Snake venom contains enzymes that hydrolyze carboxyl ester bonds. The substrates for hydrolysis are phospholipids, acetylcholine and aromatic acetate. These enzymes include three kinds: phospholipase, acetylcholinesterase and aromatic esterase. Arginine esterase in snake venom can also hydrolyze synthetic arginine or lysine, but it mainly hydrolyzes protein peptide bonds in nature, so it belongs to protease. The enzymes discussed here only act on ester substrates and cannot act on any peptide bond. Among these enzymes, the biological functions of acetylcholinesterase and phospholipase are more important and have been fully studied. Some snake venoms have strong aromatic esterase activity, which can hydrolyze p-nitrophenyl ethyl ester, a – or P-naphthalene acetate and indole ethyl ester. It is still unknown whether this activity is produced by an independent enzyme or a known side effect of carboxylesterase, let alone its biological significance. When the venom of Agkistrodon halys Japonicus was reacted with p-nitrophenyl ethyl ester and indole ethyl ester, the hydrolysates of p-nitrophenol and indole phenol were not found; On the contrary, if these esters react with cobra Zhoushan subspecies snake venom and Bungarus multicinctus snake venom, they will be quickly hydrolyzed. It is known that these cobra venoms have strong cholinesterase activity, which may be responsible for hydrolysis of the above substrates. In fact, Mclean et al. (1971) reported that many snake venoms belonging to the Cobra family can hydrolyze indole ethyl ester, naphthalene ethyl ester and butyl naphthalene ester. These snake venoms come from: cobra, black necked cobra, black lipped cobra, golden cobra, Egyptian cobra, king cobra, golden cobra mamba, black mamba and white lipped mamba (D. aw still knows eastern rhombola rattlesnake
The snake venom can hydrolyze methyl indole ethyl ester, which is the substrate for determining the cholinesterase activity in serum, but this snake venom does not show cholinesterase activity. This shows that there is an unknown esterase in cobra venom, which is different from cholinesterase. To understand the nature of this enzyme, further separation work is needed.
1、 Phospholipase A2
(I) Overview
Phospholipase is an enzyme that can hydrolyze glyceryl phosphate. There are 5 kinds of phospholipase in nature, namely phospholipase A2 and phospholipase
A. , phospholipase B, phospholipase C and phospholipase D. The snake venom mainly contains phospholipase A2 (PLA2), a few snake venoms contain phospholipase B, and other phospholipases are mainly found in animal tissues and bacteria. Fig. 3-11-4 shows the action site of these phospholipases on substrate hydrolysis.
Among phospholipases, PLA2 has been studied more. It may be the most studied enzyme in snake venom. Its substrate is the ester bond on the second position of Sn-3-glycerophosphate. This enzyme is widely found in snake venom, bee venom, scorpion venom and animal tissues, and PLA2 is abundant in four family snake venoms. Because this enzyme breaks red blood cells and causes hemolysis, it is also called “hemolysin”. Some people also call PLA2 hemolytic lecithinase.
Ludeeke first found that snake venom can produce a hemolytic compound by acting on lecithin through enzymes. Later, Delezenne et al. proved that when cobra venom acts on horse serum or yolk, it forms a hemolytic substance. It is now known that PLA2 can directly act on the phospholipids of the erythrocyte membrane, destroying the structure of the erythrocyte membrane and causing direct hemolysis; It can also act on serum or added lecithin to produce hemolytic lecithin, which acts on red blood cells to produce indirect hemolysis. Although PLA2 is abundant in the four families of snake venoms, the content of enzymes in various snake venoms is slightly different. Rattlesnake (C
Snake venom only showed weak PLA2 activity. Table 3-11-11 illustrates the comparison of PLA2 activity of 10 major venoms of venomous snakes in China.
Table 3-11-11 Comparison of phospholipase VIII activities of 10 snake venoms in China
Snake venom
Fat release
Aliphatic acid,
Cjumol/mg)
Hemolytic activity CHU50/^ g * ml)
Snake venom
Release fatty acids
(^raol/mg)
Hemolytic activity “(HU50/ftg * 1111)
Najanaja atra
9. 62
eleven
Micracephal ophis
five point one zero
kalyspallas
8. 68
two thousand and eight hundred
gracilis
V, acutus
7. 56
* * #
Ophiophagus hannah
three point eight two
one hundred and forty
Bnugarus fasctatus
7,56
two hundred and eighty
B. multicinctus
one point nine six
two hundred and eighty
Viper a russelli
seven point zero three
T, mucrosquamatus
one point eight five
Siamensis
T. stejnegeri
0. 97
（2） Separation and purification
The content of PLA2 in snake venom is large, and it is stable to heat, acid, alkali and denaturant, so that it is easy to purify and separate PLA2. The common method is to first carry out gel filtration on the crude venom, then carry out ion exchange chromatography, and the next step can be repeated. It should be noted that the freeze-drying of PLA2 after ion-exchange chromatography should not cause aggregation, because the freeze-drying process often increases the ionic strength in the system, which is an important factor causing aggregation of PLA2. In addition to the above general methods, the following methods have also been adopted: ① Wells et al. ② The substrate analogue of PLA2 was used as ligand for affinity chromatography. This ligand can bind to PLA2 in snake venom with Ca2+. EDTA is mostly used as eluent. After Ca2+is removed, the affinity between PLA2 and ligand decreases, and it can be dissociated from ligand. Others use 30% organic solution or 6mol/L urea as eluent. ③ Hydrophobic chromatography was performed with PheiiylSephar0SeCL-4B to remove trace PLA2 in cardiotoxin. ④ Anti PLA2 antibody was used as ligand to perform affinity chromatography on PLA2.
So far, a large number of snake venom PLAZ have been purified. Tu et al. (1977) listed PLA2 purified from snake venom before 1975. In recent years, a large number of articles about separation and purification of PLA2 have been reported every year. Here, we focus on the separation and purification of PLA by Chinese scholars.
Chen Yuancong et al. (1981) separated three PLA2 species from the venom of Agkistrodon halys Pallas in Zhejiang, which can be divided into acidic, neutral and alkaline PLA2 according to their isoelectric points. According to its toxicity, neutral PLA2 is more toxic, which has been identified as presynaptic neurotoxin Agkistrodotoxin. Alkaline PLA2 is less toxic, and acidic PLA2 has almost no toxicity. Wu Xiangfu et al. (1984) compared the characteristics of three PLA2s, including molecular weight, amino acid composition, N-terminal, isoelectric point, thermal stability, enzyme activity, toxicity and hemolytic activity. The results showed that they had similar molecular weight and thermal stability, but had significant differences in other aspects. In the aspect of enzyme activity, acid enzyme activity was higher than alkaline enzyme activity; The hemolytic effect of alkaline enzyme on rat red blood cells was the strongest, followed by neutral enzyme, and acid enzyme hardly hemolyzed. Therefore, it is speculated that the hemolytic effect of PLAZ is related to the charge of PLA2 molecule. Zhang Jingkang et al. (1981) have made Agkistrodotoxin crystals. Tu Guangliang et al. (1983) reported that a toxic PLA with an isoelectric point of 7. 6 was isolated and purified from the venom of Vipera rotundus from Fujian, and its physical and chemical properties, amino acid composition and the sequence of 22 amino acid residues at the N-terminal were determined. Li Yuesheng et al. (1985) isolated and purified another PLA2 from the venom of Viper rotundus in Fujian. The subunit of the PLA2 * is 13 800, the isoelectric point is 10.4, and the specific activity is 35/xnioI/miri mg。 With lecithin as the substrate, the optimal pH of the enzyme is 8.0 and the optimal temperature is 65 ° C. LD5 injected intravenously in mice. It is 0.5 ± 0.12mg/kg. This enzyme has obvious anticoagulant and hemolytic effects. The toxic PLA2 molecule consists of 123 residues of 18 kinds of amino acids. The molecule is rich in cysteine (14), aspartic acid (14) and glycine (12), but only contains one methionine, and its N-terminal is serine residue. Compared with PLA2 isolated by Tuguang, the molecular weight and the number of amino acid residues of the two isoenzymes are very similar, and the amino acid composition is also very similar, but the number of aspartic acid and proline residues is somewhat different. Guangxi king cobra snake venom contains rich PLA2. Shu Yuyan et al. (1989) isolated a PLA2 from the venom, which has a specific activity 3.6 times higher than the original venom, a molecular weight of 13000, a composition of 122 amino acid residues, an isoelectric point of 8.9, and good thermal stability. From the electron microscope observation of the effect of basic PLA2 on red blood cells, it can be seen that it has obvious effect on human red blood cell membrane, but has no obvious effect on goat red blood cells. This PLA2 has obvious retardation effect on the electrophoretic speed of red blood cells in humans, goats, rabbits and guinea pigs. Chen et al. This enzyme can inhibit platelet aggregation induced by ADP, collagen and sodium arachidonic acid. When PLA2 concentration is 10/xg/ml~lOOjug/ml, platelet aggregation is completely inhibited. If washed platelets were used as materials, PLA2 could not inhibit aggregation at the concentration of 20Mg/ml. Aspirin is an inhibitor of cyclooxygenase, which can inhibit the effect of PLA2 on platelets. PLA2 may inhibit platelet aggregation by hydrolyzing arachidonic acid to synthesize thromboxane A2. The solution conformation of PLA2 produced by Agkistrodon halys Pallas venom in Zhejiang Province was studied by means of circular dichroism, fluorescence and UV absorption. The experimental results showed that the main chain conformation of this enzyme was similar to that of the same kind of enzyme from other species and genera, the skeleton conformation had good heat resistance, and the structural change in acid environment was reversible. The combination of activator Ca2+and enzyme does not affect the environment of tryptophan residues, while the inhibitor Zn2+does the opposite. The way in which pH value of solution affects enzyme activity is different from the above reagents.
In the process of PLA2 purification of snake venom, an obvious phenomenon is that a snake venom contains two or more PLA2 elution peaks. This phenomenon can be explained as follows: ① due to the existence of isozymes; ② One kind of PLA2 is polymerized into a variety of PLA2 mixtures with various molecular weights, most of which are in the range of 9 000~40 000; ③ The combination of PLA2 and other snake venom components complicates PLA2; ④ Because the amide bond in PLA2 is hydrolyzed, the charge changes. ① And ② are common, with only a few exceptions, such as PLA2 in CrWa/w snake venom
There are two situations: ① and ②. The third condition has been found in PLA2 in the venom of the following snakes: Oxyranus scutellatus, Parademansia microlepidota, Bothrops a ^>er, Palestinian viper, sand viper, and terrible rattlesnake km。
The result of case ④ makes the migration speed of PLA2 change during electrophoresis, but the amino acid composition does not change. Peptides can be broken by hydrolysis, but generally they are still bound together by disulfide bonds. The venom of the eastern pit rattlesnake contains two forms of PLA2, called type a and type p PLA2 respectively. The difference between these two types of PLA2 is only one amino acid, that is, glutamine in one PLA2 molecule is replaced by glutamic acid in the other PLA2 molecule. Although the exact reason for this difference is not clear, it is generally believed that it is related to the deamination of PLA2. If PLA2 in the Palestinian viper venom is kept warm with the crude venom, the end groups in its enzyme molecules will become more than before. From C PLA2 isolated from snake venom has two different N-terminal, and its molecular weight is 30000. This phenomenon may be caused by the asymmetric dimer of PLA2, which is similar to the symmetric dimer formed by PLA2 in the venom of eastern diamondback rattlesnake and western diamondback rattlesnake. The Asian cobra is composed of many subspecies, some of which are not very definite in classification. For example, what used to be called the Cobra Outer Caspian subspecies is now recognized
It should be attributed to the Outer Caspian Sea Cobra. As there are many subspecies and they are mixed together, the composition of snake venom varies greatly due to different sources, and the content of PLA2 isozymes is also high. For example, cobra venom
At least 9 kinds of PLA2 isozymes of r ^ ll species were found in, and 7 kinds of PLA2 isozymes were found in the venom of cobra subspecies Caspian. Durkin et al. (1981) studied the PLA2 content and the number of isozymes in different snake venoms, including 18 cobra venoms, 3 mamba venoms, 5 viper venoms, 16 rattlesnake venoms and 3 sea snake venoms. In general, the PLA2 activity of cobra venom is high, with many isozymes. The PLA2 activity and isozymes of viper venom are medium. The PLA2 activity of mamba venom and rattlesnake venom is very low or no PLA2 activity. The PLA2 activity of sea snake venom is also low.
In recent years, it has not been reported that PLA2 in snake venom exists in the form of active dimer, such as the eastern rhombophora rattlesnake (C. snake venom contains type a and type P PLA2, both of which are composed of two identical subunits, and only dimerase has
Activity. Shen et al. also proposed that only the dimer of PLA2 of the snake venom is the active form of the enzyme. The study of spatial structure also proves that PLA2 of western diamondback rattlesnake exists in the form of dimer. Piscivorous compound
There are two different PLA ^ Ei and E2 of snake venom, in which 仏 exists in the form of dimer, the dimer is active, and its dissociated monomer is inactive. Lu Yinghua et al. (1980) further studied the physical and chemical properties and reaction kinetics of E. Jayanthi et al. (1989) isolated a basic PLA2 (VRVPL-V) from the viper venom. The molecular weight of the monomer PLA2 is 10000, which has lethal, anticoagulant and edema effects. The enzyme can polymerize polymers with different molecular weights under the condition of PH 4.8, and the degree of polymerization and molecular weight of polymers increase with the increase of temperature. The molecular weight of the polymer generated at 96 ° C is 53 100, and the PLA2 activity of this polymer increases by two
Post time: Nov-18-2022