Effects of heating on the immunogenicity and biological toxicity of Deinagkistrodon acutus venom and its fractions

Toxicon 56 (2010) 45e54

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Effects of heating on the immunogenicity and biological toxicity of Deinagkistrodon acutus venom and its fractions Ya Tang, Weihua Dong, Tianhan Kong* Department of Pathophysiology, Guangzhou Medical College, 195 Dongfeng West Road, Guangzhou, Guangdong Province 510182, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 August 2009 Received in revised form 14 November 2009 Accepted 28 January 2010 Available online 21 March 2010

To improve toxoid preparation, the effects of selective heat denaturation were assessed on Deinagkistrodon acutus venom. The venom and its fractions (peak 1 and peak 2 separated by gel filtration chromatography) were heated to various temperatures (45e70
C) for 30 min, after which protein concentration, immunoreactivity, lethality, myotoxicity and hemorrhagic and membrane lysis activities of the samples were determined. In addition, the synergistic effects of the venom fractions were evaluated by separate or simultaneous intramuscular injection in mice. The results showed that the peak 1 fraction consisted primarily of proteins in the range of 18 to 105 kDa, while the peak 2 fraction consisted primarily of proteins smaller than 21 kDa. The hemorrhagic activity, immunoreactivity, and protein concentration of heated samples were gradually reduced as the temperature increased from 25
C to 70
C. Bioactivities significantly decreased but immunoreactivity was retained when the crude venom, peak 1 fraction, or peak 2 fraction were heated to the critical temperatures of 60
C, 55
C, or 60
C, respectively. Synergistic effects of two kinds of heated fractions were observed in toxicity and antibody production after the peak 1 and peak 2 injected simultaneously or respectively. The results suggest that venom fractions heated and injected separately could significantly reduce their toxicity and enhance the neutralization of antiserum induced by them. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Deinagkistrodon acutus Snake venom Fraction Heat denaturation Immunogenicity Biological toxicity

1. Introduction Deinagkistrodon acutus (formerly Agkistrodon acutus) is a monotypic pit viper that primarily inhabits northern Vietnam, southern China (including Anhui, Zhejiang, Jiangxi, Fujian, Taiwan, Hunan, Hubei, Guizhou, Sichuan, northern Guangdong, and Guangxi), and possibly Laos. The head of D. acutus, also known as hundred-pace pit viper or hundred-pace snake, has a pronounced soft “horn” on the snout; therefore, this species is sometimes referred as a “snorkel viper”. The average venom yield from an adult (mean length, 80e100 cm) is estimated at 59e176.1 mg (dried) per bite; the median lethal dose (LD50) of crude venom injected intracutaneously into mice is 8.9 mg/kg

* Corresponding author. Tel.: þ86 20 8134 0337; fax: þ86 20 8134 0211. E-mail address: [email protected] (T. Kong). 0041-0101/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2010.01.027

(zhao, 1990). The venom possesses numerous biological activities; it is neurotoxic, cardiotoxic, myotoxic, hemotoxic, hemorrhagic, and possibly directly nephrotoxic or hepatotoxic. After being bitten, the victim rapidly develops severe systemic symptoms. The symptoms include myonecrosis, palpitation, dyspnea, hemoptysis, hematuria, hematochezia, and disseminated intravascular coagulation (Zhao and Rao, 1982; Li et al., 2000; Chen et al., 2005). Serum therapy is the primary treatment for systemic envenomation by D. acutus. Three types of D. acutus antivenom are produced and available in China: liquid antivenom from Shanghai Institute of Biological Products, lyophilized antivenom from Cheng-Du Military Area Center of Disease Prevention and Control, and lyophilized antivenom from the National Institute of Preventive Medicine, Taipei, Taiwan. Antivenom is isolated from the plasma of adult horses immunized with formaldehyde-detoxified D. acutus venom. Although formaldehyde or glutaraldehyde-treated venom

46

Y. Tang et al. / Toxicon 56 (2010) 45e54

can produce high-titer antiserum (Tan, 1983; Sadahiro et al., 1984; Chakrabarty et al., 1991), repeated immunization of the formaldehyde- or glutaraldehyde-containing solution inevitably harms the horse. Venom detoxification methods are needed that produce antivenom with higher immunogenicity and lower toxicity. Several improved techniques for venom detoxification have been reported, including titration of native venom with iodine monochloride solution (Bicalho et al., 1990), encapsulation of native crotoxin in liposomes (Freitas and Frezard, 1997), irradiation of toxic proteins with gammarays (Clissa et al., 1999; Bennacef-Heffar and LarabaDjebari, 2003), and selective heat denaturation (Saetang et al., 1998; Rangel-Santos and Mota, 2000). Because the toxic fractions of D. acutus venom are distributed primarily in the two molecular weight ranges 14e18 kDa and 25e28 kDa (Huang et al., 2006), a denaturation temperature that retains maximum immunogenicity is required. Heat denaturation is the simplest detoxification technique and it has the ability to reduce venom toxicity without altering the immunogenicity of components with molecular weights lower than 25 kDa (Saetang et al., 1998; Rangel-Santos and Mota, 2000). The aim of this study was to assess the effects of various temperatures on the precipitation, immunogenicity, biological toxicity, and interactions of different toxic components from D. acutus venom. 2. Materials and methods 2.1. Materials The D. acutus snake (90 cm; approximately 30 months old) was purchased from the Chashanfu snake farm (Guangzhou, China). The snake venom was extracted and pooled, and the venom was diluted with four volumes of distilled water. The precipitates of crude venom were removed by centrifugation at 15,000  g for 30 min at 4
C (Sigma-3K30; Laboratory Centrifuges, Germany). The crude venom was freeze-dried with a lyophilizer (Christ ALPHA 1-4 LSC, Germany), and stored in a tightly closed bottle at 4
C until use. The Sephadex G-50 chromatography column was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). 3,30 ,5,50 -Tetramethyl benzidine, Acrylamide, NN’emethylenebisacrylamide, Coomassie brilliant blue, and other reagents for gel electrophoresis were obtained from SigmaeAldrich (St Louis, MO, USA). Molecular weight marker was purchased from New England Biolabs (No.: P7708V; Massachusetts, USA) and the Bio-Rad DC Protein Assay Kit was purchased from Bio-Rad Laboratories (Cat No.: 500-0111; California, USA). Horse anti-D. acutus IgG was purchased from the Kunming Institute of Military Medicine of Centers for Disease Control (Chengdu Military Region, China). HRP-conjugated rabbit anti-horse IgG was from Otwo Biotech Inc. (Guangzhou, China). HRPconjugated goat anti-guinea pigs IgG was from Beijing Boisynthesis Biotechnology Co., Ltd. (Beijing, China). Buffers and other chemicals were all analytic reagent grade. Kunming mice weighing 18e22 g were purchased from the laboratory animal center at Sun Yat-sen University, Guangzhou City, Guangdong Province, China (SPF grade,

certification No. 2004-0011). Hartley guinea pigs weighing 320e370 g were obtained from the laboratory animal center of Guangdong Province, China (certification no. 2008-0002). 2.2. Separation of fractions D. acutus venom powder was dissolved in distilled water (400 mg/20 ml) and centrifuged at 15,000  g for 30 min. The supernatant was loaded onto a 5.5 cm  50 cm Sephadex G-50 column, and eluted by distilled water with a flow rate of 2.0 ml/min at room temperature (25
C). The eluted fractions were monitored at 280 nm, and collected at 5 min/tube. The target fractions (peak 1 and peak 2) were pooled, lyophilized, and stored in a tightly closed bottle at 4
C until use. 2.3. Heat treatment of venom and its fractions The crude venom and its fractions (0.02e4 mg/ml) were dissolved in carbonic acid buffer (pH 9.6, for ELISA) or in Ringer’s solution, and aliquots were heated at 45
C, 50
C, 55
C, 60
C, 65
C, or 70
C in a water bath for 30 min. After centrifugation, the samples were kept at 20
C until use. Samples left at room temperature (25
C) for 30 min were designated as non-heated samples. 2.4. Protein quantification of heated and non-heated samples Protein concentration of heated and non-heated samples was determined by the bicinchoninic acid (BCA) assay (Smith et al., 1985) using the DC protein assay kit. Degraded percentage (DP%) was defined as protein concentration (BCA method) determined in the heated samples (higher than 25
C) divided by the protein concentration of non-heated samples (25
C). 2.5. SDS-PAGE The non-heated and heated venom (or its fractions) were analyzed by non-reducing SDS-PAGE with 12% resolving gel and 5% stacking gel. Samples containing 10 mg (peak 1 and peak 2) or 20 mg (crude venom) protein were dissolved in 50 mM TriseHCl, pH 6.8 (containing 10% glycerol, 2% SDS, 0.1% bromophenol blue), boiled for 5 min, and loaded onto the gel. Electrophoresis was carried out at 90 V for approximately 20 min and then 110 V for 90 min. The gel was fixed for 1 h with 10% acetic acid, stained with 0.25% Commassie brilliant blue R-250, and then destained with 10% glacial acetic acid. A prestained protein marker (7e175 kDa) was used as the molecular mass marker. The molecular weights of non-heated and heated samples were calculated using a standard curve of the relationship between the log molecular weight and the electrophoretic mobility. The bands changes of heated sample was analyzed by ImageQuant TL v2005 1D Gel Analysis software (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). 2.6. Immunoreactivity of heated and non-heated samples Indirect ELISA was performed to determine sample immunoreactivity to antitoxin IgG. ELISA wells were coated

Y. Tang et al. / Toxicon 56 (2010) 45e54

with heated and non-heated venom or fractions (100 ml per well of solution containing 2 mg protein in 0.1 M carbonate buffer, pH 9.6) at 4
C overnight, then blocked by 3% bovine serum albumin (BSA), followed by the addition of horse anti-D. acutus IgG (0.01 mg/ml, diluted with antibody diluent: 0.15 M PBS containing 0.05% Tween-20 and 1% BSA). Antitoxin IgG reactivity was detected with HRPconjugated rabbit anti-horse IgG (1:3000 dilution) and substrate solution (3,30 ,5,50 -tetramethyl benzidine in 0.1 M citric acid, 0.2 M phosphate salt, 0.75% H2O2, pH 5.0). After 25 min, the reaction was stopped by adding 2 M H2SO4. Absorbance was read in a microtiter plate reader (Tecan Sunrise Absorbance Reader; Tecan Austria GmbH, Austria) at 450 nm with Magellan 3.0 software (Tecan, Austria). Absorbance values of samples were at least 2.1 times higher than control values. Absorbance percentage (AP%) of sample was defined as the absorbance of heated samples (higher than 25
C) divided by the absorbance of nonheated samples. The AP% value was used to represent the extent of the immunoreactivity. 2.7. Biological toxicity of heated and non-heated samples 2.7.1. Lethality The lethality of the heated and non-heated whole venom or its fractions was determined by intraperitoneal injection into Kunming mice (18e22 g, equal numbers of each sex). There were 10 mice in each group for each dose level (six doses/0.1 ml Ringer’s solution/10 g body weight, grouping interval in 1.25). Survival was determined after 72 h, and the LD50 was calculated according to the method of Bliss (Bliss, 1935). 2.7.2. Hemorrhagic activity The hemorrhagic activity of the heated and non-heated whole venom or its fractions was tested by the skin injection method of Kondo et al. (1960) with some modifications according to Ovadia (1987). Five different doses of each sample (non-heated venom, 0.01e15 mg/0.1 ml; heated venom, 5e400 mg/0.1 ml) diluted with Ringer’s solution were intracutaneously injected into the back skin on both sides of the vertebral column of three white mice. Animals

47

were sacrificed by ether inhalation 24 h after injection, and a red hemorrhagic spot was observed on the visceral side of the removed skin. The lowest dose of heated or non-heated sample causing a hemorrhagic spot of approximately 1 cm in diameter was defined as the minimal hemorrhagic dose (MHD). 2.7.3. Vitelline membrane lysis activity The membrane lysis activity of the venom was determined by a new method and apparatus (Patent pending; application number: 200710026832) (Fig. 1). A hen egg yolk was separated from the albumen and rolled on filter paper to remove adhering albumen. The yolk was then carefully placed into a standard 50 mm plastic dish with the ovule located in the center of the upper surface (Fig. 1A). A hole (diameter, 7 mm) was made in the center of bottom surface and a portion of the egg yolk exuded down through to form a protrusion. A filter paper disc (diameter, 5 mm), impregnated with heated or non-heated sample (1 mg/ml, dissolved in Ringer’s solution), was placed on the apex of the protrusion. As soon as the apex surface touched the filter paper disc, the yolk flow out time (YFOT) was recorded. The YFOT was defined as the time required for yolk to flow out from the apex surface (part of the vitelline membrane) through the ruptured membrane (Fig. 1B). The YFOT was used to evaluate the membrane lysis activity of the venom or its fractions. The shorter the YFOT was, the more potent the membrane lysis activity was. 2.7.4. Myotoxic activity Muscle injury in mice induced by heated and nonheated venom or its fractions was evaluated by serum creatine kinase (CK) activity (Nakada et al., 1984; Calil-Elias et al., 2002). Heated or non-heated samples (2 LD50/0.1 ml Ringer’s solution/20 g body weight) were administered intramuscularly (i.m.) to the gastrocnemius muscle of the right hind leg. Blood samples were collected 90 min after injection. Serum was separated by centrifugation (825  g at 4
C for 10 min) and stored at 20
C for subsequent determination of CK activity. CK activity was determined using the enzyme coupling method based on creatine formation through the ADP/phosphocreatine reaction

Fig. 1. An illustration of the instrument for determination of vitelline membrane lysis activity. (A) The hen egg yolk was placed into a plastic dish with a position of ovule located in the center of upper surface and a protrusion formed in the center of bottom surface. (B) The yolk flows out through the ruptured vitelline membrane.

48

Y. Tang et al. / Toxicon 56 (2010) 45e54

using a diagnostic kit (Lot no. 080901; Shanghai FudanZhangjiang Bio-Pharmaceutical Co., Ltd., China) and complete automatic biochemistry analyzer (ECHO LCD, ISE Company, Italy). CK value was expressed in international units per liter (U/L), where 1 IU is the amount that catalyzes the transformation of 1 mmol of substrate (NADPH) per min. Basal serum CK activity was determined from mice that received an injection of Ringer’s solution only. 2.8. Synergistic toxic effect of peak 1 and peak 2 2.8.1. Mean survival time Mean survival time (time between injection and death) was used to determine the synergistic toxicity of fractions (Bougis et al., 1987). Two experimental protocols were used. In protocol A (peak 1/peak 2 group), the peak 1 fraction (1 LD50/0.1 ml Ringer’s solution/20 g body weight) was injected i.m. into the gastrocnemius muscle of the right hind leg, and the peak 2 fraction (1 LD50/0.1 ml) was injected into the gastrocnemius muscle of the left leg. In protocol B (peak 1 þ peak 2 group), the peak 1 fraction was mixed with the peak 2 fraction in equal lethal doses (1 LD50/0.1 ml), and the same mixture was injected i.m. into the gastrocnemius muscle of both hind legs (0.1 ml per side). After injection, each animal’s survival time was recorded immediately after respiration ceased. 2.8.2. Myotoxic activity Experimental protocols (injection sites and doses of peak 1 and peak 2 fractions) were same as above (see Section 2.8.1). Blood samples were collected 90 min after injection of fractions, and the serum CK activities were detected using the same procedure as above (see discussion in Section 2.7.4).

2.9. Polyclonal antibodies to D. acutus venom Hartley guinea pigs, male, weighing 320e370 g, were divided into three groups (n ¼ 5). The antigens in a dose of 1/16e1/8LD50 (Table 3) were dissolved in Ringer’s solution and emulsified with an equal volume of Freund’s adjuvant (FA) to a final volume of 0.45e0.5 ml. Animals of group 1 were immunized with the mixture of non-heated crude venom and FA, group 2 with the mixture of two kinds of heated fractions and FA (peak 1 þ peak 2 þ FA), and group 3 with two kinds of mixtures (peak 1 þ FA , peak 2 þ FA) which were injected respectively in different sites. Each animal was injected i.m. for the first immunization. The second and third booster immunization were injected s.c. on days 7 and 14 after first immunization. Serum antibody titers were evaluated at 7 days post third immunization by ELISA (see Section 2.6). ELISA wells were coated with nonheated crude venom. Antiserum IgG reactivity was detected with HRP-conjugated goat anti-guinea pig IgG (1:3000 dilution). 2.10. Protective ability of the antiserum Neutralization potency of the antiserum was determined in vivo using mice. Hartley guinea pigs serum at doses ranging from 12.5 to 200 ml was mixed with 112.8 mg of crude venom dissolved in 50 ml of Ringer’s solution. The volume was made up to 250 ml with Ringer’s solution, and allowed to incubate at 37
C for 30 min after which they were injected i.p. into mice (five groups, each of 5 mice, 250 ml/20 g mouse). Control mice were injected with the same dose of venom in Ringer’s solution. The numbers of surviving animals were recorded 24 h after injection and the antisera neutralizing capacity was determined by

Fig. 2. Fractions of D. acutus venom separated by Gel filtration and analyzed by SDS-PAGE. D. acutus venom dissolved in distilled water (400 mg/20 ml) were loaded on to a 5.5 cm  50 cm Sephadex G-50 column, and eluted out by distilled water with a flow rate of 2.0 ml/min. Two different fractions were named as peak 1 and peak 2, respectively. Insert: SDS-PAGE analysis of D. acutus venom and its fractions, with 12% resolving gel and 5% stacking gel. Lane: (1) peak 1; (2) peak 2 and (3) crude venom. Numbers (kDa) on the left margin correspond to the position of molecular weight markers. According to their relative mobilities, the molecular weight of proteins in peak 2 was mainly lower than 21 kDa (:).

Y. Tang et al. / Toxicon 56 (2010) 45e54

probit analysis with median effective expressed as ml serum/mg venom.

dose

(ED50)

2.11. Statistical analysis Results were presented as mean  standard deviation. The significance of difference between two independent samples was evaluated by Student’s t-test. In all analyses, the level of statistical significance chosen was P < 0.05. 3. Results

49

and named peak 1 and peak 2 (Fig. 2). Venom exhibited at least 13 distinct bands on non-reducing SDS-PAGE, ranging from 12 to 105 kDa (Fig. 2). Comparing the proteins of peak 1 with peak 2, the higher molecular weight bands (>21 kDa) were located only in peak 1, while the lower molecular weight bands (<21 kDa) were mainly located in peak 2 (Fig. 2). The relationship between the log of molecular mass markers and their electrophoretic mobilities demonstrated good linearity.

3.2. Effects of heat treatment on the hemorrhagic activity and immunoreactivity of venom and its fractions

3.1. Fractions of D. acutus The proteins in the crude venom were distributed primarily in two ranges : 14e18 kDa and 23e40 kDa. Thus gel filtration chromatography was chosen for the initial separation of the crude venom. The venom of D. acutus was separated into two fractions with a Sephadex G-50 column

The hemorrhagic activity (measured as MHD) and immunoreactivity (AP%) of venom and its fractions were gradually reduced as temperature increased from 25
C to 70
C (Fig. 3). A critical temperature phenomenon was apparent in heat denaturation; above the critical temperature, the hemorrhagic activity and AP% of the heated

Fig. 3. Effect of heat treatment on the hemorrhagic activities (MHD) and immunoreactivities (AP%) of venom and its fractions. MHD: minimal hemorrhagic dose, the lowest dose of sample causing hemorrhagic spot of approximately 1 cm in diameter; AP%: absorbance of samples heated at various temperature divided by the absorbance of sample non-heated. The value of AP% was used to measure immunoreactivities. Data are reported as means  standard deviation. *P < 0.05, **P < 0.01, compared with the previous (lower) temperature spot. (A) MHD and AP% of heated venom, (B) MHD and AP% of heated peak 1, and (C) MHD and AP% of heated peak 2.

50

Y. Tang et al. / Toxicon 56 (2010) 45e54

Table 1 Effects of heating on the total protein changes of samples. Sample

Heating (
C)

Venom Venom Venom Venom Peak 1 Peak 1 Peak 1 Peak 2 Peak 2 Peak 2

25 50 55 60 25 50 55 25 50 60

observed for larger molecule weight proteins (>21 kDa) than for smaller proteins (<21 kDa; Fig. 4).

BCA assay (DP%)a 1 mg/ml

2 mg/ml

100.0 96.5 87.7 85.9 100.0 98.5 90.8 100.0 99.3 80.2

100.0 94.8 80.2 74.2 100.0 100.5 89.1 100.0 103.3 82.2

a DP%, degraded percentage, the protein concentration (BCA assay) determined in the heated samples (higher than 25
C) divided by the protein concentration of non-heated samples (25
C).

samples were significantly reduced (P < 0.05). The critical temperatures for hemorrhagic activity were 60
C, 55
C, or 60
C for crude venom, peak 1 fraction, or peak 2 fraction respectively, and the critical temperatures for AP% were 65
C, 60
C, or 65
C, respectively (5
C higher than hemorrhagic activity critical temperatures). Because the critical temperature for hemorrhagic activity was chosen for preparation of toxoid, the immunoreactivity of sample was retained. Therefore, the optimizing heating set points of crude venom, peak 1 fraction, and peak 2 fraction were approximately 60
C (Fig. 3A), 55
C (Fig. 3B), and 60
C (Fig. 3C).

3.4. Effects of heat treatment on the bioactivities of venom and its fractions Compared with non-heated venom and its fractions, the biological toxicity (lethal, hemorrhagic, vitelline membrane lysis, and myotoxic activities) of venom and its fractions heated to the critical temperature for 30 min was significantly decreased (Table 2). The LD50 of the heated peak 1 and peak 2 fractions were decreased by 7.1- and 6.0-fold, hemorrhagic activities by 8.5- and 7.8-fold, vitelline membrane lysis activities (YFOT) by 4.9- and 4.4-fold, and myotoxic activities (CK) by 2.2- and 1.9-fold, respectively. Temperature and bioactivities of venom and its fractions were negatively correlated (see Table 2). 3.5. Synergistic toxic effect of fractions The synergistic toxic effect of fractions (peak 1 and peak 2) was determined by comparing the serum CK and mean survival time between the two treatment groups (peak 1/ peak 2 vs peak 1 þ peak 2). After injection, mean survival time of the peak 1 þ peak 2 group was shorter than the peak 1/peak 2 group. Similarly, higher myotoxic activity (higher serum CK value) was observed in peak 1 þ peak 2 group (Fig. 5). 3.6. Production of neutralizing antibodies

3.3. Effects of heat treatment on the protein quantity of venom and its fractions The protein quantity of venom and its fractions was progressively reduced as heat increased from 25
C to 60
C. The proportion of degraded protein (DP%) determined by the BCA method (Table 1). Greater changes in band density and mobility at 50
C, 55
C, and 60
C were

The antibodies elicited by heated fractions and nonheated crude venom were able to recognize the native venom in ELISA (Table 3), but the antiserum of crude venom had higher antibody titers than other two groups, and the peak 1/peak 2 group have a higher titer than peak 1 þ peak 2 group. However, the antiserum from peak 1/ peak 2 group was the most effective in neutralizing the

Fig. 4. The heated and non-heated venom or fractions analyzed by non-reducing SDS-PAGE. The proteins were stained with coomassie brilliant blue. (1) nonheated venom (25
C); (2) venom heated at 50
C; (3) venom heated at 55
C; (4) venom heated at 60
C; (5) non-heated peak 1 (25
C); (6) peak 1 heated at 50
C; (7) peak 1 heated at 55
C; (8) non-heated peak 2 (25
C); (9) peak 2 heated at 50
C and (10) peak 2 heated at 60
C. Numbers on the left correspond to the position of molecular weight markers.

35.79 (30.93e41.40) 88.18  19.65** 290.70  179.62** 2313.44  811.90* 25.62 (22.96e30.59) 115.77  28.86* 395.00  204.26* 2303.77  304.19*

5.96 (5.34e6.66) 11.26  1.13 65.90  62.88 4491.16  326.85

4. Discussion

*P < 0.05, **P < 0.01, compared with the non-heated venom (25
C). a Figures in parentheses are 95% confidence limits. b Minimal hemorrhagic dose. c Yolk flow out time. d Creatine kinase.

32.10 (30.09e34.24) 130.33  36.25** 534.8  286.06** 2174.50  496.19** 12.69 (10.24e15.73) 45.57  5.59** 191.00  102.16** 3024.17  1245.61* 2.82 (2.34e3.4)a 10.74  1.46 77.78  44.51 4162.09  849.89 LD50(mg/kg) MHDb (ug), N ¼ 30 YFOTc (s), N ¼ 10 CKd (U/L), N ¼ 10

51

lethality induced by the crude venom at the tested dose (112.8 mg/20 g mouse).

3.59 (2.76e4.88) 13.49  1.70 80.30  40.44 5160.85  460.09

Peak 2 Peak 1

25
C 60
C 55
C

Venom

25
C

Table 2 Effects of heat treatment on the bioactivities of venom and its fractions at critical temperature.

55
C

25
C

60
C

Y. Tang et al. / Toxicon 56 (2010) 45e54

The venom of D. acutus contains a mixture of enzymes and polypeptides produced by the venom glands. Huang et al. (2006) reported that 128 protein spots of the D. acutus venom proteome were detected on 2-D gel by silver staining. Of these proteins, 73 proteins were acidic (pI < 6), 35 were neutral (pI ¼ 6e8), and 20 were alkaline (pI > 8). Among the detected proteins, the 24 most abundant proteins (12.1e105.2 kDa) were distributed primarily in two molecular weight ranges: 14e18 kDa and 25e28 kDa. In the past 30 years, more than thirty components of D. acutus venom have been isolated and characterized for their pharmacological and biochemical properties, such as L-amino acid oxidase (Zhang and Wu, 2008), thrombin-like enzyme (Liu et al., 1999), anticoagulation factors (Xu et al., 2000), fibrinogenase (Wang et al., 2004), hyaluronidase (Xu et al., 1982), C-type lectin (Li et al., 2005), metalloproteinases (Mori et al., 1984; Wang, 2007), phospholipase A2 [PLA2] (Chen et al., 2004), disintegrins (Tsai et al., 2000). In this study, the venom of D. acutus was separated into two fractions by gel filtration chromatography. The results from non-reducing SDS-PAGE showed that peak 1 consisted primarily of middle and high molecular weight proteins ranging from 18 to 105 kDa, while peak 2 consisted primarily of proteins smaller than 21 kDa. According to the purified proteins of D. acutus venom reported before, peak 1 may include more than 20 hemorrhagic toxins, several metalloproteinases, and thrombin-like enzymes; peak 2 appears to include mainly myotoxic and neurotoxic proteins and several types of secretory PLA2. To improve antivenom production and extend the life of the immunized animals, several methods have been developed to decrease snake venom toxicity. Low-dose gamma radiation (1e10 kGy) from a 60Co source has been demonstrated to be effective. After irradiation with the optimal dose of 2-kGy gamma rays, the toxicities of irradiated venom were 2.7-fold (Clissa et al., 1999) or 9-fold (Bennacef-Heffar and Laraba-Djebari, 2003) less than the native venom, while its immunogenicity was almost intact. However, compared with gamma radiation, heating venom provides a simpler and less expensive technology for toxoid preparation. Protein thermostability is markedly different among different types of venoms or venom fractions. Ownby et al. (1994) reported that only five of 28 snake venoms (seven Agkistrodon, six Bothrops, 13 Crotalus, one Sistrurus, and one Bitis) retained proteolytic activity when heated to 100
C for 5 min; however, most of the venoms retained significant hemorrhagic activity. The hemorrhagic activity of one fraction isolated from Atractaspis venom by gel filtration was destroyed when incubated at 56
C for 20 min (Ovadia, 1987). Three main hemorrhagins (BaH l, BH2, and BH3) isolated from Bothrops asper venom were completely deactivated at 60
C for 30 min (Borkow et al., 1993). On the other hand, no significant differences in the immunogenicity were observed between non-heated and heated

52

Y. Tang et al. / Toxicon 56 (2010) 45e54

Fig. 5. Determination of the synergistic effect of peak 1 and peak 2. Peak 1/peak 2, two fractions injected i.m. respectively; peak 1 þ peak 2, two fractions injected i.m. together; *P < 0.05, compared with the group of peak 1/peak 2.

Crotalus durissus terrificus venom at 56
C, 70
C, or 100
C (Rangel-Santos and Mota, 2000). Our study showed that the toxicities, protein quantity, immunoreactivity and immunogenicity of D. acutus venom and its fractions (peak 1 and peak 2) were progressively lost following temperature elevation. The heat denaturation of larger molecule weight proteins in peak 1 was more apparent than the denaturation of proteins in peak 2. To generate a high-titer immunoglobulin against snake venom, retaining sufficient immunogenicity is the most important consideration in choosing the temperature for detoxification. According to our results, the optimal denaturation temperatures for crude venom, peak 1 fraction, and peak 2 fraction were 60
C, 55
C, and 60
C, respectively. Maintaining the optimal temperature for 30 min appears to significantly reduce toxicity (lethality, myotoxicity, hemorrhagic activity, and vitelline membrane lysis activity) of venom and its fractions without significantly altering immunoreactivity. Though the titer of antiserum induced by heated fractions (peak 1 þ peak 2, peak 1/peak 2) was lower than that induced by crude venom, the ED50 Value of peak 1/peak 2 group was much lower than other two groups. This means

the antiserum from peak 1/peak 2 group was the most effective in neutralizing the lethality induced by the crude venom. Avian egg yolk is particularly useful for monitoring PLA and lipase activity (Tan and Tan, 1988; Abousalham and Verger, 2000), detecting sperm and Salmonella penetration (Takeuchi et al., 2001; Murase et al., 2006), measuring lethal effects of non-neurotoxic venoms (Sells et al., 1998), and extracting specific anti-snake venom immunoglobulin in yolk (IgY) antibodies (De Almeida et al., 2008). The avian egg-envelope (i.e., the perivitelline layer) has been reported to be morphologically homologous to mammalian zona pellucida (Bakst and Howarth, 1977). The egg-envelope of the chicken is composed of several major components, including a 42-kDa glycoprotein (Takeuchi et al., 1999), 97-kDa glycoprotein (Takeuchi et al., 2001), and the novel ZP-glycoprotein (Okumura et al., 2004). When the isolated egg-envelope was incubated with sperm acrosome for 40 min, holes were produced in the egg-envelope, suggesting local glycoprotein degradation of the egg-envelope matrix with acrosomal proteases (Takeuchi et al., 2001). The acrosome is a cap-like structure derived from the Golgi Apparatus containing several hydrolase enzymes, including

Table 3 Comparing the neutralizing capacities and titers of guinea pig anti-D. acutus venom serum raised with crude venom or heated fractions. Group (n ¼ 5)

1 2 3 a b c d e f g

Antigen

Crude venom Peak 1 þ peak 2a Peak 1/peak 2b

Venom dose (mg/kg) First immunize

Second immunize

Third immunize

CFAc

IFAg

IFA

0.23d 1.03e þ 1.44f 1.03/1.44

0.23 1.03 þ 1.44 1.03/1.44

0.23 1.03 þ 1.44 1.03/1.44

ED50 (ml serum/mg crude venom)

Antibody titers (Log10)

1.71 (1.31e2.24) 0.88 (0.31e1.29) 0.45 (0.23e0.79)

5.99  0.24 5.32  0.12 5.76  0.15

Two kinds of heated fractions and freund’s adjuvant were mixed together. Two kinds of heated fractions were mixed with freund’s adjuvant respectively, then injected respectively in different positions. Complete freund’s adjuvant. 1/8LD50. 1/16LD50. 1/16LD50. Incomplete freund’s adjuvant.

Y. Tang et al. / Toxicon 56 (2010) 45e54

secretory PLA2 (Lessig et al., 2008), serine proteases (Yamagata et al., 1999), pancreatic trypsin (Ohmura et al., 1999), hyaluronidases (Kim et al., 2005), and matrix metalloproteinases (Buchman-Shaked et al., 2002). These enzymes (also called acrosin) are released during the sperm acrosome reaction to break down of the eggenvelope and allow penetration (Koyanagi et al., 1988). The egg-envelope of Xenopus could be dissolved by the hatching enzyme (60 kDa), a trypsin-type protease or a Zn2þ metalloprotease (Fan and Katagiri, 2001). The digestive enzymes in snake venom of D. acutus are abundant and besides the homologues of acrosomal enzymes, consist of enzymes such as PLA2s, serine proteases, hyaluronidases, and metalloproteinases. In the present study, the peak 2 fraction was found to exhibit a stronger vitelline membrane lysis activity than crude venom or the peak 1 fraction. It has been suggested that PLA2s found mainly in the peak 2 fraction may be critical for destruction of the plasma membrane. The synergistic effect of cobra PLA2 and cardiotoxins in promoting erythrocyte hemolysis (Louw and Visser, 1978), blocking nerve conduction (Chang et al., 1972), and decreasing median survival time (Bougis et al., 1987; Tan and Armugam, 1990) has been demonstrated. BaH1, BH2, and BH3, three metal-dependent hemorrhagins, have been isolated from the venom of B. asper. They contained more than 50% of the total hemorrhagic activity of the crude venom when combined, but lose almost half of their activity if BH2 and BH3 are separated from BaH1 (Borkow et al., 1993). This synergistic effect between the three fractions could explain the appearance of several hemorrhagins of snake venoms which appear to act on different substrates and enhance the hemorrhagic activity. In the present study, a significant decrease in median survival time (P < 0.05) and elevation in CK value (P < 0.05) were observed in the peak 1 þ peak 2 group (two fractions injected as a mixture) compared to the peak 1/peak 2 group (the two fractions injected separately) (Fig. 5). The results strongly suggested a synergism between the peak 1 and peak 2 fractions. Our experimental results also show that the titer and neutralization of antiserum induced by peak 1/ peak 2 group was higher than that induced by peak 1 þ peak 2 group. Further work should be carried out to determine the synergistic effect of the hemorrhagic toxin and the neurotoxin of D. acutus venom. Acknowledgements This work was supported by Natural Science Foundation of Guangdong Grant 20-4102537 and a grant from the Technology Bureau of Guangzhou City for Guangzhou City Biotoxin Key Laboratory No. 2005-6 to Tianhan Kong. We thank Huang Shao, Zhong Weigao and Cui Chaowei for their excellent technical assistances in the animal experiment and snake venom isolation. Conflict of interest statement The authors declare that there are no conflicts of interest.

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