Source of carbohydrate in a toxic protein from Indian cobra (Naja naja naja) venom

Tox7ron, Vol. 19, No . 3, pp. 431 436. 1981 . Printed in Greet Britain.

0011-0I01/81~030431-05 S02.OQ0 ® 1981 Pergnmon Prea Ltd.

SHORT COMMUNICATION SOURCE OF CARBOHYDRATE IN A TOXIC PROTEIN FROM INDIAN COBRA (NAJA NAJA NAJA) VENOM A. K. CHARLES*

and A .

P . JOSHI

C.S .LR. Centre for Biochemicals, V.P. Chest Institute Building, University of Delhi Campus, Delhi 110007, India (Accepted jar publication 16 December 1980) A. K. CHARLES end A. P. Josttt .

Sourtx of carbohydrate in a toxic protein from Indian cobra (Naja noja raja) venom. Toxicon 19, 431X36,1981 .-The principal neurotoxin of Indian cobra venom has been investigated to locate the source of carbohydrate present in it . The data indicate that the neurotoxin is not a glycoprotein, but contaminated with extraneous carbohydrate particles which mighthave originated from thecelluloseor dextran medium employed in thepurification procedures.

of snake venoms has been mainly attributed to the biologically potent polypeptide toxins (LEE,1971,1972) and enzymatic proteins (MELDRUM,1965). Some studies (BRAGANCA and PATEL, 1965 ; KABARA, 1970) have indicated that the carbohydrate moiety associated with Naja raja raja (Indian cobra) neurotoxins is essential for the neurotoxicity . This postulate was questioned by LIN and LEE (1971) using purified neurotoxins from the venoms of Naja raja afro (Formosan cobra) and Bungarus multicinctus (Formosan krait) . They demonstrated that these toxins were not glycoproteins, but suggested that the traces of carbohydrate in these toxins might be due to contamination with nucleosides. We have recently reported the biochemical properties of a N. raja raja neurotoxin (NT fraction) which produces neuromuscular block in the rat phrenic nerve diaphragm preparation (CHARLES el al., 1981) and respiratory arrest in cats and rabbits (CHARLES and DESHPANDE, 1981). In view ofthe above contradictions, we attempted systematically to analyze our toxin and present the analytical data on its carbohydrate moiety . The major neurotoxic (NT) fraction of N. raja raja venom was purified to homogeneity using the procedures described by CHARLES et al. (1981). Total carbohydrate (RoE,1955) and N-acetylneuraminic acid (WARREN, 1959) were determined according to the methods indicated. For the separation of neutral reducing sugars, NT protein (20 mg) was hydrolyzed with 1 N H2S0~ in sealed tubes for 8 hr at 100°C. The diluted hydrolyzate was chromatographed on columns (0~5 x 15 cm) of Dowex 50 X4 (H+ ) and Dowex 1 X8 (formate -), eluted with distilled water and evaporated to dryness as described by SPIRO and $PIRO (1965). The neutral sugar content in the residue was determined colorimetrically (SOMOCiYL, 1952) and by paper chromatography (descending) using nbutanol : ethanol : water (10 :1 : 2, v/v) system for 96 hr. The spots were detected with THE LETHALITY

' Present address: Department of Biochemistry, School of Dentistry, University of Maryland, Baltimore, Maryland 21201, U.S.A. To whom reprint requests should be addreaced. 431

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ammoniacal silver nitrate (TREVELYAN Ct a1., 1950). In order to identify aminosugars, NT protein (10 mg) was hydrolyzed with 2 N HCl under the conditions mentioned above. The hydrolyzate was concentrated and passed through Dower 50 X4 resin column (0~5 x 15 cm). The aminosugars were eluted with 2 N HCl and were determined by the Elson-Morgan reaction (ELSON and MORGAN, 1933). The glucose content of the residue was estimated after qualitatively converting the polymeric unit of the carbohydrate using amyloglucosidase (EC 3.2 .1 .3) (LEE and WHELAN, 1966). The resulting glucose was estimated by the glucose oxidase (EC 1.1 .3 .4) method (DnHLQvisT,1961) . The u.v. absorption spectrum (230-340 nm) of the toxic protein was measured in a Beckman DU spectrophotometer. Table 1 shows the carbohydrate content of the purified toxin and the molar ratios of glucose to toxin under different analytical conditions . The presence of reducing sugar indicates that the anthrone reacted carbohydrate moiety could possibly be a polysaccharide. Subsequently, paper chromatographic analysis of the hydrolyzate revealed the presence of a sugar that corresponded to the authentic n-glucose spot (Fig . 1) . Mannose, galactose, deoxyribose and fucose were not detected in neurotoxin hydrolyzate. The above findings necessitated further analysis of the protein to confirm if the detected sugar was glucose. The values obtained for glucose content by the glucose oxidase method and the amount of reducing sugar estimated by Somogyi's method were approximately the same, suggesting that the reducing sugar is indeed glucose. The difference in the total carbohydrate and glucose contents could be due to the loss occurring during the experimental procedures. The molar ratios of glucose to toxin were estimated to be far less than one. The calculation was based on the assumption that the neurotoxin is a glycoprotein and the carbohydrate is glucose (049/). The u.v . absorption spectrum showed no absorption maxima at 260 nm implying that the toxin is not contaminated with nucleosides or nucleotides. According to BRAGANCA end PATEL (1965) the carbohydrate moiety is an essential part of the active centers of neurotoxins. On the contrary, while examining the validity of this speculation, Lix and LEE (1971) have shown that neither cobra neurotoxin (N . raja atra) nor a- and ß-bungarotoxins (B . multicinctus) are glycoproteins. Their argument was based on the very low molar ratio ( < 1) values obtained for carbohydrate to neurotoxic protein. If the toxins were glycoproteins, the glucose to protein ratio wouldhave been equal to or more than one. The molar ratio (0 .172) for our neurotoxin (N. naja raja) was also less than one ;its value being similar to the one calculated for a-bungarotoxin (018). The above workers explained the presence of carbohydrate residues in their toxins as due to contamination with nucleosides . However, our findings do not support their explanation because our neurotoxin lacked an absorption maxima at 260nm ; an absorption maxima at this wavelength is a characteristic of nucleosides . Moreover, during the purification of the toxin used in this study, the elHuent from CM cellulose column was routinely monitored at 260 and 280 nm in order to see if any of the lethal fractions were contaminated with nucleosides (CHARLES et al., 1981). The elution profile showed no absorption maxima at 260nm for any of the lethal fractions, thus ruling out possible nucleoside wntamination . Both chromatographic and other specific assay methods revealed the presence of a reducing neutral sugar (glucose) possibly derived from a polymeric carbohydrate molecule . The problem then is to locate the source of glucose in the protein hydrolyzate. KARLSSON and ERKER (1972) offered a possible clue for the presence of extraneous carbohydrate in their toxins . They suggested that the source of carbohydrate could be either sephadex or cellulose columns. In view of our experimental results, we tend to support this speculation since the

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~a~ipin

6

s 7

FIG. 1. PAPER CHROMATOGRAM OF NEUROTOXIN HYDROLYZATE. The neutral sugar containing fraction was prepared according to the method of SPtRG and SPIRO (1965) . The toxin hydrolyzate and authentic standards (50 kg each) were chromatographed using the descending technique (n-butanol : ethanol : water, 10 : 1 :2). 1, neurotoxin hydrolyzate; 2, deoxyribose ; 3, ribose ; 4, galactose; 5, mannose ; 6, glucose and 7, fucose.

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TABLE I. CARBOHYDRATE CONTENT OF NAJA naja naja VENOM NEUROTOXIN Carbohydrate content % (mg/g protein) Total carbohydrate Neutral sugar Glucose (glucose oxidase) Amyloglucosidase treated Amyloglucosidase untreated N-Acetylneuraminic acid Aminosugars

Molar ratio

491 t 166 396 ± 013

049 040

0172 0140

387 f 010 066 t 003 Not detected Not detected

039 0-07

0137 0-024

The values are expressed as mean f S.E.M . of four dete.minations. Molarratio is calculated alter converting ~ carbohydrate contents into molar ratio of glucose to neurotoxic protein on the basis of their respective molecular weights. Glu = 180 ; NT = 6300 (CHARLES et AI,1981). Protein wasmeasured according to the method of LOWRY et al. (1951) .

glucose in the protein hydrolyzate could have originated from either of the above materials used in our purification procedures. In this context, a study ~OSHIMA end IWANAGA, 1969) on the carbohydrate composition of 15 snake venons, both Viperidae and Elapidae, deserves further attention. The only neutral sugars identified in all these venons were galactose, fucose and mannose. In none of the cases was glucose detected . This is in favor of the view that glucose is not a part of the toxin molecule and therefore, the speculation on the glycoprotein nature of cobra venom neurotoxins may not be valid. Acknowledgements-Part of this study was supported by the Council of Scientific and Industrial Research, New Delhi. A.K .C. was a recipient of C.S .L R. senior research fellowship.

REFERENCES BRAOANCA, B. M. and PATEL, N. T. (1%5) Glycoproteins as components of the lethal fractions in cobra venom (Naja naja). Can. J. Btochem. 1i, 915 . CEiARLES, A. K. and DE.4HPANDE, S. S. (1981) Peripheral versus centralaction ofa toxin from Indian cobra(Naja naja naja) venom. Toxicon 19, 305-317. CHARLES, A. K., GANOALS, S. V. and JosHt, A. P. (1981) Biochemical characterization of a toxin from Indian cobra (Naja naja naja) venom. Toxicon 19, 295-303. DAHLQVISI', A. (1%1) Determination of maltase and isomaltase activities with a glucose-oxidase reagent. Biocrem. J. 80, 547. ELSON, L. A. and MORGAN, W. T. J. (1933) A colorimetric method for the determination of glucosamine and chondrosamine. Biocrem. J. 27, 1824. KAHARA, J. J. (1970) Chemistry of Naja naja neurotoxin . Toxkon 8, 137. KARLSSON, E. SIId EAKER, D. (1972) Isolation of the principal neurotoxina of Naja naja subspecies from theAsian mainland. Toxicon 1Q 217. LEE, C. Y. (1971) Mode of action ofcobra venom and itspurified toxins. In : Newopoisons, Vol. l, p. 21(SIMrsoN, L. L., Ed.) . Plenum Press, New York. LEE, C. Y. (1972) Chemistry and pharmacology of polypeptide toxins in snake venons . A. Rev. Prarmac. 12, 265. LEE, E. Y. C. and WHELAN, W. J. (1966) Enzymatic methods for the mictodetermination of glycogen and amylopectin, and their unit chain length . Arcrs Biodran. Bioprys. 116, 162. LtN, S. S. and LEE, C. Y. (1971) Are neurotoxins from elapid venons glycoproteina7 Toxicon 9, 295. Lowev, O. H., ROSENBROUOH, N. J, FARR, A. L. and RANDALL, R. J. (1951) Protein measurement with the Folie Phenol Reagent. J. bfol. Crem. 193, 265. MELDRUM, B. S. (1%5) The actions of snake venons on nerve and muscle. The pharmacology of phoapholipase A and polypeptide toxine. Prarmac. Rev. 17, 393. OSHIMA, G. and IWANAGA, S. (1%9) Occurrence of glycoproteine in various snake venons Toxicon 7, 235. ROE, J. H. (1955) Thedetermination of sugar in bloodand spinal fluidwith enthrone reagent. J. biol. Chan. 212, 335. SOMOOYI, M. (1952) Notes on sugar determination . J. biol. Crem. 195, 19. SPIRO, R. G. and $PIRO, M. J. (1%5) The carbohydrate composition of the thyroglobulina from several species. J. biol. Chem. 210, 997.

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TREVELYAN, W. E. PROCCER, D . P . eIId HARRISON, J . S . (1950) DCtOCtiOn Oi BügaiB OII papCr CbIOInBtOgrBpby.

Noture, Lord . 166, 444.

WARREN, L . (1959) The thiobarbituric acid assay of sialic acid. J. 6iol. Chem. 234, 1971 .