S.H.O.; THOMAZINI-SANTOS, I.A.; MENDES-GIANNINI, M.J.S.;. TOSCANO, E.; BARRAVIERA, B. A new fibrin sealant from Crotalus durissus terrificus venom: ...
PURIFICATION AND CHARACTERIZATION OF SERINE PROTEASES FROM BOTHROPS SNAKE VENOMS Heleno, M.A.G.1,2; Santos, L.D.1,2; Lourenço Jr., A.1; Barros, L.C.1; Ferreira Jr., R.S.1,2; Barraviera, B1,2. 1Center for the Study of Venoms and Venomous Animals (CEVAP) and 2Postgraduate Course in Tropical Diseases, Botucatu Medical School, São Paulo State University (UNESP), Botucatu, SP, Brazil
Introduction
Discussion
Results
Snake venoms are important sources of substances with a variety of pharmacological activities. Among the different proteins present in these venoms, snake venom serine proteases (SVSPs) have important effects on the hemostatic system that influence the hemodynamic properties of blood. Bothrops genus snakes presented their venom richly composed of SVSPs thrombin-like, and the isolation of these enzymes is of great interest. In 1994, the Center for the Study of Venoms and Venomous Animals (CEVAP) - UNESP standardized the fibrin sealant derived from snake venom, replacing the bovine thrombin by gyroxin thrombin-like enzyme (TLE) from Crotalus durissus terrificus (Rattlesnake) and human plasma fibrinogen by buffaloes cryoprecipitate. Despite chromatographic techniques for the purification of gyroxin be well grounded in the literature, that income is considered low. Thus, in addition to gyroxin, other thrombin-like enzymes could be employed in the composition of the new fibrin sealant after being standardized to the purifying and chromatographic performance and widely evaluated for biological activities. Therefore, it is extremely important that in our lab is deployed, standardized and validated a method for the chromatographic purification of other thrombin-like enzymes such as found in Bothrops snake venoms. Thus a two-step chromatographic procedure was developed to routinely purify serine proteases from five Bothrops snake venoms (Bothrops alternatus, B. jararaca, B. jararacussu, B. moojeni and B. pauloensis).
Purpose The aim of this work was to certify a methodology to isolate TLEs from five Bothrops snake venoms (Bothrops alternatus, B. jararaca, B. jararacussu, B. moojeni and B. pauloensis), evaluate income and identify them, searching new candidate molecules to the sealant composition.
Sample
Table 1. Bothrops snakes crude venom total mass applied to the liquid chromatography column, obtained mass of each venom fraction containing serine protease and the yield in the isolation process. Total venom mass
Protein dosage (mg) of
Yield (%) of serine protease-
applied (mg) in first step
serine protease-containing
containing fractions isolated after
liquid chromatography
fractions after first step
first step liquid chromatography
venom
liquid chromatography
B. alternatus
500
14,6
2,92
B. jararaca
500
44,8
8,96
B. jararacussu
500
132,8
26,56
B. moojeni
500
32
6,4
B. pauloensis
500
53,8
10,76
Conclusions
Table 2 - Data of peptides identified by mass spectrometry
The venoms were obtained from the "pool" of venoms extracted from adult Bothrops snakes (Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni and Bothrops pauloensis), of both sexes, individually microchipping, created and maintained in the CEVAP´s Serpentarium, located at Fazenda Experimental Lageado, UNESP, Botucatu.
Methods
Sample
Protein Name (snake databank)
Access Code
Taxonomy
BaIII-4
serine proteinase 3
gi|338855336
BaIII-5
GPV-PA
gi|380875424
BaIII-6
GPVPA
gi|380875424
BaIII-7
Protein C activator/ SVSP GPV-PA
gi|461511
The venoms were fractionated by liquid chromatography (process under patent) followed by reversed-phase liquid chromatography. The enzymes were characterized using SDS-PAGE and in-gel protein digestion coupled to LC-MS/MS and Mascot protein identification, and their coagulant activity was assessed. TLEs from B. moojeni and B. alternatus were isolated in two chromatographic steps with good protein yield, while those of B. jararaca, B. jararacussu and B. pauloensis required additional steps.
Results
A
Peptides *
560.7975
29719
4
39/41
553.276
29094
2
R.AAKPELPATSRTLCAGIL EGGK.G K.TLNEDEQTR.D
44/45
553.271
29094
2
K.TLNEDEQTR.D
27/27
500.2358
2247
3
-VGGDECNINEHR.S
49/38
553.2734
29094
2
K.TLNEDEQTR.D
114/49
605.8241
25883
2
U K.INILDHAVCR.A
Bothrops jararaca
88/49
605.8234
25883
3
K.INILDHAVCR.A
BmIII-4
gi|62464
106/47
708.8507
28854
3
K.HAGSVANYDEVVR.Y
BmIII-5
Batroxobin
gi|62464
Bothrops atrox Bothrops atrox
142/53
708.8471
28854
3
K.HAGSVANYDEVVR.Y
BjuIII-5
Chain B, Crotoxin B
gi|171848868
67/36
443.2122
14988
2
K.SGYITCGK.G
BjuIII-5
C-type lectin
gi|32396016
93/42
787.3726
15524
2
K.EFCVELVSNTGYR.L
BjuIII-6
gi|82466483
159/70
570.7832
28685
2
K.VSDYTEWIK.S
gi|82466483
Bothrops asper
160/61
603.8152
28685
2
R.IMGWGTISPTK.E
BjuIII-7
Thrombin-like enzyme Thrombin-like enzyme C-type lectin
Crotalus d. terrificus Bothrops jararacussu Bothrops asper
gi|32396016
232/57
787.3724
15524
2
K.EFCVELVSNTGYR.L
BjuIII-7
c-type lectin
gi|32396016
288/72
696.3325
15524
2
K.GQSEVWIGLCDK.K
BjuIII-8
c-type lectin
gi|32396016
191/65
696.3330
15524
2
K.GQSEVWIGLCDK.K
BjuIII-8
c-type lectin
gi|32396016
121/67
696.3366
15524
2
K.GQSEVWIGLCDK.K
BjIII-3
C- type lectin
gi|41353970
Bothrops jararacussu Bothrops jararacussu Bothrops jararacussu Bothrops jararacussu Bothrops insularis
67/52
525.5780
19137
3
K.EFCVELVSDTGYR.L
BjIII-3 BjIII-4
C- type lectin
gi|41353970
Bothrops insularis
36/36
525.5787
19137
3
K.EFCVELVSDTGYR.L
BjIII-4
serine proteinase 3
gi|338855336
22/23
560.8028
29719
4
BjIII-7
GPV-PA
gi|380875424
41/41
553.2719
29094
2
R.AAKPELPATSRTLCAGIL EGGK.G K.TLNEDEQTR.D
BjIII-7
GPV-PA
gi|380875424
58/59
553.2709
29094
2
K.TLNEDEQTR.D
BjIII-8
-
-
Crotalus adamanteus Trimeresurus albolabris Trimeresurus albolabris -
-
-
-
-
----------
BjIII-8
serine proteinase 3
gi|338855336
57/57
560.7957
29719
4
BjIII-9
-
-
Crotalus adamanteus -
-
-
-
-
R.AAKPELPATSRTLCAGIL EGGK.G ----------
BjIII-9
GPV-PA
gi|380875424
33/33
553.2595
29094
2
K.TLNEDEQTR.D
BjIII-10
GPV-PA
gi|380875424
37/37
553.2718
29094
2
K.TLNEDEQTR.D
BjIII-10
GPV-PA
gi|380875424
37/37
553.2718
29094
2
K.TLNEDEQTR.D
BpIII-8
serine proteinase precursor C-type lectin BpLec
gi|20069139
Trimeresurus albolabris Trimeresurus albolabris Trimeresurus albolabris Bothrops insularis
97/60
559.2789
29023
2
R.SVANDDEVIR.Y
85/51
811.8798
16726
2
K.EFCVELVSYTGYR.L
46/43
559.2787
29023
2
R.SVANDDEVIR.Y
gi|380875424
serine proteinase precursor serine proteinase precursor serine proteinase precursor c-type Lectin
gi|20069139
Bothrops pauloensis Bothrops insularis
gi|20069139
Bothrops insularis
25/25
471.7817
29023
3
R.IYLGIHAR.S
gi|20069139
Bothrops insularis
45/42
471.781
29023
3
R.IYLGIHAR.S
gi|32396016
162/60
811.8794
16726
2
K.EFCVELVSYTGYR.L
gi|151499825
85/52
796.8926
29815
2
R.TLCAGVLQGGIDTCK.R
68/61
559.2781
29023
2
R.SVANDDEVIR.Y
BpIII-10
thrombin-like serine protease 1 serine proteinase precursor C-type lectin
Bothrops jararacussu Trimeresurus albolabris Bothrops insularis
171/67
811.8813
16726
2
K.EFCVELVSYTGYR.L
BpIII-10
C-type lectin
gi|527504051
109/56
811.8781
16726
2
K.EFCVELVSYTGYR.L
BpIII-12
c-type lectin
gi|32396016
67/37
696.3376
15524
2
K.GQSEVWIGLCDK.K
BpIII-12
Serine proteinase 7
gi|521752312
Bothrops pauloensis Bothrops pauloensis Bothrops jararacussu Crotalus horridus
174/60
644.8352
28818
2
K.NFQMQLGVHSK.K
BpIII-12
C-type lectin
gi|32396016
66/35
639.7945
15524
2
R.LWNDQVCESK.N
BpIII-13
Thrombin-like enzyme Thrombin-like enzyme C-type lectin
gi|82466483
Bothrops jararacussu Bothrops asper
39/33
570.7793
28685
2
K.VSDYTEWIK.S
gi|82466483
Bothrops asper
98/53
430.2220
28685
3
K.NFQMQLGVHSK.K
gi|527504051
157/51
639.7919
16726
2
R.LWNDQVCESK.N
gi|114837
Bothrops pauloensis Bothrops atrox
65/51
481.7519
28854
2
R.EAYNGLPAK.T
gi|82466483
Bothrops asper
73/66
644.8283
28685
2
K.NFQMQLGVHSK.K
-
-
-
-
-
-
----------
gi|298351882
Bothrops alternatus -
32/32
596.8121
28654
2
R.VMGWGTISPTK.V
-
-
-
-
----------
BpIII-9 BpIII-9 BpIII-9 BpIII-10 BpIII-10
BpIII-13 BpIII-13 BpIII-14 BpIII-14 BpIII-14
D
Charge (+)
gi|999161
BmIII-3
BpIII-9
C
Mass
gi|999161
BpIII-8
B
Observed Ion
PABJ /serine proteinase PABJ /serine proteinase Batroxobin
BmIII-2
BjuIII-6
1- Isolation of TLE serine proteases from five Bothrops venoms.
Mascot /Ion Scores 50/43
Crotalus adamanteus Trimeresurus albolabris Trimeresurus albolabris Agkistrodon bilineatus Trimeresurus albolabris Bothrops jararaca
BaIII-8
BpIII-16 BpIII-16
Thrombin-like enzyme batroxobin Thrombin-like enzyme Thrombin-like enzyme bhalternin -
gi|527504051
gi|20069139 gi|527504051
-
Many serine proteases have been purified and characterized from B. moojeni, for example, the clotting enzyme Batroxobin (Lochnit and Geyer, 1995), three serine proteinases, MSP1, MSP2 (Serrano et al., 1993), MOO3 (Oliveira et al., 1999); BJ-48 from B. jararacussu (Guedes et al., 2008), Bhalternin from B. alternatus (Costa et al., 2010), BpSP-I from B. pauloensis (Costa et al., 2009) and through a combination of three chromatographic steps, two serine proteases from B. moojeni venom have been isolated by Oliveira et al. in 2013. We presented in this work, a protocol to obtain serine proteases from B. alternatus and B. moojeni venoms with high degree of purity, as showed by LC-MS and SDS-PAGE, suitable for structural and other biophysical and biochemical studies, while for the other Bothrops snake venoms assessed, the purification processes still are in development. Data of peptides identified by mass spectrometry showed their partial sequences sharing high identity with other SVSPs.
All isolated TLEs showed molecular mass of 30-40 kDa, and their partial sequences sharing high identity with venom plasminogen activator, platelet-aggregating enzyme, lectins and SVSPs, according Mascot database search. Individual ions scores > 34 in table 2 indicate identity or extensive homology (p
