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A nucleoside isolated from the venom of Bungarus multicinctus
Toxecon, 1963, Vol. 3, pp. 1-4. Pvatmon piesi Ltd., Printed in Great Hdtain
A NUCLEOSIDE ISOLATED FROM THE VENOM OF BUNGARUS MULTICINCTUS* A-Li Wsr and C. Y. L~ Pharmacological Institute, College of Medicine, National Taiwan University, Taipei,Taiwaa, China (Acceptedforpublication 11 March 196
Abetrod-During isolation ofneurotoxins from the venom of Brargarua ~nultlctnctua by means ofzone electrophoresis on starch, a non-proteinfraction was found in additionto the 4protein fractions obtained previously. It shawl maximum absorbance at 2S2 mEt, positive Molisch's testfor sugar and orcinol test for pwtoae, but negative Loring's teat for phosphate. It shows tl~ soma Rf value as guaaosine on thepaper~hromatogram, and its spectr8l data at various pH vahres am also identical with those of guanosine. Itscontent in the venom was mated to be 1 "1 f 0"14 per cwt. So far no biological effects could be attributed to the presence of guanosine in the venom. DuR1Na isolation of Bungarotoxins from the venom of Btasgarus enalticfnctus by means of zone electrophoresis on starch, a non-protein substance was obtained along with the 4 protein fractions described previously by CxANa and L~ [1]. It was detected by measuring the absorbance of each fraction at 278 m~. It showed maximum absorbance at 252 mw, positive Molisch's test for sugar and orcinol test for pentose, but negative Loring's test for phosphate. Upon dialysis, it passed through the cellulose membrane. From the results ofthe above tests it was suspected to be a nucleoside. When developed under n-butanol-isobutyric acid-NH,OH-H,O system, its Rp value was just the same as that of guanosine. Moreover, its spectral data at various pH values were found to be practically identical with those of guanosine. Thus, the nucleoside isolated from the venom was identified as guanosine. The guanosine content in the venom was estimated by co-zone electrophoresis, in which a certain amount of guanosine was added to the venom. By comparing the concentration of the eluate from the co-zone electrophoresis with that from the zone electrophoresis of the venom alone, it was estimated to be 1 "1 ~ 0 "14 per cent of the venom. FISCfmt and Döx~L [2] reported that adenosine was found in the venoms of Bias arietans and Dendraspis viridis and that the blood pressure lowering effect of the venoms might be partly due to the presence of adenosine. So far as our tests show the guanosine fraction, as well as pure guanosine, had no appreciable effect on the blood pressure of an anesthetized cat, even at a dose as large as 1 mg/kg of body weight . Thus, no biological effect could be attributed to the presence of guanosine in this venom. *This investigation was supported by the U.S. Arngy Medical Research aad Development Command, Department ofthe Armar, under Research Cirant No. DA-MD-49-193-GA-01108.
2
A-LI WEI aad C. Y. LEE EXPERIMENTAL
Venom
The venom of Bungarus multicinctus used in this study was freshly collected in this laboratory and stored in dried state in a vacuum desiocator . Starch-zone electrophoresis
The same electrophoresic conditions as those described by CHANO and hea [I] were followed . A typical electrophoresic pattern obtained by measuring the absorbance at 278 m~. is illustrated in Fig. 1. The venom migrated towards the cathode and separated into 5 fractions, the first of which disappeared when tested with Folin reagent. For the sake of convenience, the first fraction was designated as fraction A.
l. ZONE ELeCfAOPxOnFCia ON STARCH OF Tää VENOM OF HIJNOAalJ3 MULTICINCTUS. Potato starch moistenedwith sa acetate buffer, pH S'0, ionic strength 0"OS, plus NaCI, ionicstrength0'OS, was packed into a semicylindrical glass trough . Tweaty ßve mg of ande venom ores applied at the arrow 1S cm apart from the anode end. Electrophoresis was performed in a r~rigerator at 0~°C. An average potential difference of 170 V between tl~ two ends of the trough was applied for 30 hr. Fraction Ais Folin-negative whereasfractions I-IV areFolio-positive .
FIQ .
2. C~rANOEB OF CONCENTRATION OF FAAC'ITON A DUAIIVO DIALYSL9. Ten ml of the ehrate from fraction A was dialyaod for 3"S hr against S00ml distilled orator, at 29°C . FIa .
A Nucleoside isolated ~ Sorbe Veaoa
3
Dialysis Ten millilitres of the eluate from fraction Awere dialyzed at room temperature(29°C) for 3 "5 hr against 500 ml distilled water in a cellulose dialyzer tubing. As shown in Fig. 2, the substance passed through the membrane . Absorptton spectrum
The ultraviolet absorption spectra were obtained with a Beckmann DU spectrophotometer. The readings were taken at intervals of 2 mW in the range of 210 to 300 m~. As shown in Table 1, the spoctral data of fraction A at various pH values were practically identical with those of authentic guanosine. These data are also in good agreement with those described by VOLgIN and Cotflv [3] and Bocg et al. [4]. TwatB. 1.
Cb1lPAkmON oF "r~ aractnwt, nwTw ~tweBN
sonsple
auw
mewcnoa w wrro wvt~rrtc
pH
a max (mp)
zsol26o
2eo126o
290/260
Fraction A
1 6 10 "5
256 252 258-266
0"95 1 "14 0"87
0"66 0"68 0"61
0"49 0"31 0" 16
Guaaosine (authentic)
1 6 10 "5
256 252 260-266
0"93 1 " 13 0"86
0"67 0 "65 0"63
0"48 0"28 0"13
Testsfor the presence ofsugar andphosphate
The positive Molisch's and orcinol tests [5] indicate the presence of pentose in the molecule. For detection of phosphate, the method of Ficke and Subbarrow modified by Loien~a et al. [6] was followed. No phosphate was detected in fraction A ar in the venom even at an amount as large as 100 mg. Paper-chromatography
The unknown substance was applied along with adenosine and gtumosine respectively on strips of Whatman No. 1 paper. The chromatogram was developed under n-butanolisobutyric acid -25 per cent NH,OH-H,O (75 :37"5 :2 "5:25 v/v) system by ascending method fôr 3 "5 hr at 25°C . After drying in open air, spots were located by the ultraviolet lamp . The unknown substance had the same Rf value (0 "27) as that of guanosine, whereas that of adenosine was quite different (0 " 54). Quantitative determfnatlon
A mixture of guanosine and the venom was subjected to zone-electrophoresis : The guanosine concentration of eluate of fraction A from the oo-zone electrophoresis was compared to that from the electrophoresis with venom of same weight alone. The guanosine oontent(X) in the venom was calc~ilated from the following equation X-}-G = Cl X
Cs
G is the weight (~.g) of guanosine added to the venom on zanaelectrophoresis, Cl is the
4
A-LI WEI aad C. Y. LEE
concentration of the eluate of fraction A from the co-zone eloctrophoresis, and C, is that from the electrophoresis of the venom alone. As shown in Table 2, the guanosine content in the venom was found to be 1 "1 f 0 " 14 per cent. T~ . 2. Co-zorn; a.acrxorxox~ts oF Bungarus v
wiTx oueNOSUVE
Amount of Concentration guanosine Guanosine Concentration of guanosine found in the content in Amount of of guanosine from zone elect charged venom the venom Amount of guanosine from co-zone trophoresis of Exp. No . . venom charged added electrophoresis thevenomalone X= ~~,~
W
1 2 3 Mean f s.e .
mg 28 "0 33 "4 28 "4
wg
SOD 600 700
!~g/~ 101 110 124
wg/ml 43 3S 39
wg 371 280 321
% 1 "32 0"84 1 "13 1 "1f0"14~
Toxicity in mice
Fraction A was injected subcutaneously into the backs of mice weighing 15-20 g. The concentration was adjusted so that the required dose of guanosine was contained in 0 " 1 ml saline per 10 g body weight of mice . It was found to be practically non-toxic up to 11 l~g per g body weight (equivalent to 1 mg venom per g). E ~ect on the bloodpressure of the cat
A cat weighing 2 kg was anesthetized with chloralose (80 mg/kg) intravenously. A tracheal cannula was inserted. Blood pressure was recorded by a mercury manometer connected to the right carotid artery . Injections were given through a cannula into the femoral vein. Guanosine isolated from the venom was injected at the dose of 0 " 1 mg/kg body weight, and authentic guanosine was also injected in doses up to 1 mg/kg. Neither caused any changes in the blood pressure of the cat, however. C~,ARICe et al. [7] have also reported that guanosine has no vasodepressor activity in cats at a dose of 2 mg/kg. Acknnwkd8emaus-Wewish to thank Prof. T. B. Lo, Department of Chemistryand Prof. J. C. Su, Department of Agricultural C~anistry, National Taiwan University for their invahutble advice. We are moot grateful to Miss T. J. Ct~+u for measurement of the ultraviolet absorption spectra. REFERENCES [1] Cx~rx~, C. C. and LEe, C. Y., Isolation of neurotoxins from the venom of Bwgarus multlclnctas and their modes of neuromuscular blocking action . Arch . Int. Pharnraaodyn.,144, 241, 1%3. [2] F~, F. G. and Döx~., H., Adenosin m Gift derPuffotter Bltls arletans. Z. pltyslol . Chem . (HoppeSeyler), 296, 232, 1954 . [3] Vor xnv, E. and CoAN, W. E., Estimation of nucleic acids. Meths . Blochem. Anal. (D . Gucx, ed .), 1, 287, 1954 . [4] Hocx, R. M., Lnva, N. S., M.uertl., S. A. and LnYroN, S. H., Ultraviolet absorption spectra of adenosine-5'-triphosphate and related 5'-ribonucleotides . Arch . Blochem. Blophys., 6?, 253, 1956 . [Sj BxtYc~c~e, J., Estimation of monosaccharides by the orcinol-sulphuric acid reaction . Btcehem . J., 60, 200, 1955. [6] Lonnao, H. S., Boxes, H. W., Levy, L. W., HAMMPJ . L, M . L ., Preparation and properties of the isomeric cytidylic acids derived from yeast ribonucleic acid . J. Biol. Chem. 196, 807, 1952. [7] Cuxx~, D. A., Dnvou,, J., PHII.~s, F. S. and BxowN, G. B., Enzymatic deamination and vasodepressor effects of adenosine analogs. J. Pharmarnl. Exp . 7liergp., 106, 291, 1952 .
A NUCLEOSIDE ISOLATED FROM THE VENOM OF BUNGARUS MULTICINCTUS* A-Li Wsr and C. Y. L~ Pharmacological Institute, College of Medicine, National Taiwan University, Taipei,Taiwaa, China (Acceptedforpublication 11 March 196
Abetrod-During isolation ofneurotoxins from the venom of Brargarua ~nultlctnctua by means ofzone electrophoresis on starch, a non-proteinfraction was found in additionto the 4protein fractions obtained previously. It shawl maximum absorbance at 2S2 mEt, positive Molisch's testfor sugar and orcinol test for pwtoae, but negative Loring's teat for phosphate. It shows tl~ soma Rf value as guaaosine on thepaper~hromatogram, and its spectr8l data at various pH vahres am also identical with those of guanosine. Itscontent in the venom was mated to be 1 "1 f 0"14 per cwt. So far no biological effects could be attributed to the presence of guanosine in the venom. DuR1Na isolation of Bungarotoxins from the venom of Btasgarus enalticfnctus by means of zone electrophoresis on starch, a non-protein substance was obtained along with the 4 protein fractions described previously by CxANa and L~ [1]. It was detected by measuring the absorbance of each fraction at 278 m~. It showed maximum absorbance at 252 mw, positive Molisch's test for sugar and orcinol test for pentose, but negative Loring's test for phosphate. Upon dialysis, it passed through the cellulose membrane. From the results ofthe above tests it was suspected to be a nucleoside. When developed under n-butanol-isobutyric acid-NH,OH-H,O system, its Rp value was just the same as that of guanosine. Moreover, its spectral data at various pH values were found to be practically identical with those of guanosine. Thus, the nucleoside isolated from the venom was identified as guanosine. The guanosine content in the venom was estimated by co-zone electrophoresis, in which a certain amount of guanosine was added to the venom. By comparing the concentration of the eluate from the co-zone electrophoresis with that from the zone electrophoresis of the venom alone, it was estimated to be 1 "1 ~ 0 "14 per cent of the venom. FISCfmt and Döx~L [2] reported that adenosine was found in the venoms of Bias arietans and Dendraspis viridis and that the blood pressure lowering effect of the venoms might be partly due to the presence of adenosine. So far as our tests show the guanosine fraction, as well as pure guanosine, had no appreciable effect on the blood pressure of an anesthetized cat, even at a dose as large as 1 mg/kg of body weight . Thus, no biological effect could be attributed to the presence of guanosine in this venom. *This investigation was supported by the U.S. Arngy Medical Research aad Development Command, Department ofthe Armar, under Research Cirant No. DA-MD-49-193-GA-01108.
2
A-LI WEI aad C. Y. LEE EXPERIMENTAL
Venom
The venom of Bungarus multicinctus used in this study was freshly collected in this laboratory and stored in dried state in a vacuum desiocator . Starch-zone electrophoresis
The same electrophoresic conditions as those described by CHANO and hea [I] were followed . A typical electrophoresic pattern obtained by measuring the absorbance at 278 m~. is illustrated in Fig. 1. The venom migrated towards the cathode and separated into 5 fractions, the first of which disappeared when tested with Folin reagent. For the sake of convenience, the first fraction was designated as fraction A.
l. ZONE ELeCfAOPxOnFCia ON STARCH OF Tää VENOM OF HIJNOAalJ3 MULTICINCTUS. Potato starch moistenedwith sa acetate buffer, pH S'0, ionic strength 0"OS, plus NaCI, ionicstrength0'OS, was packed into a semicylindrical glass trough . Tweaty ßve mg of ande venom ores applied at the arrow 1S cm apart from the anode end. Electrophoresis was performed in a r~rigerator at 0~°C. An average potential difference of 170 V between tl~ two ends of the trough was applied for 30 hr. Fraction Ais Folin-negative whereasfractions I-IV areFolio-positive .
FIQ .
2. C~rANOEB OF CONCENTRATION OF FAAC'ITON A DUAIIVO DIALYSL9. Ten ml of the ehrate from fraction A was dialyaod for 3"S hr against S00ml distilled orator, at 29°C . FIa .
A Nucleoside isolated ~ Sorbe Veaoa
3
Dialysis Ten millilitres of the eluate from fraction Awere dialyzed at room temperature(29°C) for 3 "5 hr against 500 ml distilled water in a cellulose dialyzer tubing. As shown in Fig. 2, the substance passed through the membrane . Absorptton spectrum
The ultraviolet absorption spectra were obtained with a Beckmann DU spectrophotometer. The readings were taken at intervals of 2 mW in the range of 210 to 300 m~. As shown in Table 1, the spoctral data of fraction A at various pH values were practically identical with those of authentic guanosine. These data are also in good agreement with those described by VOLgIN and Cotflv [3] and Bocg et al. [4]. TwatB. 1.
Cb1lPAkmON oF "r~ aractnwt, nwTw ~tweBN
sonsple
auw
mewcnoa w wrro wvt~rrtc
pH
a max (mp)
zsol26o
2eo126o
290/260
Fraction A
1 6 10 "5
256 252 258-266
0"95 1 "14 0"87
0"66 0"68 0"61
0"49 0"31 0" 16
Guaaosine (authentic)
1 6 10 "5
256 252 260-266
0"93 1 " 13 0"86
0"67 0 "65 0"63
0"48 0"28 0"13
Testsfor the presence ofsugar andphosphate
The positive Molisch's and orcinol tests [5] indicate the presence of pentose in the molecule. For detection of phosphate, the method of Ficke and Subbarrow modified by Loien~a et al. [6] was followed. No phosphate was detected in fraction A ar in the venom even at an amount as large as 100 mg. Paper-chromatography
The unknown substance was applied along with adenosine and gtumosine respectively on strips of Whatman No. 1 paper. The chromatogram was developed under n-butanolisobutyric acid -25 per cent NH,OH-H,O (75 :37"5 :2 "5:25 v/v) system by ascending method fôr 3 "5 hr at 25°C . After drying in open air, spots were located by the ultraviolet lamp . The unknown substance had the same Rf value (0 "27) as that of guanosine, whereas that of adenosine was quite different (0 " 54). Quantitative determfnatlon
A mixture of guanosine and the venom was subjected to zone-electrophoresis : The guanosine concentration of eluate of fraction A from the oo-zone electrophoresis was compared to that from the electrophoresis with venom of same weight alone. The guanosine oontent(X) in the venom was calc~ilated from the following equation X-}-G = Cl X
Cs
G is the weight (~.g) of guanosine added to the venom on zanaelectrophoresis, Cl is the
4
A-LI WEI aad C. Y. LEE
concentration of the eluate of fraction A from the co-zone eloctrophoresis, and C, is that from the electrophoresis of the venom alone. As shown in Table 2, the guanosine content in the venom was found to be 1 "1 f 0 " 14 per cent. T~ . 2. Co-zorn; a.acrxorxox~ts oF Bungarus v
wiTx oueNOSUVE
Amount of Concentration guanosine Guanosine Concentration of guanosine found in the content in Amount of of guanosine from zone elect charged venom the venom Amount of guanosine from co-zone trophoresis of Exp. No . . venom charged added electrophoresis thevenomalone X= ~~,~
W
1 2 3 Mean f s.e .
mg 28 "0 33 "4 28 "4
wg
SOD 600 700
!~g/~ 101 110 124
wg/ml 43 3S 39
wg 371 280 321
% 1 "32 0"84 1 "13 1 "1f0"14~
Toxicity in mice
Fraction A was injected subcutaneously into the backs of mice weighing 15-20 g. The concentration was adjusted so that the required dose of guanosine was contained in 0 " 1 ml saline per 10 g body weight of mice . It was found to be practically non-toxic up to 11 l~g per g body weight (equivalent to 1 mg venom per g). E ~ect on the bloodpressure of the cat
A cat weighing 2 kg was anesthetized with chloralose (80 mg/kg) intravenously. A tracheal cannula was inserted. Blood pressure was recorded by a mercury manometer connected to the right carotid artery . Injections were given through a cannula into the femoral vein. Guanosine isolated from the venom was injected at the dose of 0 " 1 mg/kg body weight, and authentic guanosine was also injected in doses up to 1 mg/kg. Neither caused any changes in the blood pressure of the cat, however. C~,ARICe et al. [7] have also reported that guanosine has no vasodepressor activity in cats at a dose of 2 mg/kg. Acknnwkd8emaus-Wewish to thank Prof. T. B. Lo, Department of Chemistryand Prof. J. C. Su, Department of Agricultural C~anistry, National Taiwan University for their invahutble advice. We are moot grateful to Miss T. J. Ct~+u for measurement of the ultraviolet absorption spectra. REFERENCES [1] Cx~rx~, C. C. and LEe, C. Y., Isolation of neurotoxins from the venom of Bwgarus multlclnctas and their modes of neuromuscular blocking action . Arch . Int. Pharnraaodyn.,144, 241, 1%3. [2] F~, F. G. and Döx~., H., Adenosin m Gift derPuffotter Bltls arletans. Z. pltyslol . Chem . (HoppeSeyler), 296, 232, 1954 . [3] Vor xnv, E. and CoAN, W. E., Estimation of nucleic acids. Meths . Blochem. Anal. (D . Gucx, ed .), 1, 287, 1954 . [4] Hocx, R. M., Lnva, N. S., M.uertl., S. A. and LnYroN, S. H., Ultraviolet absorption spectra of adenosine-5'-triphosphate and related 5'-ribonucleotides . Arch . Blochem. Blophys., 6?, 253, 1956 . [Sj BxtYc~c~e, J., Estimation of monosaccharides by the orcinol-sulphuric acid reaction . Btcehem . J., 60, 200, 1955. [6] Lonnao, H. S., Boxes, H. W., Levy, L. W., HAMMPJ . L, M . L ., Preparation and properties of the isomeric cytidylic acids derived from yeast ribonucleic acid . J. Biol. Chem. 196, 807, 1952. [7] Cuxx~, D. A., Dnvou,, J., PHII.~s, F. S. and BxowN, G. B., Enzymatic deamination and vasodepressor effects of adenosine analogs. J. Pharmarnl. Exp . 7liergp., 106, 291, 1952 .