Chem. Res. Toxicol. 1988, 1, 337-342
337
Articles Biochemical Characterization of Two Hemorrhagic Proteases from the Venom of Lachesis muta (Bushmaster) Yong-lu Ran,+Siding Zheng,' and Anthony T. Tu* Department of Biochemistry, Colorado State University, Fort Collins, Colorado 80523 Received May 2, 1988
Two hemorrhagic proteases, Lachesis hemorrhagic toxins a and b (LHTa and LHTb), were isolated from the venom of Lachesis muta, which is distributed in Central and South America. One protease showed strong hemorrhagic action, while the other showed weak hemorrhagic activity even though the two enzymes are very similar in their chemical properties. Neither enzyme hydrolyzed arginine esters, but both hydrolyzed casein and reduced fibrinogen. The Aa chain of fibrinogen was hydrolyzed first, and the BP chain was hydrolyzed later. The y chain of fibrinogen was resistant to hydrolysis. The molecular weights of LHTa and LHTb were very similar, 22 000 and 23 000, respectively. The amino acid composition of LHTa was also similar to that of LHTb, The secondary structure of LHTa as determined by Lippert's equation was 52% a helix, 17% P sheet, and 31% random coil; that of LHTb was 47% a helix, 13% sheet, and 40% random coil.
Introduction Snake venom is a mixture of different proteins (1) among which are proteolytic enzymes. The number of investigators have recently studied these proteolytic enzymes (2-4), which are involved in blood coagulation (5), anticoagulation (6),bradykinin-releasing (7) hemorrhagic action (8), and destruction of collagen and elastin (9). A venom usually contains more than one proteolytic enzyme (10, 11).
Proteolytic enzyme activity is especially pronounced in the venom of the Crotalidae family, and most of the research thus far has been focused on the venom of genus Crotalus (rattlesnakes) of North America. Compared with the Crotalus, venom of the Lachesis muta (bushmaster), which is distributed from Nicaragua to Brazil, has been less well studied. It is the largest pit viper in the world and reaches 12 ft in length. Because of its size, it can inject a large amount of venom and is therefore a significant public health problem in Central and South America. L. muta venom causes considerable damage in tissues after envenomation (12), and the presence of its hemorrhagic factor was recognized by Sanchez et al. (13). In the present investigation two closely related hemorrhagic proteases, Lachesis toxins a and b (LHTa and LHTb), were isolated from L. muta venom, and their properties were investigated.
Materials and Methods Venom. L. muta venom was obtained from the Miami Serpentarium as lyophilized venom.
* To whom correspondence should be addressed. 'On leave from the Kunming Institute of Zoology, Kungming, China. *On leave from the Laboratory of Laser Optics, Department of Physics, Fudan University, Shanghai, China.
Purification. The snake venom was fractionated by a combination of Sephadex gel, DEAE' ion-exchange column chromatography, and HPLC.' The first step of the purification was done on a Sephadex G-75-40 column (3 X 92 cm) with 0.01 M Tris buffer containing 0.1 M NaCl a t p H 7.9. The flow rate was adjusted to 9 mL/h, and every 3 mL was collected by a fraction collector. Fraction IV from the column was isolated, dialyzed, and lyophilized. The sample was further separated into three major fractions on a DEAE-Sephadex A-50 column (2.3 X 34 cm) with 0.01 M Tris buffer at pH 7.9 as the effluent solution. Again, the flow rate was adjusted to 9 mL/h, and 3-mL fractions were collected. For the gradient, the stirring bottle contained 500 mL of 0.01 M Tris buffer, pH 7.9. The reservoir contained 500 mL of 0.01 M Tris buffer, pH 7.9, containing 0.4 M NaCl. The IV-I1 and IV-I11 fractions showed proteolytic and hemorrhagic activities (Figure lB), and each of these fractions was purified separately on a DEAE-3 SW Spherogel HPLC column (0.75 X 7.5 cm). The buffer of 0.05 M sodium phosphate at pH 7.4 was employed, with 0-0.25 M NaCl in the same buffer as a gradient. The HPLC equipment used was from Beckman Instruments. The homogeneity of LHTa and LHTb was established from SDS electrophoresis using 10% acrylamide (14). Various Assay Methods. Amino acid analysis was done by hydrolyzing protein with constant-boiling HCl a t 110 OC after reducing and carboxymethylating. Amino acids were then converted to the phenyl isothiocyanate derivative and detected by absorbance a t 254 nm. For amino acid analysis HPLC was used on the CI8 reverse-phase column (0.46 X 25 cm). Tryptophan content was determined by the spectroscopic method of Edelhoch (15). Molecular weight was determined by SDS-polyacrylamide electrophoresis. Proteins used for molecular weight reference were bovine albumin (66 000), egg albumin (45 OOO), glyceraldehydeAbbreviations: HPLC, high-pressure liquid chromatography; Tris,
tris(hydroxymethy1)aminomethane;DEAE, (diethylaminoethy1)agarme; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid; TAME, @toluenesulfony1)-L-argininemethyl ester; BAEE, N-benzoyl-L-arginine ethyl ester; LHTa, Lachesis hemorrhagic toxin a; LHTb, Lachesis hemorrhagic toxin b.
0 1988 American Chemical Societv
338 Chem. Res. Toxicol., Vol. 1, No.6,1988 3-phosphate dehydrogenase (36 OOO), carbonic anhydrase (29OOO), trypsinogen (24 OOO), trypsin inhibitor (20 loo), and a-lactalbumin (14200). The isoelectric point was estimated by isoelectric focusing gel electrophoresis using ampholine of the pH range of 3.5-10.0 first. A narrower range of 5.5-8.0 was used later for more accurate measurement. Metal content was determined by inductively coupled plasma-atomic emission spectroscopy according to the method of Dahlquist and Knoll (16). Protein was assayed by using Coomassie blue dye agent. For a hemorrhagic assay of each fraction, two or three Swiss Webster mice (20-22 g) were each injected subcutaneously in the back with one dose in 0.1 mL of 0.9% saline and sacrificed after 6 h. The skin was removed, and the hemorrhagic spot was observed. Caseinolytic activity was measured by the standard method in which unreacted protein is precipitated with TCA and the absorbance of supernatant liquid is measured at 660 nm after adding the Folin reagent. Argininesterase activity was measured with (p-toluenesulfony1)-L-arginine methyl ester (TAME) and N-benzoyl-L-arginine ethyl ester (BAEE) as substrates. Activity with TAME was measured at 247 nm by the method of Tu et al. (16).Activity with BAEE was measured at 253 nm by the method of Toom et al. (18). Phospholipase A activity was measured with phosphatidylcholine as the substrate according to the method of Wells and Hanahan (19). The substrate was dissolved in 95% diethyl ether-5% methanol and was incubated with the enzyme for 10 min at 25 "C. The liberated acid was titrated with 0.02 N NaOH in 95% ethanol. Phenol red was used as an indicator. Raman Spectroscopy. Raman spectra were obtained by excitation with the 514.5-nm line (Spectra Physics, Model SP-164 argon ion laser) with a green interference filter. The spectra represent an average of 10 scans and were recorded with the use of a Spex Ramalog 5 Raman spectrometer and a Spex SCAMP data acquistion processor. In order to perform the deuterium exchange experiments, the samples were dissolved in D20 and allowed to sit a t room temperature for 2 h, whereupon they were lyophilized. This step was repeated twice, and the lyophilized sample sealed in a capillary tube was examined.
Results Isolation. Two hemorrhagic proteases were isolated from the venom of L. muta, and they were designated as Lachesis hemorrhagic toxins a and b. Both hemorrhagic proteases were isolated by a threestep purification method (Figure 1). In the third step (HPLC), a relatively sharp peak was obtained from fraction IV-II;this protease was designated LHTa (Figure IC). Fraction IV-I11 showed two major peaks on HPLC; the LHTb was obtained from the second peak at the retention time of 4.8 min (Figure 1D). The homogeneity of both enzymes was established by SDS electrophoresis and is shown in Figure 1C,D. Proteolytic Enzymes. When casein was used as a substrate, it was hydrolyzed by both LHTa and LHTb. However, neither LHTa nor LHTb hydrolyzed TAME or BAEE. TAME and BAEE are well-known synthetic substrates for trypsin. The results indicate that LHTa and LHTb are proteolytic enzymes, but they do not have trypsin-like activity. Usually all snake venoms show phospholipase A activity. In order to show that the hemorrhagic actions of LHTa and LHTb were not due to contaminating phospholipase A, the phospholipase A activity was tested with phosphatidylcholine as the substrate. Both LHTa and LHTb gave negative results, indicating that hemorrhagic activity was due to inherent protease activity and not due to phospholipase A contamination. Effect on Fibrinogen. Since fibrinogen is an important blood component responsible for blood coagulation, it is
Ran et al.
20
40
60
70
80
90
100
120 140 160
Tube number 3mVtube
20
40
60
80
100 120
Tube number
Time in minutes
140 160
180
3ml/tube
Time in minutes
Figure 1. (A) Separation of LHTa and LHTb from L. muta venom on Sephadex G75-40. (B) M i c a t i o n of LHTa and LHTb from peak IV of Sephadex G-7440 by DEAESephadex A-50. (C) Purification of LHTa from peak IV-I1 of DEAE-Sephadex A-50 by HPLC. (D) Purification of LHTb from peak IV-I11 of DEAE-Sephadex A-50 by HPLC (see Materials and Methods for details). Shading represents the fraction with hemorrhagic activity.
appropriate to use fibrinogen as a substrate. Both LHTa and LHTb hydrolyzed fibrinogen, and their hydrolysis products were compared with those of thrombin and plasmin in electrophoresis. The fibrinogen molecule consists of Aa, BP, and y chains connected by interchain disulfide bonds. Both LHTa and LHTb have strong activity on the A a chain (Figure 2). Both LHTa and LHTb hydrolyzed Aa as soon as they were incubated together. It took only about 5 min to hydrolyz the A a subchain. The BP chain was hydrolyzed more slowly by both LHTa and LHTb, although the BO band was still visible even after 120-min incubation. With much longer incubation, the B/3 chain disappeared completely, indicating that the BP chain was also degraded. Judging from the fragments, the sites of hydrolysis by LHTa and LHTb are very similar, and it seems they hydrolyzed the Aa and the BO chains at the same sites. This explains the nearly identical molecular weight patterns of the peptide fragments. The y chain seemed quite resistant to hydrolysis by both LHTa and LHTb. Plasmin hydrolyzed fibrinogen, but the hydrolysis products were different from those of thrombin (Figure 3). The results clearly indicate that proteolytic cleavage points in fibrinogen are different for plasmin and for LHTa and LHTb. Hydrolysis of fibrinogen by thrombin is shown in Figure 3b. It is well-known that thrombin hydrolyzes specific bonds and releases fibrinopeptide A from the Aa and fi-
Chem. Res. Toxicol., Vol. 1, No. 6, 1988 339
Snake Hemorrhagic Toxins a
*? -n
b
4E-
36.-
3
3
I
2924
-
36
2924-2
I
5
0
m "..-,-."...-
15 30 60 120
0
Minutes
5
15 30
6 0 120
Minutes
Figure 2. (a) Reduced SDS-polyacrylamide gel electrophoretic pattern of fibrinogen degradation products produced by LHTa. Lanes 3-7: Fibrinogen with LHTa (10 pg/mL) incubated for 5, 15,30,60, and 120 min. Lane 2 (0): Fibrinogen control. Lane 1: Protein standards with molecular weights as shown. (b) Reduced SDS-polyacrylamide gel electrophoretic pattern of fibrinogen degradation products produced by LHTb. Lanes 3-7: Fibrinogen with LHTb (10 pg/mL) incubated for 5,15, 30,60, and 120 min. Lane 2 (0): Fibrinogen control. Lane 1: Protein standards with molecular weights as shown. a
b
Table I. Amino Acid Composition and Molecular Weight of Hemorrhagic Toxins from L. muta Venom residues amino acid LHTa LHTb 27 26 Asx 23 Glx 18 18 16 Ser 12 11 GlY 7 His 8 Thr 23 25 8 8 Arg 10 9 Ala 6 5 Pro 6 6 Tyr 2 3 Met 7 5 Val 16 11 W Y S 7 7 Ile 12 12 Leu 7 5 LYS 19 15 Phe 196 204 total residues 21 911 22 992 MW based on amino acid analysis 23 000 MW based on SDS electrophoresis 22 000
Zn Ca
LHTa, mol/mol 0.756 0.280
Table 11. LHTb, mol/mol Mg 0.770 0.592 Fe
LHTa, mol/mol 0.041 0.004
LHTb, mol/mol 0.287 0.017
The MHDs for LHTa, LHTb, and crude venom were 25, 0.5, and 2 pg, respectively. Therefore, LHTa was a very weak hemorrhagic toxin, and its activity was weaker than crude venom. LHTb had a MHD of 0.5 pg, so it was four times more potent than that of crude venom (MHD = 2
66.
Aa.BpY
---L
%W'"IW*IV
5-
6-
0
5 15
MI nutes
30 60 120
0
5 15
30 60120
Minutes
Figure 3. (a) Reduced SDS-pc-jacrylamide gel electrophoretic pattern of fibrinogen degradation products produced by plasmin. Lanes 3-7: Equal volumes of 2% fibrinogen with plasmin (0.1 unit/mL) incubated for 5,15,30,60, and 120 min. Lane 2 (0): Fibrinogen control. Lane 1: Protein standards with molecular weights as shown. (b) Reduced SDS-polyacrylamide gel electrophoretic pattern of fibrinogen degradation products produced by thrombin. Lanes 3-7: Equal volumes of 2% fibrinogen with thrombin (10 units/mL) incubated for 5,10,15,30, and 120 min. Lane 2 (0): Fibrinogen control. Lane 1: Protein standards with molecular weights as shown.
brinopeptide B from the BP chains. In Figure 3b two A a chains are visible. The one with the higher molecular weight is Aal, and the lower molecular weight chain is AaB. Occasionally the A a band splits into two bands. Figure 3b shows that the Aal subchain disappeared, while the Aa2 subchain remained. The BP chain was rapidly hydrolyzed. Because the amounts of fibrinopeptides A and B released were small, they were not detected and there were clean backgrounds in the low-molecular weight regions of the gel. Hemorrhagic Activity. In order to measure hemorrhagic activity, the minimum hemorrhagic dose (MHD) was measured for LHTa and LHTb, and crude venom. The MHD is defined as the minimum amount of a hemorrhagic toxin to produce a hemorrhagic spot of 5-mm diameter by subcutaneous injection. Therefore, the smaller the MHD number, the higher the hemorrhagic activity.
Pi?).
Chemical Properties. The total amino acid residues of LHTa and LHTb were 196 and 204, respectively (Table I), a difference of only eight residues. The two toxins also contained similar numbers of many amino acid residues. The biggest difference was the number of Glx (glutamic acid plus glutamine) and half-cystine residues (Table I). The molecular weights calculated from the amino acid analysis were 21 911 and 22 992, respectively. These numbers were very similar to those obtained from SDS electrophoresis, which gave 22 OOO and 23 OOO, respectively. The isoelectric points of LHTa and LHTb determined by isoelectric focusing gel were 6.5 and 6.1, respectively. Since many venom hemorrhagic proteases contain zinc (I1,20-22), the content of zinc, calcium, magnesium, and iron was determined by inductively coupled plasma-atomic emission spectroscopy. The results were as shown in Table 11. Only zinc gave a value close to 1. The content of magnesium and iron was almost negligible. The calcium content was higher than that of magnesium and iron, but much less than that of zinc. Conformation by Laser Raman Spectroscopy. Raman spectroscopy has been used extensively for determination of protein conformation by examining both amide I and I11 frequencies. The amide I band of the a helix appears at 1645-1658 cm-l, the P sheet at 1665-1680 cm-l, the p turn at 1662-1676 cm-l and the random coil at 1660-1666 cm-l. The amide I band of LHTa showed main peaks at 1668 and 1654 cm-l (Figure 4). The 1654-cm-l peak is obviously from the a-helix conformation. The 1668-cm-' band is contributed by either the p-sheet or &turn structure or by both. The Raman spectrum of LHTb is shown in Figure 5. LHTb showed the main amide I peak at 1660 cm-l and
340 Chem. Res. Toxicol., Vol. I, No. 6, 1988
Ran et al. Table 111. Zinc Content of Hemorrhagic Toxins Shown To Be Proteolytic Enzymes Zn content, mol/mol venom (origin) name of protein ref Agkistrodon acutus AC1 proteinase 1.15 20
(Taiwan)
hemorrhagic toxin
1.0
29
0.99 0.82 0.86 0.86 1.03 1.15
1.0
11 11 11 11 11 8 10
0.76 0.77
this paper this paper
1
Crotalus atrow
hemorrhagic toxin a b C
d e
f
I
f l
WAVENUMBER Figure 4. Raman spectra of LHTa in H20 and in D20 from 700 to 1700 cm-'.
'Dar
I
1600
1400
1200
1000
800
'
WAVENUXBER Figure 5. Raman spectra of LHTb in H20 and in D20 from 700 to 1700 cm-'.
small peaks at 1650 and 1678 cm-l. The 1660-cm-' peak is about halfway between the wavenumber of a helix and that of @ sheet and @ turn. The 1650-cm-' band is from a helix. The 1678-cm-' band is from either @-sheetor @-turnstructure. Fortunately, the amide I11 band also contains structural information. Spectra of LHTa and LHTb in D,O are also shown in Figures 4 and 5. Each band in the amide I11 region was normalized against the C-H bending vibrational band at 1449 cm-' for LHTa and 1444 cm-' for LHTb. The decrease in the intensity of bands in the amide I11 region was calculated. The intensities of the 1283-, 1271-, and 1254-cm-' bands of LHTa decreased, indicating that these three bands were real amide I11 bands. With LHTb, the intensity at 1287 and 1266 cm-' decreased. The 1283- and 1271-cm-' bands of LHTa were very likely from a helix, and the 1254-cm-' band was contributed from random coil. The 1287-cm-' band of LHTb probably originated from a helix, while the 1266-cm-' band was either a helix or /3 turn or both.
C. horridus horridus hemorrhagic proteinase L. muta Lachesis hemorrhagic toxin a b
In order to determine the content of different conformations of LHTa and LHTb more quantitatively, the equation developed by Lippert et al. (23) was used. The solution of these equations indicated that LHTa contained 52% a helix, 17% @ sheet, and 31% random coil, and LHTb contained 47% a helix, 13% @ sheet, and 40% random coil. The presence of tyrosine in LHTa and LHTb is evident from the Raman spectra (Figures 4 and 5). Because of the six tyrosine residues in each toxin, the intensity ratios of the 850- and 830-cm-' bands reflect only the average state of the tyrosine microenvironment in each toxin. The corresponding tyrosine bands for LHTa were 856 and 825 em-', while those of LHTb were 850 and 833 cm-'. The intensity ratio I,o/Is30 is about the same for both toxins. Judging from the ratio of approximately 1, both LHTa and LHTb's tyrosine side chains are exposed to solvent.
Dlscussion L. muta belongs to family of Crotalidae (pit viper) and is well distributed in Central and South America. Despite the medical importance of snake poisoning due to this species, very little study of the components of the snake venom has been made. The Crotalidae toxins usually cause pronounced local tissue damage. Extensive studies have been made of the hemorrhagic, myotoxic, and other components from the venoms of North American pit vipers (24-28). It is thus of interest to find how the hemorrhagic toxins isolated from Lachesis differ from other pit viper venoms. The two hemorrhagic toxins, LHTa and LHTb, from L. muta venom showed proteolytic activity, and they both contain 1 mol of zinc/mol of protein. This is also true of many hemorrhagic toxins from other pit viper venoms (Table 111). The molecular weights of snake hemorrhagic proteases tend to fall into two classes. One type is relatively small, ranging from 22 000 to 26 000; the other is in the 50 00068000 range. The molecular weights of LHTa and LHTb are 22 000 and 23 000, respectively. L. muta hemorrhagic toxins isolated here are of the first type (13). The hemorrhagic factor isolated from Atractaspis engaddensis has a molecular weight of 50000 and therefore is of the second type of hemorrhagic toxin. One of the higher molecular weight (100000) hemorrhagic factors, LHF-1, was recently isolated by Sanchez et al. (13).
Snake Hemorrhagic Toxins Therefore, LHT-1 is different from the hemorrhagic toxins LHTa and LHTb isolated by us, although all these toxins were isolated from the same venom. Although LHTa and LHTb from the same venom are called hemorrhagic toxins because of their hemorrhagic action, they also had an effect on fibrinogen by actively degrading the A a and BO chains. But both LHTa and LHTb are different from thrombin. They did not produce a fibrin clot like thrombin, and the molecular weight of the degradation products was different from those produced by thrombin. Both LHTa and LHTb were similar in molecular weight and amino acid composition, but there was a large difference in hemorrhagic activity. It is not known why LHTa was lower in hemorrhagic activity. Many proteases have been isolated from different snake venoms. Sometimes two proteases can hydrolyze the same substrate, but one is hemorrhagic and the other is nonhemorrhagic. For instance, both hemorrhagic toxin b and atroxase from Crotalus atrox venom hydrolyzed the Aa and B/3 chains of fibrinogen and were similar in molecular weight, zinc content, and amino acid composition. However, hemorrhagic toxin b from C. atrox venom produced severe hemorrhage, while atroxase was nonhemorrhagic (30). The detailed biochemical mechanism of hemorrhage induced by snake hemorrhagic toxin has not been clarified yet. About 20 hemorrhagic toxins were isolated from venoms of different snakes, mainly from the pit viper (Crotalidae) family and to a lesser extent from the viper (Viperidae) family. Some hemorrhagic toxins are very similar to other venom proteases in molecular weight and amino acid composition. One difficulty in comparing different proteases and hemorrhagic toxins is that some investigators never examined the hemorrhagic activity of their proteases. However, most hemorrhagic toxins are different in amino acid composition among themselves. It would be helpful if one could explain the venom hemorrhagic action on a molecular basis, but at the present state of available knowledge it precludes such a feat. None of the hemorrhagic toxins have been identified as to their amino acid sequence. Once this information is available about the many hemorrhagic toxins, an explanation of the molecular basis of hemorrhagic action is possible. In fact, this is our long-range objective.
Acknowledgment. This research was supported by NIH Merit Award R37 GM15591 (A.T.T.), a Senior Research Fellowship from the People’s Republic of China (Y.R.), and a World Bank Fellowship (S.Z.). Registry No. Proteinase, 9001-92-7.
References (1) Tu, A. T. (1977)Venoms: Chemistry and Molecular Biology, Wiley, New York. (2) Kisiel, W.,Kondo, S., Smith, K. J., McMullen, B. A., and Smith, L. F. (1987)“Characterization of a protein C activator from Agkistrodon contortrix contortrix venom”. J. Biol. Chem. 262, 12607-12613. (3) Evans, H. J. (1984)“Proteolytic activities of cobra venoms based on inactivation of a,-macroglobulin”. Biochem. Biophys. Acta 784,97-101. (4) Fress, L. F., Catanese, J., and Hirayama, T. (1983)“Analysis of the effects of snake venom proteinases on the activity of human plasma C1 esterase inhibitor, or,-antichymotrypsin and b2antiplasmin”. Biochim. Biophys. Acta 745,113-120. (5) Markland, F. S.,Kettner, C., Schiff”, S., Shaw, E., Bajwa, S. S., Reddy, K. N. N., Kirakossian, H., Patkos, G. B., Theodor, I., and Pirkle, H. (1982)“Kallikrein-likeactivity of crotalase, a snake venom enzyme that clots fibrinogen“. Prot. Natl. Acad. Sci. U. S.A. 79, 1688-1692.
Chem. Res. Toxicol., Vol. 1, No. 6, 1988 341 (6) Pandya, B. V., and Budppski, A. Z. (1984) “Anticoagulant proteases from western diamondback rattlesnake (Crotalus atrox) venom”. Biochemistry 23,460-470. (7) Bailey, G. S.,and Shipolini, R. A. (1976) “Purification and properties of a kininogenin from the venom of Vipera ammodytes Ammodytes”. Biochem. J. 153,409-414. (8) Nikai, T., Mori, N., Kishida, M., Sugihara, H., and Tu, A. T. (1984)“Isolation and biochemical characterization of hemorrhagic toxin f from the venom of Crotalus atrox (western diamondback rattlesnake)”. Arch. Biochem. Biophys. 231,309-319. (9) Webb, Z.,Banda, M. J., McKerrow, J. H., and Sandhaus, R. A. (1982)“Elastases and Elastin degradation”. J. Znuest. Dermatol. 79, 1545-1595. (10) Civello, D. J., Duong, H. L., and Geren, C. R. (1983)“Isolation and characterization of a hemorrhagic proteinase from timber rattlesnake venom”. Biochemistry 22,749-461. (11) Bjarnason, J. B., and Tu, A. T. (1978)‘Hemorrhagic toxins from western diamondback rattlesnake (Crotalus atrox) venoms: isolation and characterization of five toxins and the role of zinc in hemorrhagic toxin e”. Biochemistry 17,3395-3404. (12) Lomonte, B. (1985)“Edema-forming activity of bushmaster (Lachesis muta stenophrys) and Central American rattlesnake (Crotalus durissus durissus) venoms and neutralization by a polyvalent antivenom”. Toxicon 23, 173-176. (13) Sanchez, E. F., Magalhaes, A., and Dimiz, C. R. (1987) “Purification of a hemorrhagic factor (LHF-I) from the venom of the bushmaster snake, Lachesis muta muta”. Toxicon 25, 611-619. (14) Laemmli, U.K. (1970)“Cleavage of structural proteins during the assembly of the head of bacteriophage T4”. Nature (London) 227,680-685. (15) Edelhoch, G. (1967)“Spectroscopic determination of tryptophan and tyrosine in proteins“. Biochemistry 6,1948-1954. (16) Dahlquist, R. L., and Knoll, J. W. (1978)“Inductively coupled plasma-atomic emission spectrometry: analysis of biological materials and soils for major, trace, and ultra-trace elementa”. Appl. Spectrosc. 32,1-29. (17) Tu, A. T., Chua, A., and James, G. P. (1966)”Proteolytic enzyme activities in a variety of snake venoms“. Toxicol. Appl. Pharmacol. 8,218-233. (18) Tu, A. T., and Toom, P. M. (1971)“Enzymatic and biological studies of cholera (Vibrio cholerae) toxin”. Experientia 27, 858-859. (19) Wells, M. A., and Hanahan, D. J. (1969)“Studies on phospholipase A. I. Isolation and characterization of two enzymes from Crotalus adamanteus venomn. Biochemistry 8,414-424. (20) Nikai, T., Ishizaki, H., Tu, A. T., and Sugihara, H. (1982) “Presence of zinc in proteolytic hemorrhagic toxin isolated from Agkistrodon mutus venom”. Comp. Biochem. Physiol., C: Comp. Pharmacol. Toxicol. 72C,103-106. (21) Tu, A. T. (1983) “Local tissue damaging (hemorrhage and myonecrosis) toxins from rattlesnake and other pit vipers”. Toxin Reu. 2,205-234. (22) Mori, N., Nikai, T., Sugihara, H., and Tu, A. T. (1987) “Biochemical characterization of hemorrhagic toxins with fibrinogenase activity isolated from Crotalus ruber ruber venom”. Arch. Biochem. Biophys. 253, 108-121. (23) Lippert, J. L., Tyminski, D., and Desmeulee, P. J. (1976) “Determination of the secondary structure of proteins by laser Raman spectroscopy”. J. Am. Chem. SOC. 98,7075-7080. (24) Civello, D. J., Moran, J. B., and Geren, C. R. (1983)“Substrate specificity of a hemorrhagic proteinase from timber rattlesnake venom”. Biochemistry 22,755-762. (25) Tu, A. T., Nikai, T., and Baker, J. 0. (1982)“Proteolytic specificity of hemorrhagic toxin a isolated from westem diamondback rattlesnake (Crotalus atrox) venom”. Biochemistry 20, 7004-7009. (26) Ownby, C. L., Odell, G. V., Woods, W. M., and Colberg, T. R. (1983) “Ability of antiserum to myotoxin a from prairie rattlesnake (Crotalus uiridis uiridis) venom to neutralize local myotoxicity and lethal effects of myotoxin a and homologous crude venom”. Toxicon 21,35-45. (27) Henderson, J. T.,Nieman, R. A., and Bieber, A. L. (1987) “Assignment of the aromatic ‘H NMR resonances of myotoxin a isolated from the venom of Crotalus uiridis uiridis”. Biochim. Biophys. Acta 914,152-161. (28) Ovadia, M.(1987)“Isolation and characterization of a hemorrhagic factor from the venom of the snake Atractaspis engaddensis (Atractaspididae)”. Toxicon 25,621-630.
342 Chem. Res. Toxicol., Vol. 1, No. 6, 1988 (29) Zang, J., Chen, Z., He, Y.,and Xu, X. (1984) "Effect of calcium on proteolytic activity and conformation of hemorrhagic toxin I from five pace snake (Agkistrodon acutus) venom". Toxicon 22, 931-935.
Ran et al. (30) Willis, T. W., and Tu, A. T. (1988) "Purification and biochemical characterization of atroxase a nonhemorrhagic fibrinolytic protease from western diamondback rattlesnake venom". Biochemistry 27, 4769-4777.
