The Experts below are selected from a list of 12942 Experts worldwide ranked by ideXlab platform
John Markwell - One of the best experts on this subject based on the ideXlab platform.
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stability of the allergenic soybean kunitz trypsin inhibitor
Biochimica et Biophysica Acta, 2004Co-Authors: Robin Roychaudhuri, Michael G Zeece, Gautam Sarath, John MarkwellAbstract:The soybean Kunitz trypsin inhibitor (SKTI) is a 21.5 kDa allergenic protein that belongs to the family of all antiparallel β-sheet proteins that are highly resistant to thermal and chemical denaturation. Spectroscopic and biochemical techniques such as circular dichroism (CD), ANS fluorescence and proteolysis were used to study its molecular structure under denaturing conditions such as acid and heat to which these allergens are commonly exposed during food processing. Reduction of native SKTI leads to its complete and rapid proteolysis by pepsin in simulated gastric fluid (SGF). Limited proteolysis with chymotrypsin during Renaturation after heating showed that the native structure reforms at around 60 °C reversing the denaturation. CD spectra revealed that under acid denaturing conditions, SKTI shows major changes in conformation, indicating the possibility of a molten structure. The existence of this intermediate was established by ANS fluorescence studies at different concentrations of HCl. The remarkable stability of SKTI to both thermal and acid denaturation may be important for its role as a food allergen.
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reversible denaturation of the soybean kunitz trypsin inhibitor
Archives of Biochemistry and Biophysics, 2003Co-Authors: Robin Roychaudhuri, Michael G Zeece, Gautam Sarath, John MarkwellAbstract:Abstract The soybean Kunitz trypsin inhibitor (SKTI) is a β-sheet protein with unusual stability to chemical and thermal denaturation. Different spectroscopic criteria were used to follow the thermal denaturation and Renaturation of SKTI. Upon heating to 70 °C, changes in UV difference spectra showed increased absorbance at 292 and 297 nm, attributable to perturbation of aromatic residues. Cooling the protein resulted in restoration of the native spectrum unless reduced with dithiothreitol. Far- and near-UV CD spectra also indicate thermal unfolding involving the core tryptophan and tyrosine residues. Both CD and UV-absorbance data suggest a two-state transition with the midpoint at approximately 65 °C. CD data along with the increased fluorescence intensity of the reporter fluorophore, 1-anilino-8-naphthalenesulfonate with SKTI, between 60 and 70 °C, are consistent with a transition of the native inhibitor to an alternate conformation with a more molten state. Even after heating to 90 °C, subsequent cooling of SKTI resulted in >90% of native trypsin inhibition potential. These results indicate that thermal denaturation of SKTI is readily reversible to the native form upon cooling and may provide a useful system for future protein folding studies in the class of disordered β-sheet proteins.
Robin Roychaudhuri - One of the best experts on this subject based on the ideXlab platform.
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stability of the allergenic soybean kunitz trypsin inhibitor
Biochimica et Biophysica Acta, 2004Co-Authors: Robin Roychaudhuri, Michael G Zeece, Gautam Sarath, John MarkwellAbstract:The soybean Kunitz trypsin inhibitor (SKTI) is a 21.5 kDa allergenic protein that belongs to the family of all antiparallel β-sheet proteins that are highly resistant to thermal and chemical denaturation. Spectroscopic and biochemical techniques such as circular dichroism (CD), ANS fluorescence and proteolysis were used to study its molecular structure under denaturing conditions such as acid and heat to which these allergens are commonly exposed during food processing. Reduction of native SKTI leads to its complete and rapid proteolysis by pepsin in simulated gastric fluid (SGF). Limited proteolysis with chymotrypsin during Renaturation after heating showed that the native structure reforms at around 60 °C reversing the denaturation. CD spectra revealed that under acid denaturing conditions, SKTI shows major changes in conformation, indicating the possibility of a molten structure. The existence of this intermediate was established by ANS fluorescence studies at different concentrations of HCl. The remarkable stability of SKTI to both thermal and acid denaturation may be important for its role as a food allergen.
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reversible denaturation of the soybean kunitz trypsin inhibitor
Archives of Biochemistry and Biophysics, 2003Co-Authors: Robin Roychaudhuri, Michael G Zeece, Gautam Sarath, John MarkwellAbstract:Abstract The soybean Kunitz trypsin inhibitor (SKTI) is a β-sheet protein with unusual stability to chemical and thermal denaturation. Different spectroscopic criteria were used to follow the thermal denaturation and Renaturation of SKTI. Upon heating to 70 °C, changes in UV difference spectra showed increased absorbance at 292 and 297 nm, attributable to perturbation of aromatic residues. Cooling the protein resulted in restoration of the native spectrum unless reduced with dithiothreitol. Far- and near-UV CD spectra also indicate thermal unfolding involving the core tryptophan and tyrosine residues. Both CD and UV-absorbance data suggest a two-state transition with the midpoint at approximately 65 °C. CD data along with the increased fluorescence intensity of the reporter fluorophore, 1-anilino-8-naphthalenesulfonate with SKTI, between 60 and 70 °C, are consistent with a transition of the native inhibitor to an alternate conformation with a more molten state. Even after heating to 90 °C, subsequent cooling of SKTI resulted in >90% of native trypsin inhibition potential. These results indicate that thermal denaturation of SKTI is readily reversible to the native form upon cooling and may provide a useful system for future protein folding studies in the class of disordered β-sheet proteins.
Gautam Sarath - One of the best experts on this subject based on the ideXlab platform.
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stability of the allergenic soybean kunitz trypsin inhibitor
Biochimica et Biophysica Acta, 2004Co-Authors: Robin Roychaudhuri, Michael G Zeece, Gautam Sarath, John MarkwellAbstract:The soybean Kunitz trypsin inhibitor (SKTI) is a 21.5 kDa allergenic protein that belongs to the family of all antiparallel β-sheet proteins that are highly resistant to thermal and chemical denaturation. Spectroscopic and biochemical techniques such as circular dichroism (CD), ANS fluorescence and proteolysis were used to study its molecular structure under denaturing conditions such as acid and heat to which these allergens are commonly exposed during food processing. Reduction of native SKTI leads to its complete and rapid proteolysis by pepsin in simulated gastric fluid (SGF). Limited proteolysis with chymotrypsin during Renaturation after heating showed that the native structure reforms at around 60 °C reversing the denaturation. CD spectra revealed that under acid denaturing conditions, SKTI shows major changes in conformation, indicating the possibility of a molten structure. The existence of this intermediate was established by ANS fluorescence studies at different concentrations of HCl. The remarkable stability of SKTI to both thermal and acid denaturation may be important for its role as a food allergen.
-
reversible denaturation of the soybean kunitz trypsin inhibitor
Archives of Biochemistry and Biophysics, 2003Co-Authors: Robin Roychaudhuri, Michael G Zeece, Gautam Sarath, John MarkwellAbstract:Abstract The soybean Kunitz trypsin inhibitor (SKTI) is a β-sheet protein with unusual stability to chemical and thermal denaturation. Different spectroscopic criteria were used to follow the thermal denaturation and Renaturation of SKTI. Upon heating to 70 °C, changes in UV difference spectra showed increased absorbance at 292 and 297 nm, attributable to perturbation of aromatic residues. Cooling the protein resulted in restoration of the native spectrum unless reduced with dithiothreitol. Far- and near-UV CD spectra also indicate thermal unfolding involving the core tryptophan and tyrosine residues. Both CD and UV-absorbance data suggest a two-state transition with the midpoint at approximately 65 °C. CD data along with the increased fluorescence intensity of the reporter fluorophore, 1-anilino-8-naphthalenesulfonate with SKTI, between 60 and 70 °C, are consistent with a transition of the native inhibitor to an alternate conformation with a more molten state. Even after heating to 90 °C, subsequent cooling of SKTI resulted in >90% of native trypsin inhibition potential. These results indicate that thermal denaturation of SKTI is readily reversible to the native form upon cooling and may provide a useful system for future protein folding studies in the class of disordered β-sheet proteins.
Michael G Zeece - One of the best experts on this subject based on the ideXlab platform.
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stability of the allergenic soybean kunitz trypsin inhibitor
Biochimica et Biophysica Acta, 2004Co-Authors: Robin Roychaudhuri, Michael G Zeece, Gautam Sarath, John MarkwellAbstract:The soybean Kunitz trypsin inhibitor (SKTI) is a 21.5 kDa allergenic protein that belongs to the family of all antiparallel β-sheet proteins that are highly resistant to thermal and chemical denaturation. Spectroscopic and biochemical techniques such as circular dichroism (CD), ANS fluorescence and proteolysis were used to study its molecular structure under denaturing conditions such as acid and heat to which these allergens are commonly exposed during food processing. Reduction of native SKTI leads to its complete and rapid proteolysis by pepsin in simulated gastric fluid (SGF). Limited proteolysis with chymotrypsin during Renaturation after heating showed that the native structure reforms at around 60 °C reversing the denaturation. CD spectra revealed that under acid denaturing conditions, SKTI shows major changes in conformation, indicating the possibility of a molten structure. The existence of this intermediate was established by ANS fluorescence studies at different concentrations of HCl. The remarkable stability of SKTI to both thermal and acid denaturation may be important for its role as a food allergen.
-
reversible denaturation of the soybean kunitz trypsin inhibitor
Archives of Biochemistry and Biophysics, 2003Co-Authors: Robin Roychaudhuri, Michael G Zeece, Gautam Sarath, John MarkwellAbstract:Abstract The soybean Kunitz trypsin inhibitor (SKTI) is a β-sheet protein with unusual stability to chemical and thermal denaturation. Different spectroscopic criteria were used to follow the thermal denaturation and Renaturation of SKTI. Upon heating to 70 °C, changes in UV difference spectra showed increased absorbance at 292 and 297 nm, attributable to perturbation of aromatic residues. Cooling the protein resulted in restoration of the native spectrum unless reduced with dithiothreitol. Far- and near-UV CD spectra also indicate thermal unfolding involving the core tryptophan and tyrosine residues. Both CD and UV-absorbance data suggest a two-state transition with the midpoint at approximately 65 °C. CD data along with the increased fluorescence intensity of the reporter fluorophore, 1-anilino-8-naphthalenesulfonate with SKTI, between 60 and 70 °C, are consistent with a transition of the native inhibitor to an alternate conformation with a more molten state. Even after heating to 90 °C, subsequent cooling of SKTI resulted in >90% of native trypsin inhibition potential. These results indicate that thermal denaturation of SKTI is readily reversible to the native form upon cooling and may provide a useful system for future protein folding studies in the class of disordered β-sheet proteins.
George M Whitesides - One of the best experts on this subject based on the ideXlab platform.
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peracetylated bovine carbonic anhydrase bca ac18 is kinetically more stable than native bca to sodium dodecyl sulfate
Journal of Physical Chemistry B, 2006Co-Authors: Irina Gitlin, Katherine L Gudiksen, George M WhitesidesAbstract:Bovine carbonic anhydrase (BCA) and its derivative with all lysine groups acetylated (BCA-Ac18) have different stabilities toward denaturation by sodium dodecyl sulfate (SDS). This difference is kinetic: BCA-Ac18 denatures more slowly than BCA by several orders of magnitude over concentrations of SDS ranging from 2.5 to 10 mM. The rates of Renaturation of BCA-Ac18 are greater than those of BCA, when these proteins are allowed to refold from a denatured state ([SDS] = 10 mM) to a folded state ([SDS] = 0.1 to 1.5 mM). On Renaturation, the yields of the correctly folded protein (either BCA or BCA-Ac18) decrease with increasing concentration of SDS. At intermediate concentrations of SDS (from 0.7 to 2 mM for BCA, and from 1.5 to 2 mM for BCA-Ac18), both unfolding and refolding of the proteins are too slow to be observed; an alternative processprobably aggregationcompetes with refolding of the denatured proteins at those intermediate concentrations. Because it is experimentally impractical to prove equilibriu...
