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Francis S Markland - One of the best experts on this subject based on the ideXlab platform.
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Fibrolase trials and tribulations
Toxins, 2010Co-Authors: Francis S Markland, Steve SwensonAbstract:Fibrolase is the fibrinolytic enzyme isolated from Agkistrodon contortrix contortrix (southern copperhead snake) venom. The enzyme was purified by a three-step HPLC procedure and was shown to be homogeneous by standard criteria including reverse phase HPLC, molecular sieve chromatography and SDS-PAGE. The purified enzyme is a zinc metalloproteinase containing one mole of zinc. It is composed of 203 amino acids with a blocked amino-terminus due to cyclization of the terminal Gln residue. Fibrolase shares a significant degree of homology with enzymes of the reprolysin sub-family of metalloproteinases including an active site homology of close to 100%; it is rapidly inhibited by chelating agents such as EDTA, and by alpha2-macroglobulin (α2Μ). The enzyme is a direct-acting thrombolytic agent and does not rely on plasminogen for clot dissolution. Fibrolase rapidly cleaves the A(α)-chain of fibrinogen and the B(β)-chain at a slower rate; it has no activity on the γ-chain. The enzyme exhibits the same specificity with fibrin, cleaving the α-chain more rapidly than the β-chain. Fibrolase was shown to have very effective thrombolytic activity in a reoccluding carotid arterial thrombosis model in the canine. A recombinant version of the enzyme was made in yeast by Amgen, Inc. (Thousand Oaks, CA, USA) and called alfimeprase. Alfimeprase is identical to Fibrolase except for a two amino acid truncation at the amino-terminus and the insertion of a new amino-terminal amino acid in the truncated protein; these changes lead to a more stable enzyme for prolonged storage. Alfimeprase was taken into clinical trials by Nuvelo, Inc. (San Carlos, CA), which licensed the enzyme from Amgen. Alfimeprase was successful in Phase I and II clinical trials for peripheral arterial occlusion (PAO) and central venous access device (CVAD) occlusion. However, in Phase III trials alfimeprase did not meet the expected end points in either PAO or CVAD occlusion and in a Phaase II stroke trial, and Nuvelo dropped further development in 2008.
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Fibrolase and its evolution to clinical trials a long and winding road
2010Co-Authors: Francis S Markland, Stephen SwensonAbstract:Fibrolase is the fibrinolytic proteinase isolated from Agkistrodon contortrix contortrix (southern copperhead snake) venom. The enzyme was purified by a three-step HPLC procedure and was shown to be homogeneous by standard criteria. The purified enzyme is inhibited by EDTA and other chelating agents and is a zinc metalloproteinase containing one mole of zinc per molecule. The enzyme is also rapidly inhibited by alpha2-macroglobulin (α2 M). Fibrolase is composed of 203 amino acids with a blocked amino-terminus due to cyclization of the terminal Gln residue. The enzyme is a direct-acting thrombolytic agent and does not rely on plasminogen for clot dissolution. Fibrolase rapidly cleaves the A(α)-chain of fibrinogen and the B(β)-chain at a slower rate; it has no activity on the γ-chain. The enzyme exhibits the same specificity with fibrin. Fibrolase was shown to have very effective thrombolytic activity in a reoccluding carotid arterial thrombosis model in the canine. A recombinant version of the enzyme was made in yeast by Amgen, Inc. (Thousand Oaks, CA, USA) and called alfimeprase. Alfimeprase is identical to Fibrolase except for a two amino acid truncation at the amino-terminus and the insertion of a new amino-terminal amino acid in the truncated protein; these changes lead to a more stable enzyme for prolonged storage. Twenty years after it was first purified alfimeprase was taken into clinical trials by Nuvelo, Inc. (San Carlos, CA), which licensed the enzyme from Amgen. Alfimeprase was successful in Phase I and II clinical trials for peripheral arterial occlusion (PAO) and central venous access device (CVAD) occlusion. However, in Phase III trials alfimeprase did not meet the expected end points in either PAO or CVAD occlusion and in a Phase II stroke trial and Nuvelo dropped further development in 2008.
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snake venom fibrin ogen olytic enzymes
Toxicon, 2005Co-Authors: Steve Swenson, Francis S MarklandAbstract:Abstract Snake venoms contain a number of serine and metalloproteinases and included among these are the fibrin(ogen)olytic proteinases. Some years ago it was postulated that the fibrin(ogen)olytic enzymes may be clinically useful. Over the past 150 years a substantial body of literature has been generated on the identification and characterization of fibrin(ogen)olytic enzymes from a broad spectrum of snake species. In this review we describe the two different classes of fibrin(ogen)olytic enzymes isolated from snake venom and we summarize a number of studies aimed at characterizing the purified enzymes and/or their derivatives. Two distinct classes of venom fibrin(ogen)olytic enzymes have been previously identified, the metalloproteinases and serine proteinases. These two classes of proteinases differ in their mechanism of action and they target different amino acid sequences in fibrin(ogen), but each perform the same role in nature. When a snake envenomates its prey it needs a mechanism to facilitate the spread of the toxic components throughout the circulation. Fibrin(ogen)olytic enzymes break down fibrin rich clots and help to prevent further clot formation by their action on fibrinogen. This characteristic feature has led to development of fibrin(ogen)olytic snake venom enzymes as potential clinical agents to treat occlusive thrombi. Fibrolase, a fibrinolytic metalloproteinase isolated from Agkistrodon contortrix contortrix venom and the serine β-fibrinogenolytic proteinase from Vipera lebetina have been chosen as representative enzymes from the two classes, and their biochemical and physiochemical properties will be described in detail. Finally, the characterization and development of alfimeprase, a recombinant fibrinolytic enzyme derived from Fibrolase, as a clinical agent is described citing the progression from the laboratory bench to its current status as having successfully completed Phase II clinical trials.
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Three-dimensional structure of Fibrolase, the fibrinolytic enzyme from southern copperhead venom, modeled from the x-ray structure of adamalysin II and atrolysin C
AAPS PharmSci, 2001Co-Authors: Michael B. Bolger, Steve Swenson, Francis S MarklandAbstract:The fibrinolytic enzyme from southern copperhead snake venom, Fibrolase, contains 1 mole of zine per mole of protein, belongs to the major family of metalloproteinases known as the metzincins, and has been shown to degrade fibrin clots in vitro and in vivo. The purpose of this study was to develop a 3-dimensional model of Fibrolase to investigate the geometry of conserved and variable sequences between members of the snake venom metalloproteinases. When compared to atrolysin C (form D) or adamalysin II (metzincins with completely different substrate specificity), Fibrolase has approximately 60% overall sequence identity and nearly 100% sequence similarity in the active site. We used the crystal structure of adamalysin II to build a 3-dimensional homology model of Fibrolase. Three disulfide bonds were constructed (the highly conserved disulfide bond [118–198] was maintained from the adamalysin II structure and 2 new disulfide bonds were introduced between residues 158–182 and 160–165). We used Sculpt 2.5 and HyperChem 5.0 to “dock” a substrate fragment octapeptide (HTEKLVTS), and a water molecule into the active site cleft. We calculated the differential average homology profile for Fibrolase compared to 8 hemorrhagic and 5 nonhemorrhagic metzincins. We then determined the sequence regions that might be responsible for their substrate specificity. Our 3-dimensional homology model shows that the variable sequences lie on the periphery of the identified active site region containing the His triangle; this indicates that substrate specificity may depend on surface residues that are not directly associated with the active site.
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chimeric derivative of Fibrolase a fibrinolytic enzyme from southern copperhead venom possesses inhibitory activity on platelet aggregation
Archives of Biochemistry and Biophysics, 2000Co-Authors: Stephen Swenson, Larry R Bush, Francis S MarklandAbstract:Abstract Fibrolase, a metalloproteinase isolated from the venom of Agkistrodon contortrix contortrix (southern copperhead snake), is a direct acting fibrinolytic enzyme that has been used to digest occlusive blood clots in animal models. The snake venom enzyme directly degrades fibrin associated with platelet rich blood clots and does not rely on plasminogen activation. Rethrombosis is a serious complication that is experienced in a significant percentage of patients treated with thrombolytic agents to remove occlusive vascular thrombi. The involvement of platelets in the initiation of rethrombosis is well known. Arg–Gly–Asp(RGD)-containing agents have been shown to inhibit rethrombosis following thrombus dissolution by plasminogen activators. In an effort to create a more effective fibrinolytic enzyme and to target the enzyme to platelet-rich thrombi, thereby decreasing the potential for rethrombosis, a chimeric derivative of Fibrolase has been produced. This report describes the construction and biochemical characterization of the chimeric enzyme and an evaluation of its in vitro activities. The chimera was formed by covalently incorporating an RGD-like peptide into Fibrolase. The site of peptide attachment was determined to be a single lysine residue remote from the enzymes active site. Covalent modification of Fibrolase with the RGD-like peptide did not inhibit either fibrinolytic activity of the enzyme nor platelet aggregation inhibitory activity of the peptide. The chimera not only retained the same level of enzymatic activity as native Fibrolase, but also acquired the ability to inhibit platelet aggregation by binding to the fibrinogen receptor (integrin αIIbβ3) on platelets.
Mark C Manning - One of the best experts on this subject based on the ideXlab platform.
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sequence analysis of Fibrolase a fibrinolytic metalloproteinase from agkistrodon contortrix contortrix
Toxicon, 1995Co-Authors: Mark C ManningAbstract:Fibrolase is a small (203 amino acids), nonhemorrhagic, fibrinolytic enzyme from the venom of Agkistrodon contortrix contortrix (southern copperhead). While the chemical and physical properties of the protein have been extensively studied, its overall globular structure is unknown. By comparison with homologous metalloproteinases and snake toxins, the catalytic zinc binding site of Fibrolase has been identified, as well as a potential binding site for calcium, which has not been recognized before. The positions of the major secondary structural features are predicted, and found to be similar to other structurally characterized metalloproteinases, while the positions of the three intramolecular disulfide bonds are also postulated. Finally, Fibrolase is reported to be nonhemorrhagic and earlier work on hemorrhagic enzymes from snake venoms identified six amino acids which might be responsible for hemorrhagic activity. It is shown here that most of these residues occur in Fibrolase, and yet it is nonhemorrhagic in its activity. Altogether, this work demonstrates the utility of sequence analysis methods in the characterization of the structure of venom-derived proteins.
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Fibrolase a fibrinolytic protein from snake venom
Pharmaceutical biotechnology, 1993Co-Authors: D Pretzer, B Schulteis, C D Smith, James W Mitchell, Mark C ManningAbstract:Fibrolase is an active fibrinolytic agent and possesses potential for use in thrombolytic therapy. Its mode of action had been characterized, both in vitro and in vivo. Possessing three disulfide bonds, native Fibrolase is nonglycosylated and binds an intrinsic zinc atom. The zinc is essential for retention of activity and structural integrity. In solution, Fibrolase is sensitive to changes in pH and temperature (Pretzer et al., 1991). At neutral to basic pH (pH 5-9), the solubility and stability of Fibrolase is nearly constant. Little structural variation can be detected by CD spectroscopy. However, decrease in pH below 5 leads to a pronounced reduction in both the solubility and activity of Fibrolase. At pH 3 and below, the solubility of Fibrolase returns but the activity does not. This solubility profile is unusual in that the minimal solubility is well removed from the pI (which is 6.7). It is proposed that the behavior of Fibrolase with variation in pH can be understood in terms of capacity to bind zinc. At pH 5 to 9, the protein binds zinc and the structure and activity are preserved. Near pH 5, the histidine residues which serve as ligands for the zinc become protonated and zinc binding is lost. Loss of zinc leads to local unfolding of a helical segment of Fibrolase, exposing hydrophobic groups which allow the protein to rapidly aggregate. At lower pH values (1-3), the protein again adopts a more globular structure, similar to molten globule states, and the solubility increases. However, without the zinc, Fibrolase remains inactive. Changes in pH also affect thermal stability. The Tm for Fibrolase moves from 50 degrees C at pH 8 to 43 degrees C at pH 5. Increases in temperature also lead to removal of the zinc ion, again producing a partially denatured protein with a marked tendency to aggregate. In both cases (decrease in pH and increase in temperature), analysis of the CD spectra indicates that the protein has primarily lost alpha-helical secondary structure. A major change in structure can also be observed using NMR spectroscopy. At temperatures below 35 degrees C, the globular structure of Fibrolase remains intact, although some increase in chain mobility can be noted with increased temperature. Upon melting, numerous signals collapse as the protein unfolds. Transition temperatures (Tm) as measured by CD and NMR are in good agreement. Similar structural changes can be induced by adding zinc chelators such as EDTA and DTT. This leads to complete loss of activity at EDTA concentrations above 1.0 mM.(ABSTRACT TRUNCATED AT 400 WORDS)
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effect of zinc binding on the structure and stability of Fibrolase a fibrinolytic protein from snake venom
Pharmaceutical Research, 1992Co-Authors: D Pretzer, B Schulteis, C D Smith, James W Mitchell, Mark C ManningAbstract:Fibrolase is a metalloprotease with potential use as a fibrinolytic agent. Loss of the intrinsic zinc atom leads to a rapid decrease in enzymatic activity. Circular dichroism measurements indicate that there is a partial unfolding of an α-helical section of the protein concomitant with the loss of zinc. Removal of zinc can be affected by elevated temperatures, acidic pH values, and addition of chelating agents. At low molar concentrations, both ethylenediaminetet-raacetic acid (EDTA) and dithiothreitol (DTT) were found to remove zinc efficiently. Analysis of the sequence of Fibrolase identified a segment which possessed a high degree of homology with the metal binding site of other zinc proteases, such as thermolysin and the collagenases. However, the putative zinc binding site in Fibrolase lacks the additional glutamate ligand found in thermolysin and subtilisin. This sequence is also predicted to adopt an α-helical conformation. Together, these data indicate that there is a well-defined metal binding site in Fibrolase and that metal binding is the most important factor governing the stability of this protein.
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Stability of the Thrombolytic Protein Fibrolase: Effect of Temperature and pH on Activity and Conformation
Pharmaceutical Research, 1991Co-Authors: D Pretzer, James W Mitchell, Brenda S. Schulteis, Christopher D. Smith, David G. Vander Velde, Mark C ManningAbstract:The effect of temperature and pH on the activity and conformation of the thrombolytic protein Fibrolase was examined. Fibrolase maintained proteolytic activity over 10 days at room temperature (∼22°C). At 37°C, greater than 50% of the proteolytic activity was lost within 2 days and no activity remained after 10 days. Circular dichroism (CD) spectra at elevated temperatures showed that alphahelical structure was lost in a cooperative transition ( T _m of 50°C at pH 8). Structural changes were detected by NMR prior to unfolding which were not observable by CD, and the T _m determined by NMR was 46°C at pD 8. The effect of pH on the proteolytic activity and structure of Fibrolase was examined over the pH range from 1 to 10. Activity was maintained at neutral to alkaline pH values from pH 6.5 to pH 10.0 but decreased substantially in acidic media. While CD spectra indicated little variation in secondary structure over the pH range 5 to 9, significant differences were noted at pH 2 to 3. The melting temperature of Fibrolase decreased to 43°C at pH 5. Protein concentrations determined over the pH range 1 to 10 showed an apparent solubility minimum at pH 5.0, which did not correspond to the isoelectric point of 6.5. Explanations for these observations are proposed.
Steve Swenson - One of the best experts on this subject based on the ideXlab platform.
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Fibrolase trials and tribulations
Toxins, 2010Co-Authors: Francis S Markland, Steve SwensonAbstract:Fibrolase is the fibrinolytic enzyme isolated from Agkistrodon contortrix contortrix (southern copperhead snake) venom. The enzyme was purified by a three-step HPLC procedure and was shown to be homogeneous by standard criteria including reverse phase HPLC, molecular sieve chromatography and SDS-PAGE. The purified enzyme is a zinc metalloproteinase containing one mole of zinc. It is composed of 203 amino acids with a blocked amino-terminus due to cyclization of the terminal Gln residue. Fibrolase shares a significant degree of homology with enzymes of the reprolysin sub-family of metalloproteinases including an active site homology of close to 100%; it is rapidly inhibited by chelating agents such as EDTA, and by alpha2-macroglobulin (α2Μ). The enzyme is a direct-acting thrombolytic agent and does not rely on plasminogen for clot dissolution. Fibrolase rapidly cleaves the A(α)-chain of fibrinogen and the B(β)-chain at a slower rate; it has no activity on the γ-chain. The enzyme exhibits the same specificity with fibrin, cleaving the α-chain more rapidly than the β-chain. Fibrolase was shown to have very effective thrombolytic activity in a reoccluding carotid arterial thrombosis model in the canine. A recombinant version of the enzyme was made in yeast by Amgen, Inc. (Thousand Oaks, CA, USA) and called alfimeprase. Alfimeprase is identical to Fibrolase except for a two amino acid truncation at the amino-terminus and the insertion of a new amino-terminal amino acid in the truncated protein; these changes lead to a more stable enzyme for prolonged storage. Alfimeprase was taken into clinical trials by Nuvelo, Inc. (San Carlos, CA), which licensed the enzyme from Amgen. Alfimeprase was successful in Phase I and II clinical trials for peripheral arterial occlusion (PAO) and central venous access device (CVAD) occlusion. However, in Phase III trials alfimeprase did not meet the expected end points in either PAO or CVAD occlusion and in a Phaase II stroke trial, and Nuvelo dropped further development in 2008.
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Three-Dimensional Structure of Fibrolase, the Fibrinolytic Enzyme from Southern Copperhead Venom, Modeled from the X-Ray Structure of Adamalysin
2008Co-Authors: Atrolysin C, Michael B. Bolger, Steve Swenson, Francis S. MarklAbstract:ABSTRACT The fibrinolytic enzyme from southern copperhead snake venom, Fibrolase, contains 1 mole of zinc per mole of protein, belongs to the major family of metalloproteinases known as the metzincins, and has been shown to degrade fibrin clots in vitro and in vivo. The purpose of this study was to develop a 3-dimensional model of Fibrolase to investigate the geometry of conserved and variable sequences between members of the snake venom metalloproteinases. When compared to atrolysin C (form D) or adamalysin II (metzincins with completely different substrate specificity), Fibrolase has approximately 60 % overall sequence identity and nearly 100 % sequence similarity in the active site. We used the crystal structure of adamalysin II to build a 3-dimensional homology model of Fibrolase. Three disulfide bonds were constructed (the highly conserved disulfide bond [118-198] was maintained from the adamalysin II structure and 2 new disulfide bonds were introduced between residues 158-182 and 160-165). We used Sculpt 2.5 and HyperChem 5.0 to "dock " a substrate fragment octapeptide (HTEKLVTS), and a water molecule into the active site cleft. We calculated the differential average homology profile for Fibrolase compared to 8 hemorrhagic and 5 nonhemorrhagic metzincins. We then determined the sequence regions that might be responsible for their substrate specificity. Our 3dimensional homology model shows that the variable sequences lie on the periphery of the identified active site region containing the His triangle; this indicates that substrate specificity may depend on surface residues that are not directly associated with the active site
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snake venom fibrin ogen olytic enzymes
Toxicon, 2005Co-Authors: Steve Swenson, Francis S MarklandAbstract:Abstract Snake venoms contain a number of serine and metalloproteinases and included among these are the fibrin(ogen)olytic proteinases. Some years ago it was postulated that the fibrin(ogen)olytic enzymes may be clinically useful. Over the past 150 years a substantial body of literature has been generated on the identification and characterization of fibrin(ogen)olytic enzymes from a broad spectrum of snake species. In this review we describe the two different classes of fibrin(ogen)olytic enzymes isolated from snake venom and we summarize a number of studies aimed at characterizing the purified enzymes and/or their derivatives. Two distinct classes of venom fibrin(ogen)olytic enzymes have been previously identified, the metalloproteinases and serine proteinases. These two classes of proteinases differ in their mechanism of action and they target different amino acid sequences in fibrin(ogen), but each perform the same role in nature. When a snake envenomates its prey it needs a mechanism to facilitate the spread of the toxic components throughout the circulation. Fibrin(ogen)olytic enzymes break down fibrin rich clots and help to prevent further clot formation by their action on fibrinogen. This characteristic feature has led to development of fibrin(ogen)olytic snake venom enzymes as potential clinical agents to treat occlusive thrombi. Fibrolase, a fibrinolytic metalloproteinase isolated from Agkistrodon contortrix contortrix venom and the serine β-fibrinogenolytic proteinase from Vipera lebetina have been chosen as representative enzymes from the two classes, and their biochemical and physiochemical properties will be described in detail. Finally, the characterization and development of alfimeprase, a recombinant fibrinolytic enzyme derived from Fibrolase, as a clinical agent is described citing the progression from the laboratory bench to its current status as having successfully completed Phase II clinical trials.
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Three-dimensional structure of Fibrolase, the fibrinolytic enzyme from southern copperhead venom, modeled from the x-ray structure of adamalysin II and atrolysin C
AAPS PharmSci, 2001Co-Authors: Michael B. Bolger, Steve Swenson, Francis S MarklandAbstract:The fibrinolytic enzyme from southern copperhead snake venom, Fibrolase, contains 1 mole of zine per mole of protein, belongs to the major family of metalloproteinases known as the metzincins, and has been shown to degrade fibrin clots in vitro and in vivo. The purpose of this study was to develop a 3-dimensional model of Fibrolase to investigate the geometry of conserved and variable sequences between members of the snake venom metalloproteinases. When compared to atrolysin C (form D) or adamalysin II (metzincins with completely different substrate specificity), Fibrolase has approximately 60% overall sequence identity and nearly 100% sequence similarity in the active site. We used the crystal structure of adamalysin II to build a 3-dimensional homology model of Fibrolase. Three disulfide bonds were constructed (the highly conserved disulfide bond [118–198] was maintained from the adamalysin II structure and 2 new disulfide bonds were introduced between residues 158–182 and 160–165). We used Sculpt 2.5 and HyperChem 5.0 to “dock” a substrate fragment octapeptide (HTEKLVTS), and a water molecule into the active site cleft. We calculated the differential average homology profile for Fibrolase compared to 8 hemorrhagic and 5 nonhemorrhagic metzincins. We then determined the sequence regions that might be responsible for their substrate specificity. Our 3-dimensional homology model shows that the variable sequences lie on the periphery of the identified active site region containing the His triangle; this indicates that substrate specificity may depend on surface residues that are not directly associated with the active site.
D Pretzer - One of the best experts on this subject based on the ideXlab platform.
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Fibrolase a fibrinolytic protein from snake venom
Pharmaceutical biotechnology, 1993Co-Authors: D Pretzer, B Schulteis, C D Smith, James W Mitchell, Mark C ManningAbstract:Fibrolase is an active fibrinolytic agent and possesses potential for use in thrombolytic therapy. Its mode of action had been characterized, both in vitro and in vivo. Possessing three disulfide bonds, native Fibrolase is nonglycosylated and binds an intrinsic zinc atom. The zinc is essential for retention of activity and structural integrity. In solution, Fibrolase is sensitive to changes in pH and temperature (Pretzer et al., 1991). At neutral to basic pH (pH 5-9), the solubility and stability of Fibrolase is nearly constant. Little structural variation can be detected by CD spectroscopy. However, decrease in pH below 5 leads to a pronounced reduction in both the solubility and activity of Fibrolase. At pH 3 and below, the solubility of Fibrolase returns but the activity does not. This solubility profile is unusual in that the minimal solubility is well removed from the pI (which is 6.7). It is proposed that the behavior of Fibrolase with variation in pH can be understood in terms of capacity to bind zinc. At pH 5 to 9, the protein binds zinc and the structure and activity are preserved. Near pH 5, the histidine residues which serve as ligands for the zinc become protonated and zinc binding is lost. Loss of zinc leads to local unfolding of a helical segment of Fibrolase, exposing hydrophobic groups which allow the protein to rapidly aggregate. At lower pH values (1-3), the protein again adopts a more globular structure, similar to molten globule states, and the solubility increases. However, without the zinc, Fibrolase remains inactive. Changes in pH also affect thermal stability. The Tm for Fibrolase moves from 50 degrees C at pH 8 to 43 degrees C at pH 5. Increases in temperature also lead to removal of the zinc ion, again producing a partially denatured protein with a marked tendency to aggregate. In both cases (decrease in pH and increase in temperature), analysis of the CD spectra indicates that the protein has primarily lost alpha-helical secondary structure. A major change in structure can also be observed using NMR spectroscopy. At temperatures below 35 degrees C, the globular structure of Fibrolase remains intact, although some increase in chain mobility can be noted with increased temperature. Upon melting, numerous signals collapse as the protein unfolds. Transition temperatures (Tm) as measured by CD and NMR are in good agreement. Similar structural changes can be induced by adding zinc chelators such as EDTA and DTT. This leads to complete loss of activity at EDTA concentrations above 1.0 mM.(ABSTRACT TRUNCATED AT 400 WORDS)
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effect of zinc binding on the structure and stability of Fibrolase a fibrinolytic protein from snake venom
Pharmaceutical Research, 1992Co-Authors: D Pretzer, B Schulteis, C D Smith, James W Mitchell, Mark C ManningAbstract:Fibrolase is a metalloprotease with potential use as a fibrinolytic agent. Loss of the intrinsic zinc atom leads to a rapid decrease in enzymatic activity. Circular dichroism measurements indicate that there is a partial unfolding of an α-helical section of the protein concomitant with the loss of zinc. Removal of zinc can be affected by elevated temperatures, acidic pH values, and addition of chelating agents. At low molar concentrations, both ethylenediaminetet-raacetic acid (EDTA) and dithiothreitol (DTT) were found to remove zinc efficiently. Analysis of the sequence of Fibrolase identified a segment which possessed a high degree of homology with the metal binding site of other zinc proteases, such as thermolysin and the collagenases. However, the putative zinc binding site in Fibrolase lacks the additional glutamate ligand found in thermolysin and subtilisin. This sequence is also predicted to adopt an α-helical conformation. Together, these data indicate that there is a well-defined metal binding site in Fibrolase and that metal binding is the most important factor governing the stability of this protein.
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Stability of the Thrombolytic Protein Fibrolase: Effect of Temperature and pH on Activity and Conformation
Pharmaceutical Research, 1991Co-Authors: D Pretzer, James W Mitchell, Brenda S. Schulteis, Christopher D. Smith, David G. Vander Velde, Mark C ManningAbstract:The effect of temperature and pH on the activity and conformation of the thrombolytic protein Fibrolase was examined. Fibrolase maintained proteolytic activity over 10 days at room temperature (∼22°C). At 37°C, greater than 50% of the proteolytic activity was lost within 2 days and no activity remained after 10 days. Circular dichroism (CD) spectra at elevated temperatures showed that alphahelical structure was lost in a cooperative transition ( T _m of 50°C at pH 8). Structural changes were detected by NMR prior to unfolding which were not observable by CD, and the T _m determined by NMR was 46°C at pD 8. The effect of pH on the proteolytic activity and structure of Fibrolase was examined over the pH range from 1 to 10. Activity was maintained at neutral to alkaline pH values from pH 6.5 to pH 10.0 but decreased substantially in acidic media. While CD spectra indicated little variation in secondary structure over the pH range 5 to 9, significant differences were noted at pH 2 to 3. The melting temperature of Fibrolase decreased to 43°C at pH 5. Protein concentrations determined over the pH range 1 to 10 showed an apparent solubility minimum at pH 5.0, which did not correspond to the isoelectric point of 6.5. Explanations for these observations are proposed.
Alex L. Guan - One of the best experts on this subject based on the ideXlab platform.
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Immunological properties of the fibrinolytic enzyme (Fibrolase) from southern copperhead (Agkistrodon contortrix contortrix) venom and its purification by immunoaffinity chromatography
Toxicon, 1991Co-Authors: Alex L. Guan, Francis S MarklandAbstract:H. M. Chen, A. L. Guan and F. S. Markland, Jr. Immunological properties of the fibrinolytic enzyme (Fibrolase) from southern copperhead (Agkistrodon contortrix contortrix) venom and its purification by immunoaffinity chromatography. Toxicon29, 683–694.—An antibody to the fibrinolytic enzyme in southern copperhead venom was produced by immunizing rabbits with chromatographically purified enzyme. The antibody was purified from rabbit blood by ammonium sulfate fractionation and protein-A affinity chromatography. The purified antibody reacted only with the fibrinolytic enzyme in southern copperhead venom as demonstrated by immunodiffusion and immunoelectrophoresis. Western immunoblotting revealed that several snake venoms, including Agkistrodon piscivorus conanti, Crotalus atrox, Crotalus basiliscus basiliscus, and Bothrops asper, cross-reacted with the antibody to varying degrees. However, Deinagkistrodon acutus showed no cross-reaction. Immobilized antibody has been used, in combination with molecular sieve chromatography, to purify the fibrinolytic enzyme from southern copperhead venom. In this two-step purification procedure, the enzyme was purified in good yield within two days. The specific activity of the enzyme purified by the immunoaffinity chromatography procedure is comparable with that of enzyme purified by a four-step chromatographic procedure. The mol. wt of the purified enzyme is approximately 23,000–24,000 as determined by SDS-PAGE. Interestingly, the enzyme purified by this two-step immunoaffinity chromatography procedure possesses virtually no hemorrhagic activity.