Snake venom muaj enzymes uas hydrolyze carboxyl ester bonds.Cov substrates rau hydrolysis yog phospholipids, acetylcholine thiab aromatic acetate.Cov enzymes no suav nrog peb hom: phospholipase, acetylcholinesterase thiab aromatic esterase.Arginine esterase nyob rau hauv nab venom kuj tuaj yeem hydrolyze hluavtaws arginine los yog lysine, tab sis nws tsuas yog hydrolyzes protein peptide bonds nyob rau hauv cov xwm, yog li nws belongs rau protease.Cov enzymes tau tham ntawm no tsuas yog ua rau ester substrates thiab tsis tuaj yeem ua rau ib qho peptide daim ntawv cog lus.Ntawm cov enzymes no, kev ua haujlwm lom neeg ntawm acetylcholinesterase thiab phospholipase tseem ceeb dua thiab tau kawm tag nrho.Qee cov nab venoms muaj zog muaj zog esterase kev ua si, uas muaj peev xwm hydrolyze p-nitrophenyl ethyl ester, a – los yog P-naphthalene acetate thiab indole ethyl ester.Nws tseem tsis tau paub meej tias qhov kev ua haujlwm no yog tsim los ntawm kev ywj pheej enzyme lossis kev paub txog kev phiv ntawm carboxylesterase, cia nws nyob ib leeg nws qhov tseem ceeb ntawm kev lom neeg.Thaum cov venom ntawm Agkistrodon halys Japonicus tau hnov mob nrog p-nitrophenyl ethyl ester thiab indole ethyl ester, cov hydrolysates ntawm p-nitrophenol thiab indole phenol tsis pom;Ntawm qhov tsis sib xws, yog tias cov esters no hnov mob nrog cobra Zhoushan subspecies nab venom thiab Bungarus multicinctus nab venom, lawv yuav sai sai hydrolyzed.Nws yog lub npe hu hais tias cov cobra venoms muaj zog cholinesterase kev ua si, uas tej zaum yuav lub luag hauj lwm rau hydrolysis ntawm cov saum toj no substrates.Qhov tseeb, Mclean et al.(1971) tau tshaj tawm tias ntau tus nab venoms ntawm Cobra tsev neeg tuaj yeem hydrolyze indole ethyl ester, naphthalene ethyl ester thiab butyl naphthalene ester.Cov nab venoms no tuaj ntawm: cobra, dub necked cobra, dub lipped cobra, golden cobra, Egyptian cobra, huab tais cobra, golden cobra mamba, dub mamba thiab dawb lipped mamba (D. aw tseem paub sab hnub tuaj rhombola rattlesnake.
Tus nab venom tuaj yeem hydrolyze methyl indole ethyl ester, uas yog lub substrate rau kev txiav txim siab cholinesterase kev ua hauv cov ntshav, tab sis tus nab venom no tsis qhia txog kev ua cholinesterase.Qhov no qhia tau hais tias muaj ib qho tsis paub esterase hauv cobra venom, uas txawv ntawm cholinesterase.Txhawm rau kom nkag siab qhov xwm txheej ntawm cov enzyme no, kev ua haujlwm sib cais ntxiv yog xav tau.
1, Phospholipase A2
(I) Txheej txheem cej luam
Phospholipase yog ib qho enzyme uas tuaj yeem hydrolyze glyceryl phosphate.Muaj 5 hom phospholipase hauv qhov xwm txheej, uas yog phospholipase A2 thiab phospholipase
A. , phospholipase B, phospholipase C thiab phospholipase D. Cov nab venom mas muaj phospholipase A2 (PLA2), ob peb nab venoms muaj phospholipase B, thiab lwm yam phospholipases mas muaj nyob rau hauv cov tsiaj cov ntaub so ntswg thiab kab mob.Daim duab 3-11-4 qhia qhov chaw ua haujlwm ntawm cov phospholipases ntawm substrate hydrolysis.
Ntawm phospholipases, PLA2 tau kawm ntau dua.Tej zaum nws yuav yog qhov kev tshawb fawb tshaj plaws enzyme hauv nab venom.Nws cov substrate yog ester daim ntawv cog lus ntawm ob txoj hauj lwm ntawm Sn-3-glycerophosphate.Cov enzyme no muaj dav nyob rau hauv nab venom, muv venom, scorpion venom thiab tsiaj cov ntaub so ntswg, thiab PLA2 muaj ntau nyob rau hauv plaub tsev neeg nab venoms.Vim tias cov enzyme no ua rau cov qe ntshav liab thiab ua rau hemolysis, nws tseem hu ua "hemolysin".Qee tus neeg kuj hu ua PLA2 hemolytic lecithinase.
Ludeeke thawj zaug pom tias nab venom tuaj yeem tsim cov tshuaj hemolytic los ntawm kev ua ntawm lecithin los ntawm enzymes.Tom qab ntawd, Delezenne et al.ua pov thawj tias thaum cobra venom ua rau nees ntshav los yog yolk, nws tsim cov tshuaj hemolytic.Tam sim no paub tias PLA2 tuaj yeem ua ncaj qha rau ntawm phospholipids ntawm erythrocyte membrane, rhuav tshem cov qauv ntawm erythrocyte membrane thiab ua rau cov hemolysis ncaj qha;Nws tuaj yeem ua rau cov ntshav los yog ntxiv cov lecithin los tsim hemolytic lecithin, uas ua rau cov qe ntshav liab los tsim cov hemolysis tsis ncaj.Txawm hais tias PLA2 muaj ntau nyob rau hauv plaub tsev neeg ntawm nab venoms, cov ntsiab lus ntawm enzymes nyob rau hauv ntau yam nab venoms txawv me ntsis.Rattlesnake (C
Snake venom tsuas pom tsis muaj zog PLA2 kev ua haujlwm.Table 3-11-11 qhia txog kev sib piv ntawm PLA2 kev ua ntawm 10 tus kab mob loj ntawm cov nab venomous hauv Suav teb.
Table 3-11-11 Kev sib piv ntawm phospholipase VIII kev ua ub no ntawm 10 nab venoms hauv Suav teb
Nab venom
Rog tso
Aliphatic acid,
Cjumol / mg)
Hemolytic kev ua si CHU50 / ^ g * ml)
Nab venom
Tso cov fatty acids
(^raol/mg)
Hemolytic kev ua si "(HU50 / ftg * 1111)
Najanaja atra
9.62 ib
kaum ib
Micracephal ophis
tsib point ib xoom
kalyspallas
8.68 ib
ob txhiab yim puas
gracilis
V, ua
7.56 ib
* * #
Ophiophagus hannah
peb point yim ob
ib puas plaub caug
Bnugarus fasctatus
7,56 ib
ob puas yim caum
B. multicinctus
ib point cuaj rau
ob puas yim caum
Viper thiab russelli
xya point zero peb
T, mucrosquamatus
ib point yim tsib
Siamensis
T. stejnegeri
0.97 ib
(2) Kev sib cais thiab kev huv
Cov ntsiab lus ntawm PLA2 hauv nab venom yog qhov loj, thiab nws ruaj khov rau cua sov, kua qaub, alkali thiab denaturant, yog li nws yooj yim los ntxuav thiab cais PLA2.Txoj hauv kev zoo tshaj plaws yog ua ntej nqa gel pom ntawm cov venom crude, tom qab ntawd nqa tawm ion pauv chromatography, thiab cov kauj ruam tom ntej tuaj yeem rov ua dua.Nws yuav tsum tau muab sau tseg hais tias lub khov-qhuav ntawm PLA2 tom qab ion-exchange chromatography yuav tsum tsis txhob ua aggregation, vim hais tias lub freeze-drying txheej txheem feem ntau tsub kom lub ionic zog nyob rau hauv lub system, uas yog ib qho tseem ceeb ua rau aggregation ntawm PLA2.Ntxiv rau cov txheej txheem saum toj no, cov txheej txheem hauv qab no kuj tau txais: ① Wells et al.② Lub substrate analogue ntawm PLA2 tau siv los ua ligand rau affinity chromatography.Cov ligand no tuaj yeem khi rau PLA2 hauv nab venom nrog Ca2+.EDTA feem ntau yog siv los ua eluent.Tom qab Ca2 + raug tshem tawm, qhov kev sib raug zoo ntawm PLA2 thiab ligand txo qis, thiab nws tuaj yeem cuam tshuam los ntawm ligand.Lwm tus siv 30% organic tov los yog 6mol / L urea raws li eluent.③ Hydrophobic chromatography tau ua nrog PheiiylSephar0SeCL-4B kom tshem tawm kab PLA2 hauv cardiotoxin.④ Anti PLA2 antibody tau siv los ua ligand los ua affinity chromatography ntawm PLA2.
Txog tam sim no, ntau tus nab venom PLAZ tau ua kom huv.Tau et al.(1977) teev PLA2 purified los ntawm nab venom ua ntej 1975. Nyob rau hauv xyoo tas los no, ib tug loj tus naj npawb ntawm cov khoom hais txog kev sib cais thiab purification ntawm PLA2 tau raug tshaj tawm txhua xyoo.Ntawm no, peb tsom mus rau kev sib cais thiab kev huv ntawm PLA los ntawm cov kws tshawb fawb Suav.
Chen Yuancong et al.(1981) cais peb hom PLA2 los ntawm venom ntawm Agkistrodon halys Pallas hauv Zhejiang, uas tuaj yeem muab faib ua acidic, nruab nrab thiab alkaline PLA2 raws li lawv cov ntsiab lus isoelectric.Raws li nws cov tshuaj toxicity, nruab nrab PLA2 yog cov tshuaj lom ntau dua, uas tau pom tias yog presynaptic neurotoxin Agkistrodotoxin.Alkaline PLA2 tsis muaj tshuaj lom, thiab acidic PLA2 yuav luag tsis muaj tshuaj lom.Wu Xiangfu et al.(1984) piv cov yam ntxwv ntawm peb PLA2s, suav nrog molecular hnyav, amino acid muaj pes tsawg leeg, N-terminal, isoelectric point, thermal stability, enzyme kev ua, toxicity thiab hemolytic kev ua si.Cov txiaj ntsig tau pom tias lawv muaj qhov hnyav molecular zoo sib xws thiab thermal stability, tab sis muaj qhov sib txawv tseem ceeb hauv lwm yam.Nyob rau hauv nam ntawm enzyme kev ua si, acid enzyme kev ua si yog siab tshaj alkaline enzyme kev ua si;Cov nyhuv hemolytic ntawm alkaline enzyme ntawm cov qe ntshav liab yog qhov muaj zog tshaj plaws, ua raws li cov enzyme nruab nrab, thiab cov kua qaub enzyme tsis tshua muaj hemolyzed.Yog li, nws tau kwv yees tias cov nyhuv hemolytic ntawm PLAZ cuam tshuam nrog cov nqi ntawm PLA2 molecule.Zhang Jingkang et al.(1981) tau ua Agkistrodotoxin crystals.Tu Guangliang et al.(1983) tau tshaj tawm tias muaj tshuaj lom PLA nrog rau qhov isoelectric point ntawm 7. 6 raug cais tawm thiab ntxuav tawm los ntawm cov venom ntawm Vipera rotundus los ntawm Fujian, thiab nws lub cev thiab tshuaj lom neeg, amino acid muaj pes tsawg leeg thiab qhov sib lawv liag ntawm 22 amino acid residues ntawm N. -terminal raug txiav txim.Li Yuesheng et al.(1985) cais thiab ua kom huv dua lwm tus PLA2 los ntawm cov venom ntawm Viper rotundus hauv Fujian.Lub subunit ntawm PLA2 * yog 13 800, qhov taw tes isoelectric yog 10.4, thiab cov haujlwm tshwj xeeb yog 35 / xnioI / miri mg. Nrog lecithin ua lub substrate, qhov zoo tshaj pH ntawm cov enzyme yog 8.0 thiab qhov zoo tshaj plaws kub yog 65 ° C. LD5 txhaj tshuaj txhaj rau hauv cov nas.Nws yog 0.5 ± 0.12mg / kg.Cov enzyme no muaj qhov pom tseeb los tiv thaiv coagulant thiab hemolytic teebmeem.Cov tshuaj lom PLA2 molecule muaj 123 seem ntawm 18 hom amino acids.Lub molecule yog nplua nuj nyob rau hauv cysteine (14), aspartic acid (14) thiab glycine (12), tab sis tsuas muaj ib tug methionine, thiab nws N-terminal yog serine residue.Piv nrog PLA2 cais los ntawm Tuguang, qhov hnyav molecular thiab tus naj npawb ntawm cov amino acid residues ntawm ob isoenzymes zoo sib xws, thiab cov amino acid muaj pes tsawg leeg kuj zoo sib xws, tab sis tus naj npawb ntawm aspartic acid thiab proline residues txawv me ntsis.Guangxi huab tais cobra nab venom muaj nplua nuj PLA2.Shu Yuan et al.(1989) cais PLA2 los ntawm cov venom, uas muaj cov haujlwm tshwj xeeb 3.6 npaug siab dua qhov qub venom, qhov hnyav molecular ntawm 13000, muaj pes tsawg leeg ntawm 122 amino acid residues, isoelectric point ntawm 8.9, thiab zoo thermal stability.Los ntawm electron microscope soj ntsuam cov txiaj ntsig ntawm PLA2 theem pib ntawm cov qe ntshav liab, nws tuaj yeem pom tau tias nws muaj txiaj ntsig zoo rau tib neeg cov ntshav liab daim nyias nyias, tab sis tsis muaj kev cuam tshuam rau cov ntshav liab tshis.Qhov PLA2 no muaj kev cuam tshuam pom tseeb rau ntawm electrophoretic ceev ntawm cov qe ntshav liab hauv tib neeg, tshis, luav thiab guinea npua.Chen et al.Cov enzyme no tuaj yeem tiv thaiv platelet aggregation tshwm sim los ntawm ADP, collagen thiab sodium arachidonic acid.Thaum PLA2 concentration yog 10 / xg / ml ~ lOOjug / ml, platelet aggregation yog inhibited kiag li.Yog tias cov platelets ntxuav tau siv los ua cov khoom siv, PLA2 tsis tuaj yeem cuam tshuam kev sib sau ntawm qhov concentration ntawm 20Mg / ml.Aspirin yog ib qho inhibitor ntawm cyclooxygenase, uas tuaj yeem cuam tshuam cov nyhuv ntawm PLA2 ntawm platelets.PLA2 tuaj yeem inhibit platelet aggregation los ntawm hydrolyzing arachidonic acid los ua ke thromboxane A2.Kev daws teeb meem ntawm PLA2 tsim los ntawm Agkistrodon halys Pallas venom hauv Zhejiang Xeev tau kawm los ntawm kev ncig dichroism, fluorescence thiab UV nqus.Cov txiaj ntsig kev sim pom tau hais tias cov saw tseem ceeb ntawm cov enzyme no zoo ib yam li tib hom enzyme los ntawm lwm hom thiab cov noob, cov pob txha conformation muaj qhov kub zoo, thiab cov qauv hloov hauv cov kua qaub ib puag ncig yog thim rov qab.Kev sib xyaw ua ke ntawm activator Ca2 + thiab enzyme tsis cuam tshuam rau ib puag ncig ntawm tryptophan residues, thaum lub inhibitor Zn2 + ua qhov txawv.Txoj hauv kev uas tus nqi pH ntawm kev daws cuam tshuam rau kev ua enzyme yog txawv ntawm cov tshuaj reagents saum toj no.
Nyob rau hauv tus txheej txheem ntawm PLA2 purification ntawm nab venom, ib tug pom tseeb tshwm sim yog hais tias ib tug nab venom muaj ob los yog ntau tshaj PLA2 elution peaks.Qhov tshwm sim no tuaj yeem piav qhia raws li hauv qab no: ① vim muaj cov isozymes;② Ib hom PLA2 yog polymerized rau hauv ntau hom PLA2 sib xyaw nrog ntau qhov hnyav molecular, feem ntau yog nyob rau hauv thaj tsam ntawm 9 000 ~ 40 000;③ Kev sib xyaw ua ke ntawm PLA2 thiab lwm yam nab venom Cheebtsam cuam tshuam PLA2;④ Vim tias daim ntawv cog lus amide hauv PLA2 yog hydrolyzed, tus nqi hloov pauv.① Thiab ② muaj ntau, tsuas yog qee qhov kev zam, xws li PLA2 hauv CrWa/w nab venom
Muaj ob qhov xwm txheej: ① thiab ②.Qhov thib peb mob tau pom nyob rau hauv PLA2 nyob rau hauv lub venom ntawm cov nram qab no nab: Oxyranus scutellatus, Parademansia microlepidota, Bothrops a ^>er, Palestinian viper, xuab zeb viper, thiab txaus ntshai rattlesnake km.
Qhov tshwm sim ntawm rooj plaub ④ ua rau kev txav ceev ntawm PLA2 hloov pauv thaum lub sij hawm electrophoresis, tab sis cov amino acid muaj pes tsawg leeg tsis hloov.Peptides tuaj yeem tawg los ntawm hydrolysis, tab sis feem ntau lawv tseem ua ke los ntawm disulfide bonds.Cov venom ntawm lub qhov dej sab hnub tuaj muaj ob hom PLA2, hu ua hom a thiab hom p PLA2 feem.Qhov sib txawv ntawm ob hom PLA2 no tsuas yog ib qho amino acid, uas yog, glutamine nyob rau hauv ib PLA2 molecule yog hloov los ntawm glutamic acid nyob rau hauv lwm yam PLA2 molecule.Txawm hais tias qhov tseeb vim li cas rau qhov sib txawv no tsis meej, nws feem ntau ntseeg tias nws muaj feem xyuam nrog kev deamination ntawm PLA2.Yog tias PLA2 nyob rau hauv Palestinian viper venom khaws cia sov nrog cov venom crude, cov pab pawg kawg hauv nws cov enzyme molecules yuav dhau los ua ntej.Los ntawm C PLA2 cais los ntawm tus nab venom muaj ob qhov sib txawv N-terminal, thiab nws qhov hnyav molecular yog 30000. Qhov tshwm sim no yuav tshwm sim los ntawm asymmetric dimer ntawm PLA2, uas zoo ib yam li lub symmetric dimer tsim los ntawm PLA2 nyob rau hauv lub venom ntawm sab hnub tuaj pob zeb diamondback rattlesnake. thiab western diamondback rattlesnake.Asian cobra yog tsim los ntawm ntau yam subspecies, qee qhov tsis meej heev hauv kev faib tawm.Piv txwv li, dab tsi siv los hu ua Cobra Outer Caspian subspecies tam sim no tau lees paub
Nws yuav tsum raug ntaus nqi rau sab nrauv Caspian Hiav Txwv Cobra.Raws li muaj ntau yam subspecies thiab lawv tau sib xyaw ua ke, qhov sib xyaw ntawm nab venom txawv heev vim muaj ntau qhov chaw, thiab cov ntsiab lus ntawm PLA2 isozymes kuj siab.Piv txwv li, cobra venom
Yam tsawg kawg 9 hom PLA2 isozymes ntawm r^ll hom tau pom nyob rau hauv, thiab 7 hom PLA2 isozymes tau pom nyob rau hauv cov venom ntawm cobra subspecies Caspian.Durkin et al.(1981) tau kawm txog PLA2 cov ntsiab lus thiab pes tsawg tus isozymes nyob rau hauv txawv nab venoms, xws li 18 cobra venoms, 3 mamba venoms, 5 viper venoms, 16 rattlesnake venoms thiab 3 hiav txwv nab venoms.Feem ntau, PLA2 kev ua ntawm cobra venom yog siab, nrog ntau isozymes.PLA2 kev ua haujlwm thiab isozymes ntawm viper venom yog nruab nrab.PLA2 kev ua ntawm mamba venom thiab rattlesnake venom yog tsawg heev los yog tsis muaj kev ua PLA2.PLA2 kev ua ntawm hiav txwv nab venom kuj tsawg.
Nyob rau hauv xyoo tas los no, nws tsis tau tshaj tawm tias PLA2 nyob rau hauv nab venom muaj nyob rau hauv daim ntawv ntawm active dimer, xws li lub sab hnub tuaj rhombophora rattlesnake (C. nab venom muaj hom a thiab hom P PLA2, ob qho tib si yog tsim los ntawm ob lub subunits zoo tib yam. , thiab tsuas yog dimerase muaj
Kev ua si.Shen et al.kuj tau hais tias tsuas yog lub dimer ntawm PLA2 ntawm tus nab venom yog daim ntawv nquag ntawm cov enzyme.Txoj kev tshawb fawb ntawm spatial qauv kuj ua pov thawj tias PLA2 ntawm western diamondback rattlesnake muaj nyob rau hauv daim ntawv ntawm dimer.Piscivorous compound
Muaj ob qhov sib txawv PLA ^ Ei thiab E2 ntawm nab venom, nyob rau hauv uas 仏 muaj nyob rau hauv daim ntawv ntawm dimer, lub dimer yog active, thiab nws dissociated monomer yog inactive.Lu Yinghua et al.(1980) tau kawm ntxiv txog lub cev thiab tshuaj lom neeg thiab cov tshuaj tiv thaiv kinetics ntawm E. Jayanthi li al.(1989) cais ib qho yooj yim PLA2 (VRVPL-V) los ntawm viper venom.Qhov hnyav molecular ntawm monomer PLA2 yog 10000, uas muaj qhov ua rau tuag taus, tiv thaiv coagulant thiab edema.Lub enzyme tuaj yeem polymerize polymers nrog sib txawv molecular hnyav nyob rau hauv cov xwm txheej ntawm PH 4.8, thiab cov degree ntawm polymerization thiab molecular hnyav ntawm polymers nce nrog rau qhov kub thiab txias.Qhov hnyav molecular ntawm cov polymer tsim ntawm 96 ° C yog 53 100, thiab PLA2 kev ua ntawm cov polymer no nce los ntawm ob.
Lub sij hawm xa tuaj: Nov-18-2022