The Experts below are selected from a list of 213 Experts worldwide ranked by ideXlab platform
Long-sen Chang - One of the best experts on this subject based on the ideXlab platform.
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Blocking of negative charged carboxyl groups converts Naja atra neurotoxin to cardiotoxin-like protein.
International journal of biological macromolecules, 2020Co-Authors: Yi-jun Shi, Jing-ting Chiou, Liang-jun Wang, Chia-hui Huang, Yuan-chin Lee, Ying-jung Chen, Long-sen ChangAbstract:Naja atra Cobrotoxin and cardiotoxin 3 (CTX3) exhibit neurotoxicity and cytotoxicity, respectively. In the present study, we aimed to investigate whether the carboxyl groups of Cobrotoxin play a role in structural constraints, thereby preventing Cobrotoxin from exhibiting cytotoxic activity. Six of the seven carboxyl groups in Cobrotoxin were conjugated with semicarbazide. Measurement of circular dichroism spectra and Trp fluorescence quenching showed that the gross conformation of semicarbazide-modified Cobrotoxin (SEM-Cobrotoxin) and Cobrotoxin differed. In sharp contrast to Cobrotoxin, SEM-Cobrotoxin demonstrated membrane-damaging activity and cytotoxicity, which are feature more characteristic of CTX3. Furthermore, both SEM-Cobrotoxin and CTX3 induced cell death through AMPK activation. Analyses of the interaction between polydiacetylene/lipid vesicles and fluorescence-labeled lipids revealed that SEM-Cobrotoxin and Cobrotoxin adopted different membrane-bound states. The structural characteristics of SEM-Cobrotoxin were similar to those of CTX3, including trifluoroethanol (TFE)-induced structural transformation and membrane binding-induced conformational change. Conversely, Cobrotoxin was insensitive to the TFE-induced effect. Collectively, the data of this study indicate that blocking negatively charged residues confers Cobrotoxin with membrane-damaging activity and cytotoxicity. The findings also suggest that the structural constraints imposed by carboxyl groups control the functional properties of snake venom α-neurotoxins during the divergent evolution of snake venom neurotoxins and cardiotoxins.
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Role of amino and carboxyl groups of Cobrotoxin in the conformational stability and the interaction with acetylcholine receptor
International Journal of Peptide and Protein Research, 2009Co-Authors: Long-sen Chang, Chun-chang ChangAbstract:: To study the functional involvements of the common interaction of the Leu-1 alpha-amino group and Asp-58 in Cobrotoxin, the lysine epsilon-amino groups of Cobrotoxin were initially guanidinated with o-methylisourea. The alpha-amino group of Leu-1 was them modified with TNBS after the guanidination of Cobrotoxin. Both modified derivatives displayed no significant changes in the secondary structure and antigenicity of Cobrotoxin, whereas the binding affinity for nicotinic acetylcholine receptor (nAChR) was pronouncedly decreased when Leu-1 was modified. Six out of seven free carboxyl groups and the remaining buried Glu-21 carboxyl group of Cobrotoxin were modified with glycine methyl ester in the absence and presence of guanidine HCl, respectively. Alternation in the beta-sheet secondary structure of Cobrotoxin was observed with the carboxyl-group modified derivatives, which caused a decrease in the binding activity of the toxin molecule to the antibody and nAChR. Moreover, modification of the Glu-21 carboxyl group of Cobrotoxin further reduced the nAChR binding activity, while the antigenicity remained unchange. Thus, our results conclude that the Glu-21 residue and the common interaction of the terminal Leu-1 alpha-amino group and the Asp-58 carboxyl group are related to the nAChR-binding activity of Cobrotoxin, and the free carboxyl groups in Cobrotoxin are conformation-essential.
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Enrichment of the antibodies against the C-terminus of Taiwan cobra Cobrotoxin using dimeric glutaraldehyde-modified toxin as an immunogen.
Toxicon : official journal of the International Society on Toxinology, 2003Co-Authors: Long-sen Chang, Rose Lin, Ku Chung Chen, Chun-chang ChangAbstract:The repertoire of antibodies producing by immunizing rabbits with Cobrotoxin and dimeric glutaraldehyde-modified Cobrotoxin (dGA-Cobrotoxin) was analyzed by studying the immunoreactivity of the two antibody preparations toward Cobrotoxin, GA-Cobrotoxin and recombinant Cobrotoxin. The results of enzyme-linked immunoassay revealed that the two antibody preparations exhibited a higher reactivity against their cognate antigen. Moreover, different behavior was observed for the reactivity of the two antibody preparations against GA-Cobrotoxin and recombinant Cobrotoxin. Notably, distortion of disulfide linkages at the C-terminus resulted in a reduced decrease in the antigenic activity of recombinant Cobrotoxin toward anti-Cobrotoxin antibodies compared to anti-dGA-Cobrotoxin antibodies. Affinity purification of the antibodies against the C-terminus of Cobrotoxin revealed that its amount represented 77% and 35.5% of the total anti-dGA-Cobrotoxin antibodies and the total anti-Cobrotoxin antibodies, respectively. These findings suggest that the antibody preparation elicited by dGA-Cobrotoxin enriches the content of antibodies recognizes the C-terminal region of native Cobrotoxin.
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glutaraldehyde cross linking alters the environment around trp29 of Cobrotoxin and the pathway for regaining its fine structure during refolding
Journal of Peptide Research, 2001Co-Authors: Long-sen Chang, Shinne-ren Lin, Chen-chung YangAbstract:Cobrotoxin, purified from the venom of Naja naja atra (Taiwan cobra), was subjected to modification with glutaraldehyde in order to prepare intra- and intermolecule cross-linked derivatives. Monomeric and dimeric derivatives were separated from polymeric derivatives by gel filtration. The results of amino acid analysis and sequence determination revealed that only Lys residues were selectively modified by glutaraldehyde. Glutaraldehyde cross-linking was accompanied by a change in the gross conformation of Cobrotoxin as revealed by circular dichroism spectra of the modified derivatives. Compared with Cobrotoxin, Trp(29) of monomeric and dimeric derivatives was in an apolar microenvironment. This was in agreement with acrylamide quenching studies showing that the spatial position of the Trp indole ring became buried in the interior of the molecule after glutaraldehyde cross-linking. Moreover, the Trp of modified derivatives was less accessible for iodide than that observed with Cobrotoxin. Notably, disulfide reduction could not completely unfold the structure of glutaraldehyde-modified derivatives as evidenced by the results of acrylamide quenching studies and enzyme-linked immunoassay. Study of the characteristic changes in Trp fluorescence after the initiation of refolding suggested that the fine structure around Trp(29) of Cobrotoxin and glutaraldehyde-modified derivatives was formed differently. These results suggest that glutaraldehyde cross-linking leads to a change in the microenvironment of Cobrotoxin Trp(29) and alters the pathway of its fine structure formation during the refolding of Cobrotoxin.
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Refolding of Taiwan cobra neurotoxin: intramolecular cross-link affects its refolding reaction.
Archives of biochemistry and biophysics, 2001Co-Authors: Long-sen Chang, Shinne-ren Lin, Chen-chung YangAbstract:Abstract In order to explore the effect of intramolecular cross-linking in the folding reaction of Cobrotoxin from Naja naja atra (Taiwan cobra) venom, the toxin molecule was modified with glutaraldehyde (GA). The monomeric GA-modified Cobrotoxin (mGA-Cobrotoxin) was separated from the dimeric and trimeric derivatives using gel filtration. The results of electrophoretic and chromatographic analyses revealed that mGA-Cobrotoxin comprised two modified derivatives, which contained modified Lys residues at positions 26 and 27 and at positions 26, 27, and 47, respectively. Moreover, an intramolecular cross-linking of loops II and III by Lys residues was noted with the monomeric derivative containing three modified Lys residues. In sharp contrast to Cobrotoxin observations, the folding rate of mGA-Cobrotoxin decreased in the presence of GSH/GSSG, but notably increased in the absence of thiol compounds. Particularly, the accelerated effect of GSH/GSSG on the refolding reaction was affected by the presence of the intramolecular cross-link. Comparative analyses on Cobrotoxin and mGA-Cobrotoxin CD spectra revealed that modification with the GA reagent caused a change in the gross conformation of Cobrotoxin. Fluorescence measurement revealed that the stability of the microenvironment around the single Trp-29 in mGA-Cobrotoxin and unfolded mGA-Cobrotoxin was appreciably higher than in Cobrotoxin and unfolded toxin. Moreover, the ordered structure formation around Trp-29 in refolded mGA-Cobrotoxin was faster than in refolded Cobrotoxin as evidenced by fluorescence quenching studies. Taken together, these results suggest that the structural flexibility of unfolded Cobrotoxin should be favorable for the thiol catalyst to exert its action in the refolding reaction and that the Cobrotoxin refold kinetics may change after modification with GA.
Chun-chang Chang - One of the best experts on this subject based on the ideXlab platform.
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Role of amino and carboxyl groups of Cobrotoxin in the conformational stability and the interaction with acetylcholine receptor
International Journal of Peptide and Protein Research, 2009Co-Authors: Long-sen Chang, Chun-chang ChangAbstract:: To study the functional involvements of the common interaction of the Leu-1 alpha-amino group and Asp-58 in Cobrotoxin, the lysine epsilon-amino groups of Cobrotoxin were initially guanidinated with o-methylisourea. The alpha-amino group of Leu-1 was them modified with TNBS after the guanidination of Cobrotoxin. Both modified derivatives displayed no significant changes in the secondary structure and antigenicity of Cobrotoxin, whereas the binding affinity for nicotinic acetylcholine receptor (nAChR) was pronouncedly decreased when Leu-1 was modified. Six out of seven free carboxyl groups and the remaining buried Glu-21 carboxyl group of Cobrotoxin were modified with glycine methyl ester in the absence and presence of guanidine HCl, respectively. Alternation in the beta-sheet secondary structure of Cobrotoxin was observed with the carboxyl-group modified derivatives, which caused a decrease in the binding activity of the toxin molecule to the antibody and nAChR. Moreover, modification of the Glu-21 carboxyl group of Cobrotoxin further reduced the nAChR binding activity, while the antigenicity remained unchange. Thus, our results conclude that the Glu-21 residue and the common interaction of the terminal Leu-1 alpha-amino group and the Asp-58 carboxyl group are related to the nAChR-binding activity of Cobrotoxin, and the free carboxyl groups in Cobrotoxin are conformation-essential.
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Enrichment of the antibodies against the C-terminus of Taiwan cobra Cobrotoxin using dimeric glutaraldehyde-modified toxin as an immunogen.
Toxicon : official journal of the International Society on Toxinology, 2003Co-Authors: Long-sen Chang, Rose Lin, Ku Chung Chen, Chun-chang ChangAbstract:The repertoire of antibodies producing by immunizing rabbits with Cobrotoxin and dimeric glutaraldehyde-modified Cobrotoxin (dGA-Cobrotoxin) was analyzed by studying the immunoreactivity of the two antibody preparations toward Cobrotoxin, GA-Cobrotoxin and recombinant Cobrotoxin. The results of enzyme-linked immunoassay revealed that the two antibody preparations exhibited a higher reactivity against their cognate antigen. Moreover, different behavior was observed for the reactivity of the two antibody preparations against GA-Cobrotoxin and recombinant Cobrotoxin. Notably, distortion of disulfide linkages at the C-terminus resulted in a reduced decrease in the antigenic activity of recombinant Cobrotoxin toward anti-Cobrotoxin antibodies compared to anti-dGA-Cobrotoxin antibodies. Affinity purification of the antibodies against the C-terminus of Cobrotoxin revealed that its amount represented 77% and 35.5% of the total anti-dGA-Cobrotoxin antibodies and the total anti-Cobrotoxin antibodies, respectively. These findings suggest that the antibody preparation elicited by dGA-Cobrotoxin enriches the content of antibodies recognizes the C-terminal region of native Cobrotoxin.
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Disulfide Isomerization within the C-Terminus of Cobrotoxin Decelerates by Thiol Compounds and Trinitrophenylation, but Accelerates by Modification of Carboxyl Groups☆
Archives of biochemistry and biophysics, 1998Co-Authors: Long-sen Chang, Chun-chang Chang, Shinne-ren Lin, Chen-chung YangAbstract:A disulfide isomerization at the C-terminus of Cobrotoxin occurred spontaneously by dissolving in alkali buffer. Irreversible conversion of Cobrotoxin into its isomers was completely achieved within 4 days. The isomerization reaction was decelerated by thiol compounds including GSSG, GSH, cystamine, and cysteamine in a pseudo-first-order kinetic, and GSSG was the most effective one among the thiol compounds used. Moreover, the oxidized thiol compounds were always superior to reduced ones in decreasing the rate of disulfide interchange. To further assess the intrinsic elements essential for the occurrence of disulfide isomerization of Cobrotoxin, the toxin molecule was subjected to modification on its Arg, Lys, Trp, Tyr, and carboxyl groups. In sharp contrast to other modified derivatives, the isomerization reaction was decelerated by trinitrophenylation on Lys-26, Lys-27, and Lys-47, whereas it was rapidly completed after modification of carboxyl groups. Neither chemical modification nor the toxin's conformation affected the irreversibility of isomerization reaction. Thus, the observed change in the rate of disulfide isomerization reflects the involvement of Lys residues and carboxyl groups in this reaction. Although thiol compounds further decelerated the conversion of trinitrophenylated Cobrotoxin into its isomers, they did not exert a notable effect on the isomerization of carboxyl groups-modified derivative. These results clearly indicate that disulfide isomerization of Cobrotoxin is, in part, driven by the positively charged Lys residues at positions 26, 27, and 47 of the toxin molecule, and that the thiol compounds are coordinated with the negatively charged groups of Cobrotoxin to exert their inhibitory action.
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Unfolding/Folding Studies on Cobrotoxin from Taiwan Cobra Venom: pH and GSH/GSSG Govern Disulfide Isomerization at the C-Terminus
Archives of biochemistry and biophysics, 1998Co-Authors: Long-sen Chang, Shinne-ren Lin, Chun-chang ChangAbstract:Refolding of Cobrotoxin was assessed by the exposure degree of its single Trp determined by an acrylamide quenching study. The change in the accessibility of Trp for acrylamide quantitatively reflected the formation of folded Cobrotoxin, and the data were confirmed by HPLC and gel electrophoresis analyses. However, the site-specific information provided by quenching Trp fluorescence revealed that the ordered structure in the neighborhood of Trp was attained prior to the complete formation of the tertiary structure of Cobrotoxin. HPLC analyses showed that, in addition to refolded Cobrotoxin, two novel species (Cobrotoxin II and Cobrotoxin III) with isomerization of disulfide bonds at the C-terminus of the toxin molecule were produced along the folding reaction. The disulfide pairings in Cobrotoxin II and Cobrotoxin III were Cys43-Cys55 and Cys54-Cys60 and Cys43-Cys60 and Cys54-Cys55, respectively. Among the three possible two-disulfide species at the C-terminus, the disulfide linkages Cys43-Cys60 and Cys54-Cys55 of Cobrotoxin III caused a marked decrease in lethality and resulted in a conformation which was notably different from that observed with the native toxin molecule as evidenced by CD spectra. The refolding reaction was accelerated by the addition of GSH/GSSG, and the resulting products were mostly folded Cobrotoxin. However, if GSH/GSSG was not added into the initial folding materials, the yields of Cobrotoxin II and Cobrotoxin III greatly increased. The conversion of Cobrotoxin to its isomers was to be irreversible and pH-dependent: the higher the pH, the faster the rate of conversion. However, this conversion could be partly inhibited by GSH/GSSG. Cobrotoxin II and Cobrotoxin III were purified from Taiwan cobra venom as well, and their yields in comparison to that of Cobrotoxin in venom were similar to that noted with the folded products in the presence of GSH/GSSG. Moreover, the rate of disulfide isomerization was expected to be slow in venom fluid in which the pH was approximately pH 6.2. Thus, the finding that Cobrotoxin represents the predominant neurotoxin species in Taiwan cobra venom is probably associated with the synergistic effects of GSH/GSSG and pH.
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unfolding folding studies on Cobrotoxin from taiwan cobra venom ph and gsh gssg govern disulfide isomerization at the c terminus
Archives of Biochemistry and Biophysics, 1998Co-Authors: Long-sen Chang, Shinne-ren Lin, Chun-chang ChangAbstract:Refolding of Cobrotoxin was assessed by the exposure degree of its single Trp determined by an acrylamide quenching study. The change in the accessibility of Trp for acrylamide quantitatively reflected the formation of folded Cobrotoxin, and the data were confirmed by HPLC and gel electrophoresis analyses. However, the site-specific information provided by quenching Trp fluorescence revealed that the ordered structure in the neighborhood of Trp was attained prior to the complete formation of the tertiary structure of Cobrotoxin. HPLC analyses showed that, in addition to refolded Cobrotoxin, two novel species (Cobrotoxin II and Cobrotoxin III) with isomerization of disulfide bonds at the C-terminus of the toxin molecule were produced along the folding reaction. The disulfide pairings in Cobrotoxin II and Cobrotoxin III were Cys43-Cys55 and Cys54-Cys60 and Cys43-Cys60 and Cys54-Cys55, respectively. Among the three possible two-disulfide species at the C-terminus, the disulfide linkages Cys43-Cys60 and Cys54-Cys55 of Cobrotoxin III caused a marked decrease in lethality and resulted in a conformation which was notably different from that observed with the native toxin molecule as evidenced by CD spectra. The refolding reaction was accelerated by the addition of GSH/GSSG, and the resulting products were mostly folded Cobrotoxin. However, if GSH/GSSG was not added into the initial folding materials, the yields of Cobrotoxin II and Cobrotoxin III greatly increased. The conversion of Cobrotoxin to its isomers was to be irreversible and pH-dependent: the higher the pH, the faster the rate of conversion. However, this conversion could be partly inhibited by GSH/GSSG. Cobrotoxin II and Cobrotoxin III were purified from Taiwan cobra venom as well, and their yields in comparison to that of Cobrotoxin in venom were similar to that noted with the folded products in the presence of GSH/GSSG. Moreover, the rate of disulfide isomerization was expected to be slow in venom fluid in which the pH was approximately pH 6.2. Thus, the finding that Cobrotoxin represents the predominant neurotoxin species in Taiwan cobra venom is probably associated with the synergistic effects of GSH/GSSG and pH.
Shinne-ren Lin - One of the best experts on this subject based on the ideXlab platform.
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glutaraldehyde cross linking alters the environment around trp29 of Cobrotoxin and the pathway for regaining its fine structure during refolding
Journal of Peptide Research, 2001Co-Authors: Long-sen Chang, Shinne-ren Lin, Chen-chung YangAbstract:Cobrotoxin, purified from the venom of Naja naja atra (Taiwan cobra), was subjected to modification with glutaraldehyde in order to prepare intra- and intermolecule cross-linked derivatives. Monomeric and dimeric derivatives were separated from polymeric derivatives by gel filtration. The results of amino acid analysis and sequence determination revealed that only Lys residues were selectively modified by glutaraldehyde. Glutaraldehyde cross-linking was accompanied by a change in the gross conformation of Cobrotoxin as revealed by circular dichroism spectra of the modified derivatives. Compared with Cobrotoxin, Trp(29) of monomeric and dimeric derivatives was in an apolar microenvironment. This was in agreement with acrylamide quenching studies showing that the spatial position of the Trp indole ring became buried in the interior of the molecule after glutaraldehyde cross-linking. Moreover, the Trp of modified derivatives was less accessible for iodide than that observed with Cobrotoxin. Notably, disulfide reduction could not completely unfold the structure of glutaraldehyde-modified derivatives as evidenced by the results of acrylamide quenching studies and enzyme-linked immunoassay. Study of the characteristic changes in Trp fluorescence after the initiation of refolding suggested that the fine structure around Trp(29) of Cobrotoxin and glutaraldehyde-modified derivatives was formed differently. These results suggest that glutaraldehyde cross-linking leads to a change in the microenvironment of Cobrotoxin Trp(29) and alters the pathway of its fine structure formation during the refolding of Cobrotoxin.
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Refolding of Taiwan cobra neurotoxin: intramolecular cross-link affects its refolding reaction.
Archives of biochemistry and biophysics, 2001Co-Authors: Long-sen Chang, Shinne-ren Lin, Chen-chung YangAbstract:Abstract In order to explore the effect of intramolecular cross-linking in the folding reaction of Cobrotoxin from Naja naja atra (Taiwan cobra) venom, the toxin molecule was modified with glutaraldehyde (GA). The monomeric GA-modified Cobrotoxin (mGA-Cobrotoxin) was separated from the dimeric and trimeric derivatives using gel filtration. The results of electrophoretic and chromatographic analyses revealed that mGA-Cobrotoxin comprised two modified derivatives, which contained modified Lys residues at positions 26 and 27 and at positions 26, 27, and 47, respectively. Moreover, an intramolecular cross-linking of loops II and III by Lys residues was noted with the monomeric derivative containing three modified Lys residues. In sharp contrast to Cobrotoxin observations, the folding rate of mGA-Cobrotoxin decreased in the presence of GSH/GSSG, but notably increased in the absence of thiol compounds. Particularly, the accelerated effect of GSH/GSSG on the refolding reaction was affected by the presence of the intramolecular cross-link. Comparative analyses on Cobrotoxin and mGA-Cobrotoxin CD spectra revealed that modification with the GA reagent caused a change in the gross conformation of Cobrotoxin. Fluorescence measurement revealed that the stability of the microenvironment around the single Trp-29 in mGA-Cobrotoxin and unfolded mGA-Cobrotoxin was appreciably higher than in Cobrotoxin and unfolded toxin. Moreover, the ordered structure formation around Trp-29 in refolded mGA-Cobrotoxin was faster than in refolded Cobrotoxin as evidenced by fluorescence quenching studies. Taken together, these results suggest that the structural flexibility of unfolded Cobrotoxin should be favorable for the thiol catalyst to exert its action in the refolding reaction and that the Cobrotoxin refold kinetics may change after modification with GA.
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Glutaraldehyde cross‐linking alters the environment around Trp29 of Cobrotoxin and the pathway for regaining its fine structure during refolding
The journal of peptide research : official journal of the American Peptide Society, 2001Co-Authors: Long-sen Chang, Shinne-ren Lin, Chen-chung YangAbstract:Cobrotoxin, purified from the venom of Naja naja atra (Taiwan cobra), was subjected to modification with glutaraldehyde in order to prepare intra- and intermolecule cross-linked derivatives. Monomeric and dimeric derivatives were separated from polymeric derivatives by gel filtration. The results of amino acid analysis and sequence determination revealed that only Lys residues were selectively modified by glutaraldehyde. Glutaraldehyde cross-linking was accompanied by a change in the gross conformation of Cobrotoxin as revealed by circular dichroism spectra of the modified derivatives. Compared with Cobrotoxin, Trp(29) of monomeric and dimeric derivatives was in an apolar microenvironment. This was in agreement with acrylamide quenching studies showing that the spatial position of the Trp indole ring became buried in the interior of the molecule after glutaraldehyde cross-linking. Moreover, the Trp of modified derivatives was less accessible for iodide than that observed with Cobrotoxin. Notably, disulfide reduction could not completely unfold the structure of glutaraldehyde-modified derivatives as evidenced by the results of acrylamide quenching studies and enzyme-linked immunoassay. Study of the characteristic changes in Trp fluorescence after the initiation of refolding suggested that the fine structure around Trp(29) of Cobrotoxin and glutaraldehyde-modified derivatives was formed differently. These results suggest that glutaraldehyde cross-linking leads to a change in the microenvironment of Cobrotoxin Trp(29) and alters the pathway of its fine structure formation during the refolding of Cobrotoxin.
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Structure-function studies on Taiwan cobra long neurotoxin homolog.
Biochimica et Biophysica Acta, 2000Co-Authors: Long-sen Chang, Shinne-ren Lin, Jeh-jeng Wang, Hsien-bin HuangAbstract:Abstract A novel long neurotoxin homolog was purified from Naja naja atra (Taiwan cobra) venom using the combination of ion exchange chromatography and reverse phase high performance liquid chromatography. The determined protein sequence was essentially the same as that deduced from the cDNA amplified by reverse transcriptase-polymerase chain reaction. The long neurotoxin homolog exhibited an activity that inhibited acetylcholine-induced muscle contractions, as with N. naja atra Cobrotoxin. The degree of inhibition caused by the addition of long neurotoxin homolog was approximately 70% of that observed with the addition of Cobrotoxin. Unlike the well-known short and long neurotoxins, this neurotoxin homolog contained two additional cysteine residues forming a disulfide linkage in the N-terminal region. Circular dichroism measurement and computer models of the neurotoxin reveal that its secondary structure was not abundant in β-sheet as noted with short and long neurotoxins. This less ordered structure may be associated with the lower activity noted with the long neurotoxin homolog. Together with the finding that the known long neurotoxin homologs exclusively appear in the venoms of the Naja and Bungarus genera, the long neurotoxin homologs should represent an evolutionary branch from the long and short neurotoxins in the Elapidae family.
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Disulfide Isomerization within the C-Terminus of Cobrotoxin Decelerates by Thiol Compounds and Trinitrophenylation, but Accelerates by Modification of Carboxyl Groups☆
Archives of biochemistry and biophysics, 1998Co-Authors: Long-sen Chang, Chun-chang Chang, Shinne-ren Lin, Chen-chung YangAbstract:A disulfide isomerization at the C-terminus of Cobrotoxin occurred spontaneously by dissolving in alkali buffer. Irreversible conversion of Cobrotoxin into its isomers was completely achieved within 4 days. The isomerization reaction was decelerated by thiol compounds including GSSG, GSH, cystamine, and cysteamine in a pseudo-first-order kinetic, and GSSG was the most effective one among the thiol compounds used. Moreover, the oxidized thiol compounds were always superior to reduced ones in decreasing the rate of disulfide interchange. To further assess the intrinsic elements essential for the occurrence of disulfide isomerization of Cobrotoxin, the toxin molecule was subjected to modification on its Arg, Lys, Trp, Tyr, and carboxyl groups. In sharp contrast to other modified derivatives, the isomerization reaction was decelerated by trinitrophenylation on Lys-26, Lys-27, and Lys-47, whereas it was rapidly completed after modification of carboxyl groups. Neither chemical modification nor the toxin's conformation affected the irreversibility of isomerization reaction. Thus, the observed change in the rate of disulfide isomerization reflects the involvement of Lys residues and carboxyl groups in this reaction. Although thiol compounds further decelerated the conversion of trinitrophenylated Cobrotoxin into its isomers, they did not exert a notable effect on the isomerization of carboxyl groups-modified derivative. These results clearly indicate that disulfide isomerization of Cobrotoxin is, in part, driven by the positively charged Lys residues at positions 26, 27, and 47 of the toxin molecule, and that the thiol compounds are coordinated with the negatively charged groups of Cobrotoxin to exert their inhibitory action.
Chen-chung Yang - One of the best experts on this subject based on the ideXlab platform.
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PURIFICATION AND PROPERTIES OF NON‐PRECIPITATING ANTIBODIES TO Cobrotoxin*
International journal of peptide and protein research, 2009Co-Authors: Chen-chung Yang, Chin-chen Chang, M.f. Lin, H.y. Lee, L.y. ChuangAbstract:The heterogeneity of precipitating and non-precipitating antibodies to Cobrotoxin was demonstrated by their elution pattern on Cobrotoxin-Sepharose and chromatography on DEAE-cellulose column. Gel filtration patterns on a Sepharose 6B column revealed that the soluble complexes formed from non-precipitating antibody and HNB-Cobrotoxin at a different molar ratio all emerged in the void volume, indicating that the molecular weight of the soluble complex is around 4,000,000 or larger. Unreacted free non-precipitating antibody coincided with the peak of IgG and was proved to be free from HNB-Cobrotoxin. The molar ratio of antibody to antigen for the soluble complex was found to be 0.79 to 0.97 indicating that 1.58–1.94 molecules of non-precipitating antibody are bound to HNB-Cobrotoxin instead of three molecules as in the case of precipitating antibody.
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purification and properties of non precipitating antibodies to Cobrotoxin
International Journal of Peptide and Protein Research, 2009Co-Authors: Chen-chung Yang, Chin-chen Chang, M.f. Lin, H.y. Lee, L.y. ChuangAbstract:The heterogeneity of precipitating and non-precipitating antibodies to Cobrotoxin was demonstrated by their elution pattern on Cobrotoxin-Sepharose and chromatography on DEAE-cellulose column. Gel filtration patterns on a Sepharose 6B column revealed that the soluble complexes formed from non-precipitating antibody and HNB-Cobrotoxin at a different molar ratio all emerged in the void volume, indicating that the molecular weight of the soluble complex is around 4,000,000 or larger. Unreacted free non-precipitating antibody coincided with the peak of IgG and was proved to be free from HNB-Cobrotoxin. The molar ratio of antibody to antigen for the soluble complex was found to be 0.79 to 0.97 indicating that 1.58–1.94 molecules of non-precipitating antibody are bound to HNB-Cobrotoxin instead of three molecules as in the case of precipitating antibody.
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glutaraldehyde cross linking alters the environment around trp29 of Cobrotoxin and the pathway for regaining its fine structure during refolding
Journal of Peptide Research, 2001Co-Authors: Long-sen Chang, Shinne-ren Lin, Chen-chung YangAbstract:Cobrotoxin, purified from the venom of Naja naja atra (Taiwan cobra), was subjected to modification with glutaraldehyde in order to prepare intra- and intermolecule cross-linked derivatives. Monomeric and dimeric derivatives were separated from polymeric derivatives by gel filtration. The results of amino acid analysis and sequence determination revealed that only Lys residues were selectively modified by glutaraldehyde. Glutaraldehyde cross-linking was accompanied by a change in the gross conformation of Cobrotoxin as revealed by circular dichroism spectra of the modified derivatives. Compared with Cobrotoxin, Trp(29) of monomeric and dimeric derivatives was in an apolar microenvironment. This was in agreement with acrylamide quenching studies showing that the spatial position of the Trp indole ring became buried in the interior of the molecule after glutaraldehyde cross-linking. Moreover, the Trp of modified derivatives was less accessible for iodide than that observed with Cobrotoxin. Notably, disulfide reduction could not completely unfold the structure of glutaraldehyde-modified derivatives as evidenced by the results of acrylamide quenching studies and enzyme-linked immunoassay. Study of the characteristic changes in Trp fluorescence after the initiation of refolding suggested that the fine structure around Trp(29) of Cobrotoxin and glutaraldehyde-modified derivatives was formed differently. These results suggest that glutaraldehyde cross-linking leads to a change in the microenvironment of Cobrotoxin Trp(29) and alters the pathway of its fine structure formation during the refolding of Cobrotoxin.
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Refolding of Taiwan cobra neurotoxin: intramolecular cross-link affects its refolding reaction.
Archives of biochemistry and biophysics, 2001Co-Authors: Long-sen Chang, Shinne-ren Lin, Chen-chung YangAbstract:Abstract In order to explore the effect of intramolecular cross-linking in the folding reaction of Cobrotoxin from Naja naja atra (Taiwan cobra) venom, the toxin molecule was modified with glutaraldehyde (GA). The monomeric GA-modified Cobrotoxin (mGA-Cobrotoxin) was separated from the dimeric and trimeric derivatives using gel filtration. The results of electrophoretic and chromatographic analyses revealed that mGA-Cobrotoxin comprised two modified derivatives, which contained modified Lys residues at positions 26 and 27 and at positions 26, 27, and 47, respectively. Moreover, an intramolecular cross-linking of loops II and III by Lys residues was noted with the monomeric derivative containing three modified Lys residues. In sharp contrast to Cobrotoxin observations, the folding rate of mGA-Cobrotoxin decreased in the presence of GSH/GSSG, but notably increased in the absence of thiol compounds. Particularly, the accelerated effect of GSH/GSSG on the refolding reaction was affected by the presence of the intramolecular cross-link. Comparative analyses on Cobrotoxin and mGA-Cobrotoxin CD spectra revealed that modification with the GA reagent caused a change in the gross conformation of Cobrotoxin. Fluorescence measurement revealed that the stability of the microenvironment around the single Trp-29 in mGA-Cobrotoxin and unfolded mGA-Cobrotoxin was appreciably higher than in Cobrotoxin and unfolded toxin. Moreover, the ordered structure formation around Trp-29 in refolded mGA-Cobrotoxin was faster than in refolded Cobrotoxin as evidenced by fluorescence quenching studies. Taken together, these results suggest that the structural flexibility of unfolded Cobrotoxin should be favorable for the thiol catalyst to exert its action in the refolding reaction and that the Cobrotoxin refold kinetics may change after modification with GA.
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Glutaraldehyde cross‐linking alters the environment around Trp29 of Cobrotoxin and the pathway for regaining its fine structure during refolding
The journal of peptide research : official journal of the American Peptide Society, 2001Co-Authors: Long-sen Chang, Shinne-ren Lin, Chen-chung YangAbstract:Cobrotoxin, purified from the venom of Naja naja atra (Taiwan cobra), was subjected to modification with glutaraldehyde in order to prepare intra- and intermolecule cross-linked derivatives. Monomeric and dimeric derivatives were separated from polymeric derivatives by gel filtration. The results of amino acid analysis and sequence determination revealed that only Lys residues were selectively modified by glutaraldehyde. Glutaraldehyde cross-linking was accompanied by a change in the gross conformation of Cobrotoxin as revealed by circular dichroism spectra of the modified derivatives. Compared with Cobrotoxin, Trp(29) of monomeric and dimeric derivatives was in an apolar microenvironment. This was in agreement with acrylamide quenching studies showing that the spatial position of the Trp indole ring became buried in the interior of the molecule after glutaraldehyde cross-linking. Moreover, the Trp of modified derivatives was less accessible for iodide than that observed with Cobrotoxin. Notably, disulfide reduction could not completely unfold the structure of glutaraldehyde-modified derivatives as evidenced by the results of acrylamide quenching studies and enzyme-linked immunoassay. Study of the characteristic changes in Trp fluorescence after the initiation of refolding suggested that the fine structure around Trp(29) of Cobrotoxin and glutaraldehyde-modified derivatives was formed differently. These results suggest that glutaraldehyde cross-linking leads to a change in the microenvironment of Cobrotoxin Trp(29) and alters the pathway of its fine structure formation during the refolding of Cobrotoxin.
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Cobrotoxin: structure and function.
Journal of natural toxins, 1999Co-Authors: C.c. YangAbstract:Cobrotoxin is the main neurotoxic protein isolated from the venom of Taiwan cobra Naja naja atra. It is a small, basic protein consisting of a single polypeptide chain of 62 amino acids, cross-linked by four disulfide bonds. The disulfide bonds and Tyr-25 which is buried in the molecule form a central core to maintain and stabilize the active conformation of the toxin. Selective and stepwise chemical modifications of Cobrotoxin indicate that at least two cationic groups, an epsilon-amino group of Lys-47 and a guanidino group of Arg-33, both of which are common to all known postsynaptic neurotoxins, held at a certain critical distance in the molecule, are functionally important for its neuromuscular blocking activity. The cDNA encoding Cobrotoxin was constructed from the cellular RNA isolated from the venom glands of Naja naja atra by reverse transcription polymerase chain reaction. Sequencing several clones containing about 0.5 Kb DNA inserts contained a complete and full-length reading frame of 249 base pairs covering a precursor of Cobrotoxin gene with a deduced mature protein sequence of 62 amino acids which are identical to the amino acid sequence of Cobrotoxin and a 21 amino acid segment of signal peptide. Expression of Cobrotoxin in E. coli vector generated a polypeptide which can cross-react with the antisera against the native Cobrotoxin.
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Sequence Comparison and Computer Modeling of Cardiotoxins and Cobrotoxin Isolated from Taiwan Cobra
Biochemical and biophysical research communications, 1995Co-Authors: Shyh-horng Chiou, Chin-chun Hung, Hsuan-cheng Huang, Shui-tein Chen, Kung-tsung Wang, C.c. YangAbstract:Six cardiotoxins and one neurotoxin isolated and purified from the Taiwan cobra venom (Naja naja atra) possess distinct pharmacological and biochemical properties despite the existence of a grossly similar tertiary structure among these toxins, i. e., a core consisting of a series of short loops and four disulfide bridges. A systematic structure comparison of these major toxin isoforms was made by the secondary-structure predictions together with computer model-building based on the primary sequences and the established X-ray and NMR structures of one published cardiotoxin isoform and Cobrotoxin. It is of interest to find that some defined and subtle differences can be detected upon the superposition of these three-dimensional polypeptide chains, which may reflect the intrinsic differences in the surface hydrophobicity of cardiotoxins and Cobrotoxin as revealed by hydropathy profiles of these toxins in one of three major loops. The differences seem to correlate with different inhibitory activities exhibited by cardiotoxins in contrast to the lack of activity by Cobrotoxin on protein kinase C (PKC).
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Structures in Solution of Toxins from Taiwan Cobra Venom, Naja naja atra, Derived from NMR Spectra
Journal of Toxicology: Toxin Reviews, 1994Co-Authors: R. Bhaskaran, C.c. YangAbstract:AbstractThe structures in solution of Cobrotoxin and cardiotoxins isolated from Taiwan cobra venom (Naja naja atra) were determined using 2D-NMR and simulated annealing methods. These toxins possess similar backbone conformations (a two- and a three-stranded antiparallel β sheets) with the emergence of three independent loops from the central core formed by the four disulfide bonds. Comparison of these NMR structures with the crystal structures of their homologous toxins showed good agreement except at the segments of the turns. Even though overall similarity is observed between the toxins, their functions are distinct and specific. In Cobrotoxin, the electrostatic interactions of functional sites found in loops of the three-stranded β sheet cause strong binding with the receptor. In cardiotoxins, the stretches of hydrophobic residues at the tip of the flexible loops, that are surrounded by positively charged residues are expected to penetrate the lipid phase of the membrane and to form a hydrophobic clus...