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Ion-exchange chromatographic characterization of stinging insect vespid venoms
Tonklar. Vol. 22, No. 1, pp . 154-160, 1954. Primed m Grat aritdn.
0041-0101/54 53 .00+ .00 O 1984 Pcrpmoa Prey Ltd.
ION-EXCHANGE CHROMATOGRAPHIC CHARACTERIZATION OF STINGING INSECT VESPID VENOMS R. EINARSSON and B. RENCK Pharmacia Diagnostics AB, Uppsala, Sweden (Accepted for publication 26 August 1983)
R. EINAtessoN and B. REw3c . Ion-exchange chromatographic characterization of stinging insect vespidvenoms . Toxicon 22,154-160,1984. - Paper wasp, hornets and yellow jacket vespid venoms were analysed by elaxrofocusing - electrophoresis titration curves and cation exchange chromatography on Mono STM. Important protein components were identified by zymography . The elution profiles from the cation exchange column for the different vespid venoms differed significantly, demonstrating the use ofthis technique for identification purposes . The various fractions from the ion-exchange elution profiles were collected and analysed with respect to enzymatic activity. The high capacity of the system makes it also suitable for preparative scale separation.
VENOMS of stinging insects, such as bees, yellow jackets, paper wasps and hornets are composed of proteins (enzymes), peptides and different low molecular weight components, with both pharmacological and allergenic activities (HABERMANN, 1972; KING et al., 1978) . Honey bee venom has been extensively studied by biochemical and immunological techniques while vespid venoms have been subjected to relatively few investigations . The methods used for purification and characterization of Hymenoptera venoms have mostly been based upon gel chromatography, ion-exchange chromatography, capillary isotachophoresis and various electrophoretic methods (EiNARSsoN et al., 1982; EINARSSON et al., 1981 ; HOFFMAN, 1977; KING et ál., 1976, 1978) . In this paper we report on the use of a new ion-exchange material (Mono STM cation exchanger) for characterization of stinging insect vespid venoms . Paper wasp (Polistes spp .), white faced hornet, yellow hornet (Dolichovespula arenaria) and yellow jacket (Vespula spp.) were purchased from Vespa Laboratories, Spring Mills, PA, U.S.A., as lyophilized material . The different vespid venoms were extracted from venom sacs. Electrofocusing - electrophoresis titration curves of the venoms were performed essentially according to a previously described procedure (RIGI-IM7i et al., 1979) using a broad pH interval (3 -10). The venoms were dissolved in distilled water to a concentration of 12% and then desalted on a small Sephadex G-25M column (Pharmacia Fine Chemicals). A sample of 25 jul was applied on each plate. Ion-exchange chromatography was performed on a Fast Protein Liquid Chromatography system equipped with cation exchanger Mono STM (HR 5/5), (Pharmacia Fine Chemicals) (Ftigerstam et al., 1983) . The venoms were dialyzed against the starting buffer, 50 mM Bicine [N, N-bis (2-hydroxyethyl)glycine] pH 8 .4, prior to injection. Seven milligrams of each insect venom dissolved in the starting buffer was injected onto the column . Desorption of venom components was performed by applying a linear gradient (50 mM Bicine, 350 mM NaCl, pH 8 .4) at a rate of 8 .75 mM 154
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FIG . 1 . TITRATION CURvBs OF sTINOm INsECr vEsPm vENoms . (a) Paper wasp ; (b) white faced hornet ; (c) yellow jacket ; (d) yellow hornet . The trench was filled with 25 yl (1 mg protein) of each vespid venom. The gel contained 546 acrylamide, 346 big-aaylamide and 2% Pharmalyte, pH 3 -10 . The second dimension electrophoresis was run for 30 min. 500 V/11 .5 cm, at 4°C . The two arrows and positive and negative symbols represent the direction and polarity of isoelectric focusing (IM and electrophoresis (EPH) . The arrow at the application zone denotes the pH of adsorption to the cation exchanger.
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Na'/min and a total flow rate of 1 ml/min (linear salt gradient). The eluent was monitored at 280 nm. Acid phosphatase activity was assayed by hydrolysis of the chromogenic substrate pnitrophenyl-phosphate (EINARSSON et al., 1982). Phospholipase A and B activity were measured by using agarose diffusion plates with lecithin and lysolecithin, respectively, as substrate (HABERMANN et al., 1972). Hyaluronidase was determined by using hyaluronic acid (HealonO) dispersed in agarose as substrate, essentially in accordance with a recently described procedure (RicHMAN et al., 1980). Proteolytic activity was demonstrated using radial diffusion in agarose gel with bovine serum albumin as substrate adsorbed to a polystyrene surface (WuWRöm et al., 1981). Figures la-d demonstrate titration curves of the different Hymenoptera venoms produced by electrofocusing - electrophoresis. Each venom was investigated over a broad pH range, 3 -10. The proteins were visualized by staining with Coomassie Brilliant Blue. This technique is invaluable for choosing the optimal pH in ion-exchange chromatography, since it provides us with relative pH/net charge data for all important components of the venom sample . All titration curves of the Hymenoptera venoms produced exhibit a wide variety of fine structure (many resolved protein bands) over the entire pH range. They also demonstrate the presence of several resolved protein bands at the most alkaline region (pH > 9). The titration curves of yellow jacket, yellow hornet and white faced hornet (Figs. 1a - c) do not show any drastic difference in the electrofocusing - electrophoresis pattern while that of the paper wasp (Fig . Id) shows a higher amount of neutral and acidic components compared to the other vespid venom extracts . These results are in good agreement with isotachophoretic data, where the paper wasp also exhibits a markedly different pattern compared to the other three vespid venoms (EINAPSSON et al., 1981). Figure 2a shows an ion-exchange chromatographic separation of white faced hornet venom (absorbance at 280 nm). Acid phosphatase is not adsorbed to the cation exchanger, since the isoelectric point of the enzyme is markedly less than pH 8.4. The first two eluted, partly separated peaks in the chromatogram exhibit hyaluronidase enzyme activity . After that, four main peaks follow, of which three exhibit marked shoulders. In this area phospholipase A and B activity have been identified . Figure 2b shows the chromatographic separation of yellow hornet venom. The material not adsorbed to the ion-exchanger not only exhibits acid phosphatase but also phospholipase B activity, suggesting that phospholipase B in the yellow hornet has an isoelectric point lower than pH 8.4. Hyaluronidase elutes as the first component in the form of a complex peak . After this, four other well-resolved peaks follow, but with a different pattern to that of the white faced hornet venom. The elution patterns of the white faced hornet and yellow hornet show differences which might be used for identification purposes . Figure 2c shows the chromatographic separation of yellow jacket vespid venom. Acid phosphatase activity is detected in the material not adsorbed to a Mono STA+ cation exchanger. The first eluted peak contains hyaluronidase and proteolytic activity. After these, a complex of several close peaks with phospholipase A activity appear . Finally, the chromatogram has a peak with considerable tailing, which exhibits both phospholipase A and B activity. The pronounced peak in the middle of the elution profile does not show enzyme activity. This peak has been identified as a protein and might be Antigen 5 (KING et al., 1978). Figure 2d shows the chromatographic separation of paper wasp venom and the identification of enzymatic activities in the elution profile. This elution profile differs significantly from the elution profiles of both hornets and the yellow jacket . A considerable
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ION-mcCHAME cH e~roaw+PHIC WpwawTION OF (a) WtmM FACED Ho~. (b) xm.ww Hoae zr, (c) YELL ow j~ AND (d) PAF®e wAsp, Conditions : ation co~ Mono S'M, HR 3/3 co~ mobile phase 0.03 M ~buffer, pH 8.4; linear salt Sradimt 0-0.33 M N&Q; flow rate l -Vmin; ambient tempmahue; u.v . detection at 280 am; ample size 7 ma dry w~ The linear salt Sadimt Is Murtrated me tbc dotted line. Ideatifled enzyme activities are indicted in the Qadieat ehttloa p^ . MG . 2 .
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0 .050 .0.
159
C . 12 Phosplgllpass
A " PhwphoUpaaa B " HyakroNdass " Acid phmphatave " Protease
// /
0
160
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amount of phospholipase A and B and acid phosphatase is detected in the material not adsorbed to the ion-exchanger. This is in agreement with the pattern from the titration curves, where two stained protein bands at pH 7 and 5.5 show up, while the phospholipase bands appear weaker . The presence of alkaline phosphatase enzyme activity (retention time 20 - 25 min) is somewhat puzzling, since enzyme activity either of acid or alkaline type usually has an isoelectric point in the acid or neutral pH range. At a retention time of 40 min a distorted peak containing phospholipase activity was also identified . Results obtained to date with ion-exchange chromatography using a Mono STM prepacked column and eluting with a linear gradient of sodium chloride suggests that it is possible to differentiate between the individual vespid venoms . Further, the reproducibility and high resolution of this ion-exchanger is such that each species chromatographed on various separate occasions showed peaks occurring at similar retention times, rendering this technique suitable for identification purposes . The high capacity and resolution of the ion-exchanger material makes fast protein liquid chromatography on a monodispersed ion-exchanger (Mono STM) a powerful tool in the characterization and purification of Hymenoptera stinging insect venoms . REFERENCES EINARSSON, R. and MOHERO, U. (1981) Isotachophoretic characterization of stinging insect venoms . J. Chromat. 209, 121 . EINARssoN, R., ANNERHED, A., K,UassoN, R., OLssoN, B. and RENCK, B. (1982) Crossed immunoelectrophoresis analysis of bee venom. Enzymatic identification of antigens . Allergy 37, 273. FXGERSrAM, L., SaDERwao, L., WAHLvrRÖM, L., FREDumsoN, U.-B., PLrrH, K. and WALLDPN, E. (1983) Basic principles used in the selection of MonobeadsTM ion exchangers for the separation of biopolymers. In: Protides of the Biological Fluids Vol 30, pp . 621-628, (P=Rs, H., Ed .) . Oxford: Pergamon Press. HAwRMANN, E. (1972) Bee and wasp venoms . Science 177, 314. HAaERmANN, E. and HARm, K. L. (1972) A sensitive and specific plate test for the quantitation of phospholipases . Analyt . Biochern. 50, 163 . HOFFmAN, D. R. (1977) Allergens in bee venom. III. Identification of allergen B of bee venom as an acid phosphatase. J. Allergy clip. Immun. 59, 364. KING, T. P., Sor+oTxA, A. K., KGCHOUMIAN, L. and LICHTENSrEIN, L. (1976) Allergens of honey bee venom. Archs Biochem. Blophys. 172, 661. KING, T. P., SoeoTxA, A. K., ALAGON, A., KOCHOUMIAN, L. and LICHTENsTEIN, L. (1978) Protein allergens of white-faced hornet, yellow hornet and yellow jacket venoms . Biochemistry 17, 5165 . RIcHmAN, P. G. and BAER, H. (1980) A convenient plate assay for the quantitation of hyaluronidase in hymenoptera venoms . Analyt. Biochern . 109, 376. RIGHE7TI, P. G. and GIANAzzA, E. (1979) pH-mobility curves of proteins by isoelectric focusing combined with electrophoresis at right angles . In : Electrophoresis, pp . 23-38 (RADorA, B. J., Ed.). Berlin : de Gruyter . WncsrR6M, M., ELWING, H. and LINDE, A. (1981) Determination of proteolytic activity: a sensitive and simple assay utilizing substrate adsorbed to a plastic surface and radial diffusion gel. Analyt. Biochem. 118, 240.
0041-0101/54 53 .00+ .00 O 1984 Pcrpmoa Prey Ltd.
ION-EXCHANGE CHROMATOGRAPHIC CHARACTERIZATION OF STINGING INSECT VESPID VENOMS R. EINARSSON and B. RENCK Pharmacia Diagnostics AB, Uppsala, Sweden (Accepted for publication 26 August 1983)
R. EINAtessoN and B. REw3c . Ion-exchange chromatographic characterization of stinging insect vespidvenoms . Toxicon 22,154-160,1984. - Paper wasp, hornets and yellow jacket vespid venoms were analysed by elaxrofocusing - electrophoresis titration curves and cation exchange chromatography on Mono STM. Important protein components were identified by zymography . The elution profiles from the cation exchange column for the different vespid venoms differed significantly, demonstrating the use ofthis technique for identification purposes . The various fractions from the ion-exchange elution profiles were collected and analysed with respect to enzymatic activity. The high capacity of the system makes it also suitable for preparative scale separation.
VENOMS of stinging insects, such as bees, yellow jackets, paper wasps and hornets are composed of proteins (enzymes), peptides and different low molecular weight components, with both pharmacological and allergenic activities (HABERMANN, 1972; KING et al., 1978) . Honey bee venom has been extensively studied by biochemical and immunological techniques while vespid venoms have been subjected to relatively few investigations . The methods used for purification and characterization of Hymenoptera venoms have mostly been based upon gel chromatography, ion-exchange chromatography, capillary isotachophoresis and various electrophoretic methods (EiNARSsoN et al., 1982; EINARSSON et al., 1981 ; HOFFMAN, 1977; KING et ál., 1976, 1978) . In this paper we report on the use of a new ion-exchange material (Mono STM cation exchanger) for characterization of stinging insect vespid venoms . Paper wasp (Polistes spp .), white faced hornet, yellow hornet (Dolichovespula arenaria) and yellow jacket (Vespula spp.) were purchased from Vespa Laboratories, Spring Mills, PA, U.S.A., as lyophilized material . The different vespid venoms were extracted from venom sacs. Electrofocusing - electrophoresis titration curves of the venoms were performed essentially according to a previously described procedure (RIGI-IM7i et al., 1979) using a broad pH interval (3 -10). The venoms were dissolved in distilled water to a concentration of 12% and then desalted on a small Sephadex G-25M column (Pharmacia Fine Chemicals). A sample of 25 jul was applied on each plate. Ion-exchange chromatography was performed on a Fast Protein Liquid Chromatography system equipped with cation exchanger Mono STM (HR 5/5), (Pharmacia Fine Chemicals) (Ftigerstam et al., 1983) . The venoms were dialyzed against the starting buffer, 50 mM Bicine [N, N-bis (2-hydroxyethyl)glycine] pH 8 .4, prior to injection. Seven milligrams of each insect venom dissolved in the starting buffer was injected onto the column . Desorption of venom components was performed by applying a linear gradient (50 mM Bicine, 350 mM NaCl, pH 8 .4) at a rate of 8 .75 mM 154
155
FIG . 1 . TITRATION CURvBs OF sTINOm INsECr vEsPm vENoms . (a) Paper wasp ; (b) white faced hornet ; (c) yellow jacket ; (d) yellow hornet . The trench was filled with 25 yl (1 mg protein) of each vespid venom. The gel contained 546 acrylamide, 346 big-aaylamide and 2% Pharmalyte, pH 3 -10 . The second dimension electrophoresis was run for 30 min. 500 V/11 .5 cm, at 4°C . The two arrows and positive and negative symbols represent the direction and polarity of isoelectric focusing (IM and electrophoresis (EPH) . The arrow at the application zone denotes the pH of adsorption to the cation exchanger.
Short Communications
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Na'/min and a total flow rate of 1 ml/min (linear salt gradient). The eluent was monitored at 280 nm. Acid phosphatase activity was assayed by hydrolysis of the chromogenic substrate pnitrophenyl-phosphate (EINARSSON et al., 1982). Phospholipase A and B activity were measured by using agarose diffusion plates with lecithin and lysolecithin, respectively, as substrate (HABERMANN et al., 1972). Hyaluronidase was determined by using hyaluronic acid (HealonO) dispersed in agarose as substrate, essentially in accordance with a recently described procedure (RicHMAN et al., 1980). Proteolytic activity was demonstrated using radial diffusion in agarose gel with bovine serum albumin as substrate adsorbed to a polystyrene surface (WuWRöm et al., 1981). Figures la-d demonstrate titration curves of the different Hymenoptera venoms produced by electrofocusing - electrophoresis. Each venom was investigated over a broad pH range, 3 -10. The proteins were visualized by staining with Coomassie Brilliant Blue. This technique is invaluable for choosing the optimal pH in ion-exchange chromatography, since it provides us with relative pH/net charge data for all important components of the venom sample . All titration curves of the Hymenoptera venoms produced exhibit a wide variety of fine structure (many resolved protein bands) over the entire pH range. They also demonstrate the presence of several resolved protein bands at the most alkaline region (pH > 9). The titration curves of yellow jacket, yellow hornet and white faced hornet (Figs. 1a - c) do not show any drastic difference in the electrofocusing - electrophoresis pattern while that of the paper wasp (Fig . Id) shows a higher amount of neutral and acidic components compared to the other vespid venom extracts . These results are in good agreement with isotachophoretic data, where the paper wasp also exhibits a markedly different pattern compared to the other three vespid venoms (EINAPSSON et al., 1981). Figure 2a shows an ion-exchange chromatographic separation of white faced hornet venom (absorbance at 280 nm). Acid phosphatase is not adsorbed to the cation exchanger, since the isoelectric point of the enzyme is markedly less than pH 8.4. The first two eluted, partly separated peaks in the chromatogram exhibit hyaluronidase enzyme activity . After that, four main peaks follow, of which three exhibit marked shoulders. In this area phospholipase A and B activity have been identified . Figure 2b shows the chromatographic separation of yellow hornet venom. The material not adsorbed to the ion-exchanger not only exhibits acid phosphatase but also phospholipase B activity, suggesting that phospholipase B in the yellow hornet has an isoelectric point lower than pH 8.4. Hyaluronidase elutes as the first component in the form of a complex peak . After this, four other well-resolved peaks follow, but with a different pattern to that of the white faced hornet venom. The elution patterns of the white faced hornet and yellow hornet show differences which might be used for identification purposes . Figure 2c shows the chromatographic separation of yellow jacket vespid venom. Acid phosphatase activity is detected in the material not adsorbed to a Mono STA+ cation exchanger. The first eluted peak contains hyaluronidase and proteolytic activity. After these, a complex of several close peaks with phospholipase A activity appear . Finally, the chromatogram has a peak with considerable tailing, which exhibits both phospholipase A and B activity. The pronounced peak in the middle of the elution profile does not show enzyme activity. This peak has been identified as a protein and might be Antigen 5 (KING et al., 1978). Figure 2d shows the chromatographic separation of paper wasp venom and the identification of enzymatic activities in the elution profile. This elution profile differs significantly from the elution profiles of both hornets and the yellow jacket . A considerable
Short Communications
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ION-mcCHAME cH e~roaw+PHIC WpwawTION OF (a) WtmM FACED Ho~. (b) xm.ww Hoae zr, (c) YELL ow j~ AND (d) PAF®e wAsp, Conditions : ation co~ Mono S'M, HR 3/3 co~ mobile phase 0.03 M ~buffer, pH 8.4; linear salt Sradimt 0-0.33 M N&Q; flow rate l -Vmin; ambient tempmahue; u.v . detection at 280 am; ample size 7 ma dry w~ The linear salt Sadimt Is Murtrated me tbc dotted line. Ideatifled enzyme activities are indicted in the Qadieat ehttloa p^ . MG . 2 .
Short Communications
0 .050 .0.
159
C . 12 Phosplgllpass
A " PhwphoUpaaa B " HyakroNdass " Acid phmphatave " Protease
// /
0
160
Short Communications
amount of phospholipase A and B and acid phosphatase is detected in the material not adsorbed to the ion-exchanger. This is in agreement with the pattern from the titration curves, where two stained protein bands at pH 7 and 5.5 show up, while the phospholipase bands appear weaker . The presence of alkaline phosphatase enzyme activity (retention time 20 - 25 min) is somewhat puzzling, since enzyme activity either of acid or alkaline type usually has an isoelectric point in the acid or neutral pH range. At a retention time of 40 min a distorted peak containing phospholipase activity was also identified . Results obtained to date with ion-exchange chromatography using a Mono STM prepacked column and eluting with a linear gradient of sodium chloride suggests that it is possible to differentiate between the individual vespid venoms . Further, the reproducibility and high resolution of this ion-exchanger is such that each species chromatographed on various separate occasions showed peaks occurring at similar retention times, rendering this technique suitable for identification purposes . The high capacity and resolution of the ion-exchanger material makes fast protein liquid chromatography on a monodispersed ion-exchanger (Mono STM) a powerful tool in the characterization and purification of Hymenoptera stinging insect venoms . REFERENCES EINARSSON, R. and MOHERO, U. (1981) Isotachophoretic characterization of stinging insect venoms . J. Chromat. 209, 121 . EINARssoN, R., ANNERHED, A., K,UassoN, R., OLssoN, B. and RENCK, B. (1982) Crossed immunoelectrophoresis analysis of bee venom. Enzymatic identification of antigens . Allergy 37, 273. FXGERSrAM, L., SaDERwao, L., WAHLvrRÖM, L., FREDumsoN, U.-B., PLrrH, K. and WALLDPN, E. (1983) Basic principles used in the selection of MonobeadsTM ion exchangers for the separation of biopolymers. In: Protides of the Biological Fluids Vol 30, pp . 621-628, (P=Rs, H., Ed .) . Oxford: Pergamon Press. HAwRMANN, E. (1972) Bee and wasp venoms . Science 177, 314. HAaERmANN, E. and HARm, K. L. (1972) A sensitive and specific plate test for the quantitation of phospholipases . Analyt . Biochern. 50, 163 . HOFFmAN, D. R. (1977) Allergens in bee venom. III. Identification of allergen B of bee venom as an acid phosphatase. J. Allergy clip. Immun. 59, 364. KING, T. P., Sor+oTxA, A. K., KGCHOUMIAN, L. and LICHTENSrEIN, L. (1976) Allergens of honey bee venom. Archs Biochem. Blophys. 172, 661. KING, T. P., SoeoTxA, A. K., ALAGON, A., KOCHOUMIAN, L. and LICHTENsTEIN, L. (1978) Protein allergens of white-faced hornet, yellow hornet and yellow jacket venoms . Biochemistry 17, 5165 . RIcHmAN, P. G. and BAER, H. (1980) A convenient plate assay for the quantitation of hyaluronidase in hymenoptera venoms . Analyt. Biochern . 109, 376. RIGHE7TI, P. G. and GIANAzzA, E. (1979) pH-mobility curves of proteins by isoelectric focusing combined with electrophoresis at right angles . In : Electrophoresis, pp . 23-38 (RADorA, B. J., Ed.). Berlin : de Gruyter . WncsrR6M, M., ELWING, H. and LINDE, A. (1981) Determination of proteolytic activity: a sensitive and simple assay utilizing substrate adsorbed to a plastic surface and radial diffusion gel. Analyt. Biochem. 118, 240.