteolytic activity toward hemoglobin and casein have no esterase activity and, similarly, purified enzymes that cleave BAEE do not hydrolyze hemoglobin or casein. 7 Therefore, one of the criteria for purity of leucostoma peptidase A is the absence of activity toward BAEE. s Stability. The protease is stable for several weeks either frozen or at 4°; however, prolonged storage accompanied by freezing and thawing results in some autolysis that is manifest by the appearance of one or two new bands on gel electrophoresis, even when no appreciable loss in activity is evident. Physical Properties. Sedimentation velocity studies showed s~0.w to be 2.87 S for the venom protease and the value for D=,o,,~-has been found to be 10.66 X 10 Tcm 2 see-1. From the amino acid content, the partial specific volume was calculated to be 0.711 cm3/g. Substitution of these values into the Svedberg equation gave a molecular weight of 22,500, and that obtained from the approach to equilibrium method of Archibald was 22,400-~- 1250. Leucostoma peptidase A has an isoelectric point of 6.5 at an ionic strength of 0.1. The amino acid content is half-CysG, Asp._,,,, Thrl~, Ser,~, Glul., Pro~, Glyl~, Ala11, Val~, Mets, Ile~o, Leu,.,, Tyro, PheG, Lys~o, Hisg, Arg6, and Trp4. 6,7 Metal Content. Analyses of purified leucostoma peptidase A by atomic absorption spectrophotometry reveal the presence of both Ca ~* and Zn °-+ ions in stoichiometric quantities and indicate that the enzyme is a metalloprotein containing 2 g-atoms of calcium and 1 g-atom of zinc per mole. An essential role for metal ions in catalysis is indicated by the fact that both E D T A and o-phenanthroline inhibit proteolysis. Removal of Zn ~+ by o-phenanthroline in the presence of excess Ca ~+ inhibits the action. Of leucostoma peptidase A, which can then be reactivated by the addition of Zn '-'+. Thus, zinc is the catalytically essential metal, and it is likely that the role of calcium is to stabilize the enzyme. pH Optimum. Leucostoma peptidase A exhibits maximal activity toward hemoglobin substrate at pH 8.5. Substrate Specificity. As is the case for some other purified proteases from snake venoms, leucostoma peptidase A shows little activity toward 7 p. M. Toom, T. N. Solie, and A. T. Tu, J. Biol. Chem. 245, 2549 (1970). s Venom enzymes that hydrolyze BAEE may be specialized proteases of some type, even though they show no activity toward common protein substrates.
404
ENDOPEPTIDASES
[33]
small synthetic substrates. It has no activity against any exopeptidase substrate tested, and experiments with a large number of NH~-substituted and doubly substituted dipeptides revealed that only a few were susceptible and these were hydrolyzed slowly. ~- However, the protease readily cleaved the B-chain of oxidized insulin at positions Phel-Val..,, His~-Leu6, Hislo-Leu11, Alal~-Leu15, Gly~o-Glu~I, Gly._,a-Phe.,~, Phe.,4-Phe~.~.~ The substrate specificity is obviously dictated by the identity of the residue that contributes the NH~ group to the bond, and a preference is shown for substrates having a hydrophobic residue in that position. It is likely that some minimal substrate size is required for significant rates of hydrolysis, as substrate size has been shown to be important in the action of other purified venom proteases. 9,~° 9T. Takahashi and A. Ohsaka, Biochim. Biophgs. Acta 198, 293 (1970). 10K. Mella, M. Volz, and G. Pfleiderer, Anal. Biochern. 21, 219 (1967).
[33] A e r o m o n a s
Neutral
Protease
B y STELLA H. WILKES and JOHN M. PRESCOTT
Culture filtrates of A e r o m o n a s p r o t e o l y t i c a possess unusually high activity toward casein and hemoglobin substrates, 1,~ surpassing the levels produced by several other microbial species known to yield large quantities of extracellular proteolytic enzymes. 3 Fractionation of culture filtrates has revealed that this activity is due to two endopeptidases, one of which is the subject of this chapter.
Enzyme Assays Previous work on A e r o m o n a s neutral protease in this laboratory has been done with an assay based on the procedure of Anson, 4 using denatured hemoglobin as the substrate. It is frequently desirable, however, to be able to assay proteases by monitoring the cleavage of a single peptide bond of known identity. Details are given below, therefore, for assaying the enzyme both with hemoglobin and with a doubly blocked ' J. M. Prescott and C. R. Willms, Proc. Soc. Exp. Biol. Med. 103, 410 (1960). S. H. Wilkes, B. B. Mukherjee, F. W. Wagner, and J. M. Prescott, Proc. Soc. Exp. Biol. Med. 131, 382 (1969). 3L. Keay, M. H. Moseley, R. G. Anderson, R. J. O'Connor, and B. S. Wildi, Biotechnol. Bioeng. Symp. 3, 63 (1972). 4 M. L. Anson, J. Gen. Physiol. 22, 79 (1938).
[32l
397
[32] L e u c o s t o m a P e p t i d a s e A
By JOH~ M. PRESCOTTand FREDW. WAG~ER Leucostoma peptidase A is the most abundant protease in the venom of the western cottonmouth moccasin (Agkistrodon piscivorus leucostoma) from which it can be isolated in good yield and a high degree of homogeneity. 1,2 This endopeptidase possesses some characteristics similar to those of bacterial neutral proteases, inasmuch as it is inhibited by metal complexing reagents, is a metalloprotein that contains calcium and zinc, and has a substrate specificity dictated primarily by the identity of the residue contributing the amino group to the susceptible peptide bond.
Assay Procedures Leucostoma peptidase A readily digests casein and denatured hemoglobin, either of which can be used as a substrate to assay the proteolytic activity of fractions obtained during enzyme isolation. Details for the assay with hemoglobin substrate are given below. In addition, it is also advisable to assay for esterase and arylamidase activities at each stage of the procedure in order to monitor the elimination of enzymes that accompany Ieucostoma peptidase A through part of the purification procedure. Hemoglobin Assay for Proteolytic Activity. The procedure is essentially the same as that of Anson, 3 and involves determining the increase in trichloroacetic acid-soluble products that result from digestion of urea-denatured hemoglobin. The substrate is prepared by suspending 5 g of lyophilized, dialyzed hemoglobin (Worthington) in 80 ml of water with 80 g of urea. After the mixture has been incubated at 37 ° for 1 hr, 125 ml of 0.133 M phosphate buffer (pH 8.5) and 10 g of urea are added. The mixture is permitted to stand overnight and the pH is checked to ensure that it is 8.5. Assays are run by adding 1 ml of suitably diluted enzyme solution to 5 ml of the substrate and incubating the mixture at 37 ° for 10 min. The addition of 10 ml of 5% trichloroacetic acid serves to stop the reaction and to precipitate undigested protein, which is removed by filtration through Whatman No. 3 paper. The absorbance of 1F. W. Wagner, A. M. Spiekerman, and J. M. Prescott, J. Biol. Chem. 243, 4486 (1968). "A. M. Spiekerman, K. K. Fredericks, :F. W. Wagner, and J. M. Prescott, Biochim. Biophys. Acta 293, 464 (1973). 3M. L. Anson, Y. Gen. Physiol. 22, 79 (1938).
398
ENDOPEPTIDASES
[32]
the filtrate at 280 nm is then determined against a blank made by adding trichloroaeetic acid to the substrate prior to addition of the enzyme. One unit is defined as that quantity of enzyme that produces an increase of 1.0 in al)sorbance in 10 rain. Arylamidase Assay. Although the venom of A. p. leucostoma does not contain true aminopeptidase activity, it catalyzes the hydrolysis of L-leucyl-fl-naphthylamide' and thus contains one or more enzymes that can be classified for operational convenience as arylamidases, the activities of which are assayed by the aminopeptidase assay procedure of Goldbarg and Rutenburg. '~ The reagents required are a-Leucyl-fl-naphthylamide-HC1, (Leu-NA) 0.2 mg/ml in 0.1 M citrate buffer, pH 6.7 Trichloroacetic acid, 40% (w/w) NaNO,, 0.1% Ammonium sulfamate, 0.5% 1-Naphthylethylenediamine dihydrochloride (NEDD), 0.05% in 95% ethanol Assays are run by incuhating 0.5 ml of enzyme solution with 0.5 ml of Leu-NA substrate for 30 min, and the reaction is terminated by the addition of 0.5 ml of 40% trichloroacetic acid. To the mixture is added 1 ml of the NaNO., solution and the reaction is allowed to proceed for 3 min to convert the enzymically liberated B-naphthylamine to its diazonium salt. Excess nitrous acid is neutralized by the addition of 1 ml of 0.5% ammonium sulfamate, and after 2 rain the diazonium salt is coupled to NEDD by the addition of 2 ml of the NEDD reagent. Color development is complete in 20 rain, after which absorbance is measured at 560 nm. One unit of arylamidase activity is defined as the amount of enzyme required to liberate, in 30 min, sufficient fl-naphthylamine to yield a diazotized product having an absorbance of 1.0 at 560 nm. Esterase Assay. The speetrophotometric assay of Schwert and Takenaka 6 is used to measure the increase in absorbance at 253 nm when N-benzoyl-L-arginine ethyl ester (BAEE) is hydrolyzed. The substrate consists of 0.1 mM BAEE in 5 mM Tris-HC1 buffer, pH 7.6, equilibrated at 25 °. Cuvettes containing 2.9 ml of substrate are placed in a recording spectrophotometer, and the reaction is started by thoroughly mixing 0.1 ml of enzyme with the substrate. The increase in absorbance is recorded for 2-3 min while the temperature in the cell compartment: is maintained 4 F. W. Wagner and J. M. Prescott, Comp. Biochem. Physiol. 17, 191 (1966). J. A. Goldbarg and A. M. Rutenburg, Ca~cer 11,283 (1958). 6 G. W. Schwert and Y. Takenaka, Biochim. Biophys. Acta 16, 570 (1955).
[32]
Leucostoma PEPTIDASE A
399
at 25 °. The initial velocity of the reaction is determined from the recorder tracing, and 1 unit of esterase activity is defined as the amount of enzyme that. causes an increase of 1.0 in absorbance in 1 min at 25 °.
Enzyme Isolation All chromatographic procedures are carried out at 40-5 ° in order to minimize loss of enzymic activity, and it is convenient, but by no means necessary, to have continuous monitoring of the column effluents at 280 nm. Step 1. Preparation o/ Venom /or Chromatography. Lyophilized venom of A. p. leucostoma is purchased from Miami Serpentarium, Miami, Florida, and stored at --10 ° until used. Normally, 1-5 g of the dried venom are used in an isolation experiment; the volumes suggested below are suitable for a sample of about 2 g. The venom is dissolved in 20-50 ml of 5 m M Tris-HC1 buffer, pH 8.5, and dialyzed against this buffer overnight. The somewhat turbid solution is clarified by centrifugation at 5400 g for 5-10 rain and the clear supernatant fluid is stored at 4 ° until used. Step 2. Chromatography on DEAE-Sephadex A-50. DEAE-Sephadex A-50 is suspended in distilled water and allowed to stand for several hours, after which the fine particles are decanted. The ion-exchange material is suspended in 0.5 N HC1, filtered immediately, resuspended in distilled water, then washed several times in distilled water. The DEAE-Sephadex is next suspended in 0.5 N NaOH, allowed to stand for 15-20 min then filtered and again suspended in 0.5 N N a O H for a few minutes. After filtration, the resin is washed in distilled water until the washings are neutral to pH paper. The hydroxide form of D E A E Sephadex thus prepared is suspended in 5 m M Tris-HC1 buffer, pH 8.5, and allowed to equilibrate before being poured in a 2.5 X 50 cm column which is throughly washed with the buffer at 5 °. The sample from step 1 is applied and the column is washed with the same buffer at a rate of 60 ml/hr until the unadsorbed material emerges. The chromatogram is then developed by the application of a linear gradient from 0 to 0.1 M NaC1, with the effluent being collected in 10 ml fractions at a flow rate of 50-60 ml/hr. Typically, the first cylinder of the gradient-producing apparatus contains 1 liter of the 5 m M Tris-HCl buffer, pH 8.5, and the second contains an equal amount of the same buffer that is 0.1 M in NaC1. For experiments in which less than 1 g of venom is used as the starting material, the total volume of the gradient is reduced to 600 ml and for samples over 5 g it should be increased correspondingly. In some experiments we have completed the ehromatogram by washing the column
400
ENDOPEPTIDASES
[321
sequentially with 0.2 M and with 1.0 M NaC1 as shown in Fig. 1 to ensure elution of all proteolytic activity, but this step is not essential inasmuch as leucostoma peptidase A is eluted at about 0.05 M NaC1. This fraction always contains the greatest amount of activity toward hemoglobin substrate, although a small amount of proteolytic activity emerges in the breakthrough fraction, and some lots of venom contain a third chromatographically distinct proteolytic peak. Step 3. Rechromatography on DEAE-Sephadex A-50. The protease from step 2 (fraction I in Fig. 1) is dialyzed against 5 mM borateNaOH buffer, pH 9.2, and applied to a 2.5 X 50 cm column of DEAESephadex A-50 that has been equilibrated with this buffer. The chromatogram is developed with approximately 700 ml of a linear gradient of increasing NaC1 concentration as in step 2 (0-0.1 M NaC1), with a flow rate of 60 ml/hr. Fractions of 10 ml are collected in tubes containing 1 ml of 0.1 M Na2HPO~, pH 8.0, to lower the pH of the eluate. Each tube is read at 280 nm, and every fifth fraction is assayed for the three types of enzymic activity. The proteolytic activity emerges in a prominent peak that is only partially separated from smaller protein fractions that contain esterase and arylamidase activities.
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95 I00 FRACTION NUMBER
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145 150
190 215
FIG. 1. Chromatography of c~.ude, dialyzed Agkistrodon piscivorus leucostoma venom on DEAE-Sephadex A-50 in 5 m M Tris-HC1, pH 8.5. A 2-g sample of venom was dialyzed, then applied to a 2.5 X 50 cm column and eluted at approximately 50 ml/hr into fractions containing 12 ml. - - , absorbance at 280 nm; Q, proteolytic activity toward denatured hemoglobin; A, esterase activity toward N-benzoyl-Larginine ethyl ester; [-1, arylamidase activity toward Leu-NA; - - - , NaC1 gradient. Fraction I (tubes 95-145) was selected for further purification. From F. W. Wagner, A. M. Spiekerman and J. M. Prescott, J. Biol. Chem. 243, 4486 (1968).
[32]
Leucostoma PEPTIDASE A
401
Step 4. Gel Filtration. The active material from step 3 is concentrated to approximately 5-10 ml by ultrafiltration with an Amicon UM-05 membrane under a nitrogen atmosphere of 50 psi. The concentrated sample is applied to a 2.5 X 50 cm column of Sephadex G-75 previously equilibrated with 5 mM Tris-HC1 buffer, pH 8.5, that is 0.1 M in NaC1, and eluted at a flow rate of 15-20 ml/hr until a total volume of 150-250 ml has been collected in fractions of 3-5 ml. The exact volume at which the ehromatogram is considered to be fully developed should be ascerrained by monitoring the column effluent at 280 nm, either manually or by a flow analyzer. The emergence of the proteolytie activity frequently is preceded by two relatively small components possessing esterase, but no protease, activity. The third and largest fraction is highly proteolytic but also contains some BAEE activity. In some experiments, this fraction may also be contaminated by arylamidase, and, if so, rechromatography under the same conditions is advisable. Step 5. Rechromatography on DEAE-Sephadex. The protease fraction from step 4 is dialyzed for 18 hr against 5 mM borate-NaOH buffer, pH 9.6, and applied to a 2.5 X 50 cm column of DEAE-Sephadex A-50, which has been equilibrated with the same buffer. The protease is eluted with 800-850 ml of a linear gradient of NaC1 (20 mM to 0.5 M) in the same buffer, with fractions of 6 ml collected at a rate of 15 ml/hr in tubes containing 1 ml of 0.1 M Tris-HC1 buffer, pH 7.8. Three components are separated by this procedure; the second and most prominent of these is protease, uncontaminated by esterase; the latter enzyme emerges in the third peak. Those fractions that show protease activity but no esterase are combined to form the final preparation. A summary of a purification experiment is shown in the table.
Properties Purity. Preparations of leucostoma peptidase A, purified as described above, have been found to be homogeneous by moving boundary electrophoresis at several pH values and by polyacrylamide gel electrophoresis at pH 9.5. Moreover, enzymic assays for activity toward BAEE and Leu-NA show the absence of detectable amounts of. esterase and arylamidase, respectively. The absence of esterase activity is an important criterion for purity because of the confusion generated by early workers who used unfractionated venoms as a source of enzyme in characterizing proteolytic activity. They found that not only were hemoglobin and casein rapidly hydrolyzed, but a number of venoms also cleaved such trypsin and chymotrypsin substrates as BAEE and N-acetyl-Ltyrosine ethyl ester, respectively. By analogy with the well known animal
402
ENDOPEPTIDASES
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Leucostoma PEPTIDASE A
403
enzymes, but without experimental evidence, some of these workers assumed that the proteolytic and esterolytic activities resulted from a single enzyme; this assumption led to the view that snake venoms contain "trypsin-like" and "chymotrypsin-like" proteases. Subsequently, experiments with carefully isolated proteases, including leucostoma peptidase A, showed the enzymes that are responsible for most of the proteolytic activity toward hemoglobin and casein have no esterase activity and, similarly, purified enzymes that cleave BAEE do not hydrolyze hemoglobin or casein. 7 Therefore, one of the criteria for purity of leucostoma peptidase A is the absence of activity toward BAEE. s Stability. The protease is stable for several weeks either frozen or at 4°; however, prolonged storage accompanied by freezing and thawing results in some autolysis that is manifest by the appearance of one or two new bands on gel electrophoresis, even when no appreciable loss in activity is evident. Physical Properties. Sedimentation velocity studies showed s~0.w to be 2.87 S for the venom protease and the value for D=,o,,~-has been found to be 10.66 X 10 Tcm 2 see-1. From the amino acid content, the partial specific volume was calculated to be 0.711 cm3/g. Substitution of these values into the Svedberg equation gave a molecular weight of 22,500, and that obtained from the approach to equilibrium method of Archibald was 22,400-~- 1250. Leucostoma peptidase A has an isoelectric point of 6.5 at an ionic strength of 0.1. The amino acid content is half-CysG, Asp._,,,, Thrl~, Ser,~, Glul., Pro~, Glyl~, Ala11, Val~, Mets, Ile~o, Leu,.,, Tyro, PheG, Lys~o, Hisg, Arg6, and Trp4. 6,7 Metal Content. Analyses of purified leucostoma peptidase A by atomic absorption spectrophotometry reveal the presence of both Ca ~* and Zn °-+ ions in stoichiometric quantities and indicate that the enzyme is a metalloprotein containing 2 g-atoms of calcium and 1 g-atom of zinc per mole. An essential role for metal ions in catalysis is indicated by the fact that both E D T A and o-phenanthroline inhibit proteolysis. Removal of Zn ~+ by o-phenanthroline in the presence of excess Ca ~+ inhibits the action. Of leucostoma peptidase A, which can then be reactivated by the addition of Zn '-'+. Thus, zinc is the catalytically essential metal, and it is likely that the role of calcium is to stabilize the enzyme. pH Optimum. Leucostoma peptidase A exhibits maximal activity toward hemoglobin substrate at pH 8.5. Substrate Specificity. As is the case for some other purified proteases from snake venoms, leucostoma peptidase A shows little activity toward 7 p. M. Toom, T. N. Solie, and A. T. Tu, J. Biol. Chem. 245, 2549 (1970). s Venom enzymes that hydrolyze BAEE may be specialized proteases of some type, even though they show no activity toward common protein substrates.
404
ENDOPEPTIDASES
[33]
small synthetic substrates. It has no activity against any exopeptidase substrate tested, and experiments with a large number of NH~-substituted and doubly substituted dipeptides revealed that only a few were susceptible and these were hydrolyzed slowly. ~- However, the protease readily cleaved the B-chain of oxidized insulin at positions Phel-Val..,, His~-Leu6, Hislo-Leu11, Alal~-Leu15, Gly~o-Glu~I, Gly._,a-Phe.,~, Phe.,4-Phe~.~.~ The substrate specificity is obviously dictated by the identity of the residue that contributes the NH~ group to the bond, and a preference is shown for substrates having a hydrophobic residue in that position. It is likely that some minimal substrate size is required for significant rates of hydrolysis, as substrate size has been shown to be important in the action of other purified venom proteases. 9,~° 9T. Takahashi and A. Ohsaka, Biochim. Biophgs. Acta 198, 293 (1970). 10K. Mella, M. Volz, and G. Pfleiderer, Anal. Biochern. 21, 219 (1967).
[33] A e r o m o n a s
Neutral
Protease
B y STELLA H. WILKES and JOHN M. PRESCOTT
Culture filtrates of A e r o m o n a s p r o t e o l y t i c a possess unusually high activity toward casein and hemoglobin substrates, 1,~ surpassing the levels produced by several other microbial species known to yield large quantities of extracellular proteolytic enzymes. 3 Fractionation of culture filtrates has revealed that this activity is due to two endopeptidases, one of which is the subject of this chapter.
Enzyme Assays Previous work on A e r o m o n a s neutral protease in this laboratory has been done with an assay based on the procedure of Anson, 4 using denatured hemoglobin as the substrate. It is frequently desirable, however, to be able to assay proteases by monitoring the cleavage of a single peptide bond of known identity. Details are given below, therefore, for assaying the enzyme both with hemoglobin and with a doubly blocked ' J. M. Prescott and C. R. Willms, Proc. Soc. Exp. Biol. Med. 103, 410 (1960). S. H. Wilkes, B. B. Mukherjee, F. W. Wagner, and J. M. Prescott, Proc. Soc. Exp. Biol. Med. 131, 382 (1969). 3L. Keay, M. H. Moseley, R. G. Anderson, R. J. O'Connor, and B. S. Wildi, Biotechnol. Bioeng. Symp. 3, 63 (1972). 4 M. L. Anson, J. Gen. Physiol. 22, 79 (1938).