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A Study of Ribonuclease Activity in Venom of Vietnam Cobra Thiet Van Nguyen1* and A

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A Study of Ribonuclease Activity in Venom of Vietnam Cobra Thiet Van Nguyen1* and A Nguyen and Osipov Journal of Animal Science and Technology (2017) 59:20 DOI 10.1186/s40781-017-0145-5 RESEARCH Open Access A study of ribonuclease activity in venom of vietnam cobra Thiet Van Nguyen1* and A. V. Osipov2 Abstract Background: Ribonuclease (RNase) is one of the few toxic proteins that are present constantly in snake venoms of all types. However, to date this RNase is still poorly studied in comparison not only with other toxic proteins of snake venom, but also with the enzymes of RNase group. The objective of this paper was to investigate some properties of RNase from venom of Vietnam cobra Naja atra. Methods: Kinetic methods and gel filtration chromatography were used to investigate RNase from venom of Vietnam cobra. Results: RNase from venom of Vietnam cobra Naja atra has some characteristic properties. This RNase is a thermostable enzyme and has high conformational stability. This is the only acidic enzyme of the RNase A superfamily exhibiting a high catalytic activity in the pH range of 1–4, with pHopt = 2.58 ± 0.35. Its activity is considerably reduced with increasing ionic strength of reaction mixture. Venom proteins are separated by gel filtration into four peaks with ribonucleolytic activity, which is abnormally distributed among the isoforms: only a small part of the RNase activity is present in fractions of proteins with molecular weights of 12–15 kDa and more than 30 kDa, but most of the enzyme activity is detected in fractions of polypeptides, having molecular weights of less than 9 kDa, that is unexpected. Conclusions: RNase from the venom of Vietnam cobra is a unique member of RNase A superfamily according to its acidic optimum pH (pHopt = 2.58 ± 0.35) and extremely low molecular weights of its major isoforms (approximately 8.95 kDa for RNase III and 5.93 kDa for RNase IV). Keywords: Acidic optimum pH, Conformational stability, Gel filtration, Ionic strength, Isoforms, Thermostability, Venom RNase, Vietnam cobra Background anti-cancer effects and is used to develop anti-cancer Since ancient times snake venom has been consid- drugs [4–7]. ered as the precious medicinal source. Not by Snake venom is a biological fluid having the highest chance that the symbol of medicine in all over the RNase activity [8]. RNase is one of the toxic enzymes world is the image of a snake releasing venom. that are permanently present in snake venoms of all Today we know that snake venom is the source, very kinds [9, 10]. However, up to now snake venom RNase rich in biologically active substances, such as toxins, is little studied in comparison not only with the enzymes proteins, enzymes and toxic peptides. Snake venom of RNase group, but also with the other proteins of affects many organs and organ systems in the body snake venoms. Probably this is the reason that so far we such as the nervous system, blood and cardiovascu- still do not understand the role of this enzyme in the lar system, respiratory and muscular systems... Snake toxic action of the snake venoms in general. venom is used to treat various diseases, including Moreover, snake venom RNase is a secreted cardiovascular diseases [1], hypertension [2] and enzyme,sothisRNasebelongstotheRNaseAsuper- osteoarthritis [3]. The venom of some snakes showed family. At present has been known that the RNase A superfamily is a large family of secreted RNases in * Correspondence: [email protected] 1 vertebrates from amphibians to mammals, including Institute of Biotechnology, Vietnam Academy of Science and Technology Homo sapiens (VAST), Hanoi, Vietnam species withRNaseA(anenzymefrom Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Nguyen and Osipov Journal of Animal Science and Technology (2017) 59:20 Page 2 of 9 bovine pancreas) as a prototype [11, 12]. In the last Glycine buffer (G-buffer) at pH 1.5–4.0 and in 10 mM time the main function of the enzymes of RNase A mixed buffer (M-buffer) at pH 1.0 to 9.5, prepared in the superfamily is thought to be involved in the host equal molar ratio (1: 1: 1: 1) from glycine, citrate, defense system. Many members of this superfamily phosphate and tris-HCl. possess antimicrobial [13–19], antiviral [20–23], cytotoxic [24–29] and other biological actions [26, 30–32], as ob- served for the most of RNases of the RNase A superfamily Study the effect of ionic strength in human [33–35] and for RNases of this superfamily in The influence of ionic strength on RNase activity was other organisms [13, 19, 26, 36, 37]. From this context, we studied by performing RNA hydrolysis at different con- believe that the permanent presence of RNase in snake centrations of G- and M-buffers (from 5 to 100 mM), venoms of all kinds is not accidental; and the fact that NaCl and (NH4)2SO4 (from 0 to 100 mM), MgSO4 (from snake venom is a biological fluid having very high ribonu- 0 to 4 mM) and EDTA (from 0 to 10 mM). cleolytic activity may reflect a certain role of this enzyme in the toxic effects of snake venom. Treatment with trypsin In this work, the results of studying some properties Venom RNase in the enzyme preparation E0 was incubated of RNase from the venom of Vietnam cobra will be pre- у sented. Information about these properties may help to with trypsin (at a ratio tr psin/protein in preparation E0 understand the role of RNase in general toxic effects of equal to 1/13) at 25 °C, and then the RNase activity snake venom. remaining in the reaction mixture was determined at various time intervals. Methods Materials Gel-filtration chromatography Naja atra The dry (lyophilized) venom of cobra the 140 mg of the dried venom of Vietnam cobra Naja atra (purchased from Vinh Son Snake farming village, were separated by a gel-filtration column (ø2.5 × 90 cm) Vinh Tuong District, Vinh Phuc Province) was used with Sephadex G50sf as a carrier in 0.1 M ammonium as a source of RNase. Total RNA from the yeast Torula acetate buffer pH 6.2 at a flow rate of 15 ml/h, the (purchased from Sigma) was used as a sub- volume of collected fractions was 5 ml. In the case of strate for the determination of RNase activity. Glycine gel-filtration on a Superdex 75 column (ø0.9 × 60 cm), from Prolabo, sodium citrate, citric acid, sodium different amounts of cobra venom (10-50 mg) were used chloride, sodium hydroxide, hydrochloric acid and for fractionation, and the chromatographic process was other chemicals are analytically pure. carried out in 10 mM citrate buffer pH 5.25 or in 10 mM The initial enzyme solution (E0) was prepared by dis- – phosphate buffer pH 7.4 at a flow rate of 1 ml/min, the solving 2 10 mg or more of snake venom in the appropri- volume of collected fractions was 1 ml. After chromatog- ate buffer solution, and then centrifuged at 10000 rpm for raphy, the ribonucleolytic activity was determined in all 10 min to remove the insoluble material. fractions obtained as described below. Methods Heat treatment RNase activity assay To study the thermostability of the RNase activity in RNA hydrolysis reactions were carried out in 10 mM Vietnam cobra venom, several series of enzyme samples glycine buffer pH 2.6 at room temperature for 30 s in a with concentrations of 2–10 mg/ml of lyophilized venom total volume of 1 ml of the reaction mixture [8]. The were heating in a water bath at different temperatures hydrolysis reaction is initiated by adding x μl of the from 30 to 100 °C over in the same period of time enzyme solution in the (1000 - x) μl of RNA solution (5 min). After thermal treatment, the enzyme samples prepared in glycine buffer. RNase activity was evaluated were cooled on ice and centrifuged at 10.000 rpm for by the increase in OD260 of the reaction mixture during 10 min to remove the residue of inactivated proteins and the hydrolysis of the RNA substrate. One unit of RNase then RNase activity in the supernatant liquids was activity is defined as the amount of enzyme which determined, as described below. hydrolyzes RNA causing an increase in OD260 value of the reaction mixture by 1 unit. Determination of the optimum pH For studying the influence of pH on RNase activity and determining the optimum pH (pHopt) of the enzyme, Protein determination RNA hydrolysis reactions were carried out in 10 mM The protein content in the fractions of gel filtration was Phosphate buffer (P-buffer) at pH 5.8–8.0, in 10 mM automatically recorded at a wavelength of 280 nm. Nguyen and Osipov Journal of Animal Science and Technology (2017) 59:20 Page 3 of 9 Table 1 Results of determination of ribonucleolytic activity (A, OD260) remained in the supernatant liquids after centrifugation of the heat-treated enzyme samples ToC Ribonucleolytic activity remained after heat treatment Series 1 Series 2 Series 3 Series 4 Series 5 Series 6 OD260 %OD260 %OD260 %OD260 %OD260 %OD260 % 25 0.165 100.0 0.206 100.0 0.190 100.0 0.166 100.0 0.229 100.0 0.228 100.0 30 0.214 129.3 0.254 123.6 0.239 125.9 0.193 115.9 0.259 113.0 0.250 109.5 40 0.227 137.0 0.270 131.6 0.257 135.0 0.212 127.4 0.279 121.7 0.284 124.6 50 0.239 144.5 0.289 140.7 0.262 137.9 0.224 135.2 0.297 129.2 0.299 131.4 60 0.206 124.2 0.276 134.3 0.254 133.5 0.215 129.4 0.252 109.8 0.274 120.1 70 0.200 121.0 0.241 117.2 0.215 113.2 0.189 113.8 0.221 96.1 0.231 101.4 80 0.174 105.3 0.234 113.6 0.205 107.6 0.176 105.9 0.209 91.0 0.204 89.4 90 0.169 102.4 0.223 108.7 0.203 106.7 0.166 99.9 0.203 88.7 0.198 87.1 100 0.155 93.5 0.212 103.2 0.170 89.4 0.146 88.1 0.197 85.7 0.186 81.6 Results enzyme retained approximately 82–103% of its original Thermostability of the enzyme activity.
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