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Ljuba M Mandic - One of the best experts on this subject based on the ideXlab platform.
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binding of enterolactone and enterodiol to human serum albumin increase of cysteine 34 Thiol Group reactivity
Food & Function, 2016Co-Authors: Marija Takic, Ivan D Pavicevic, Vesna Jovanovic, Jelena M Acimovic, Tamara N Uzelac, Danijela Risticmedic, Ljuba M MandicAbstract:The interaction of polyphenolic molecules with human serum albumin (HSA) could lead to changes in the reactivity of the HSA Cys34 Thiol Group (HSA-SH). The influences of enterolactone (EL) and enterodiol (ED) binding on HSA-SH reactivity in fatty acid (FA)-free HSA, and in HSA with bound stearic acid (S) in S/HSA molar ratios of 1:1 and 4:1, were investigated by the determination of the pseudo first order rate constants (k') for the Thiol reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). The binding affinities and binding sites of EL and ED were also determined, using fluorescence measurements of the intrinsic fluorescence of Trp214 and diazepam (binding site marker). EL and ED binding to HSA increased the reactivity of HSA-SH in all assayed HSA-enterolignan complexes by 9.1-33.1%. The strongest effects were obtained for FA-free HSA-enterolignan complexes. S modulated/reduced the effect of EL on HSA-SH reactivity, while its influence on the effect of ED was negligible. The binding of enterolignans to HSA was investigated: the binding constants were the highest for FA-free HSA (EL: 11.64 × 10(4) M(-1) and ED: 5.59 × 10(4) M(-1) at 37 °C) and the lowest for S/HSA 4:1-enterolignan complexes (EL: 2.43 × 10(4) M(-1) and ED: 1.92 × 10(4) M(-1)). When the S/HSA ratio was increased, the binding affinities and number of binding sites for EL and ED were decreased. At the same time, a high correlation between binding constants and increased Cys34 reactivity was found (r = 0.974). Competitive experiments using diazepam indicated that the binding of ED and of EL was located in the hydrophobic pocket of site II in HSA. Overall, it is evident that stearic acid could modulate the enterolignan effects on HSA-SH reactivity as well as their binding to HSA. This finding could be important for pharmacokinetics and the expression of enterolignan antioxidant effects in vivo after an intake of lignan rich food.
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fatty acids binding to human serum albumin changes of reactivity and glycation level of cysteine 34 free Thiol Group with methylglyoxal
Chemico-Biological Interactions, 2014Co-Authors: Ivan D Pavicevic, Vesna Jovanovic, Marija Takic, Ana Penezic, Jelena M Acimovic, Ljuba M MandicAbstract:Fatty acids (FAs) binding to human serum albumin (HSA) could lead to the changes of Cys-34 Thiol Group accessibility and reactivity, i.e. its scavenger capacity and antioxidant property. The influence of saturated, mono and poly unsaturated, and fish oil FAs binding to HSA on the carbonylation level and the reactivity of HSA-SH and HSA modified with methylglyoxal (MG-HSA-SH) was investigated. Changes of Thiol Group reactivity were followed by determination of pseudo first order rate constant (k') for Thiols reaction with 5,5'-dithiobis(2-nitrobenzoic acid). HSA changes were monitored using native PAG electrophoresis and fluorescence spectroscopy. For FA/HSA molar ratios screening, qTLC and GC were used. FAs increase Thiol Group carbonylation levels from 8% to 20%. The k' values obtained for FAs-free HSA-SH and FAs-free MG-HSA-SH are almost equal (7.5×10(-3) and 7.7×10(-3)s(-1), resp.). Binding of all FAs amplify the reactivity (k' values from 14.6×10(-3) to 26.0×10(-3)s(-1)) of HSA-SH Group for 2-3.5times in the order: palmitic, docosahexaenoic, fish oil extract, stearic, oleic, myristic and eicosapentaenoic acid, due to HSA conformational changes. FAs-bound MG-HSA-SH samples follow that pattern, but their k' values (from 9.8×10(-3) to 14.3×10(-3)s(-1)) were lower compared to unmodified HSA due to additional conformation changes of HSA molecules during carbonylation. Carbonylation level and reactivity of Cys34 Thiol Group of unmodified and carbonylated HSA depend on type of FAs bound to HSA, which implies the possibility for modulation of -SH reactivity (scavenger capacity and antioxidant property) by FAs as a supplement.
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the influence of fatty acids on determination of human serum albumin Thiol Group
Analytical Biochemistry, 2014Co-Authors: Vesna Jovanovic, Ivan D Pavicevic, Marija Takic, Jelena M Acimovic, Ana Z Penezicromanjuk, Ljuba M MandicAbstract:During investigation of the changes of the Cys34 Thiol Group of human serum albumin (HSA) (isolated by affinity chromatography with Cibacron Blue (CB)) in diabetes, we found that the HSA-SH content was higher (11-33%) than the total serum Thiol content. The influence of fatty acids (FA) binding to HSA on this discrepancy was investigated in vitro (using fluorescence and CD spectroscopy and GC) and with HSA samples from diabetic (n=20) and control Groups (n=17). HSA-bound FA determine the selection of HSA molecules by CB and enhance reactivity and/or accessibility of the SH Group. A high content of polyunsaturated FA (35.6%) leads to weaker binding of HSA molecules to CB. Rate constants of DTNB reaction with the SH Group of HSA applied to a CB column, bound-HSA and unbound-HSA fractions, were 4.8×10(-3), 21.6×10(-3), and 11.2×10(-3) s(-1), respectively. The HSA-SH Group of diabetics is more reactive compared with control individuals (rate constants 20.9×10(-3)±4.4×10(-3) vs 12.9×10(-3)±2.6×10(-3) s(-1), P<0.05). Recovery values of the SH Group obtained after chromatography of HSA with bound stearic acid ranged from 110 to 140%, while those for defatted HSA were from 98.5 to 101.7%. Thus, HSA-bound FA leads to an increase of HSA-SH content and a contribution to total serum Thiols, which make the determination of the Thiol Group unreliable.
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improving the reliability of human serum albumin Thiol Group determination
Analytical Biochemistry, 2013Co-Authors: Vesna Jovanovic, Ivan D Pavicevic, Jelena M Acimovic, Ana Z Penezicromanjuk, Ljuba M MandicAbstract:Abstract The Thiol (Cys34) content of human serum albumin (HSA-SH) decreases during oxidative and carbonyl stress and, therefore, could represent a useful parameter in clinical practice. Nevertheless, the reliability of HSA-Thiol determination with Ellman’s method depends on the purity of isolated HSA. Determination of total serum Thiols (mmol/L) and HSA-SH content (mmol -SH/mmol HSA) after HSA isolation from diabetic patient and control sera by a two-step precipitation with ammonium sulfate (AS), as well as HSA-SH contribution (%) to total serum Thiols, was assessed. Purity and yield of isolated HSA were monitored spectrophotometrically and by native polyacrylamide gel electrophoresis. Precipitation of HSA from serum via a two-step method with AS produced HSA with 91.9 ± 3.6% purity and 69.7 ± 4.4% yield, allowing for precise (relative standard deviation of 3.2%) and reliable (comparing with total serum Thiols) measurement of HSA-SH content with DTNB [5,5′-dithiobis-(2-nitrobenzoic acid)]. The content of the HSA-SH Group in patients with type 2 diabetes was significantly ( P
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the role of the Thiol Group in protein modification with methylglyoxal
Journal of The Serbian Chemical Society, 2009Co-Authors: Jelena M Acimovic, Bojana Stanimirovic, Ljuba M MandicAbstract:Methylglyoxal is a highly reactive α-oxoaldehyde with elevated production in hyperglycemia. It reacts with nucleophilic Lys and Arg side-chains and N-terminal amino Groups causing protein modification. In the present study, the importance of the reaction of the Cys Thiol Group with methylglyoxal in protein modification, the competitiveness of this reaction with those of amino and guanidine Groups, the time course of these reactions and their role and contribution to protein cross-linking were investigated. Human and bovine serum albumins were used as model systems. It was found that despite the very low levels of Thiol Groups on the surface of the examined protein molecules (approx. 80 times lower than those of amino and guanidino Groups), a very high percentage of it reacts (25-85 %). The amount of reacted Thiol Groups and the rate of the reaction, the time for the reaction to reach equilibrium, the formation of a stable product and the contribution of Thiol Groups to protein cross-linking depend on the methylglyoxal concentration. The product formed in the reaction of Thiol and an insufficient quantity of methylglyoxal (compared to the concentrations of the Groups accessible for modification) participates to a significant extent (4 %) to protein cross-linking. Metformin applied in equimolar concentration with methylglyoxal prevents its reaction with amino and guanidino Groups but, however, not with Thiol Groups.
Amir Abbas Rafati - One of the best experts on this subject based on the ideXlab platform.
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chemically modified silica gel with Thiol Group as an adsorbent for retention of some toxic soft metal ions from water and industrial effluent
Chemical Engineering Journal, 2011Co-Authors: Mojgan Najafi, Rahele Rostamian, Amir Abbas RafatiAbstract:Abstract In this study, functionalized silica with mercaptopropyl Groups were synthesized. These materials have been characterized by powder X-ray diffraction and FT-IR spectroscopy. We have shown that silica gel (SG) particles with a surface functionalization of Thiol Group (S–H) are an effective adsorbent material for toxic soft metal ions such as Hg2+, Pb2+ and Cd2+, which effectively bind to the Thiol ligands. The ability of the Thiol modified silica gel (TMSG) to remove heavy metal ions was studied by the method of continuous variation of two process variables: Initial heavy metal concentration (mg L−1) in distilled water and the contact time of adsorbent and solutions in water and industrial effluent. The experimental data were fitted into three kinetic models: Lagergren, pseudo-second order and Elovich. The experimental isotherm data were analyzed using Langmuir, Freundlich, Sips, Redlich–Peterson and Temkin isotherms. Six error functions were used to treat the adsorption data using nonlinear optimization techniques for evaluating the fit of the model equations. Error analysis showed that the Langmuir and Redlich–Peterson isotherms best fit the ions adsorption data on TMSG and the pseudo-second order kinetic model described the adsorption process.
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chemically modified silica gel with Thiol Group as an adsorbent for retention of some toxic soft metal ions from water and industrial effluent
Chemical Engineering Journal, 2011Co-Authors: Mojgan Najafi, Rahele Rostamian, Amir Abbas RafatiAbstract:Abstract In this study, functionalized silica with mercaptopropyl Groups were synthesized. These materials have been characterized by powder X-ray diffraction and FT-IR spectroscopy. We have shown that silica gel (SG) particles with a surface functionalization of Thiol Group (S–H) are an effective adsorbent material for toxic soft metal ions such as Hg2+, Pb2+ and Cd2+, which effectively bind to the Thiol ligands. The ability of the Thiol modified silica gel (TMSG) to remove heavy metal ions was studied by the method of continuous variation of two process variables: Initial heavy metal concentration (mg L−1) in distilled water and the contact time of adsorbent and solutions in water and industrial effluent. The experimental data were fitted into three kinetic models: Lagergren, pseudo-second order and Elovich. The experimental isotherm data were analyzed using Langmuir, Freundlich, Sips, Redlich–Peterson and Temkin isotherms. Six error functions were used to treat the adsorption data using nonlinear optimization techniques for evaluating the fit of the model equations. Error analysis showed that the Langmuir and Redlich–Peterson isotherms best fit the ions adsorption data on TMSG and the pseudo-second order kinetic model described the adsorption process.
Vesna Jovanovic - One of the best experts on this subject based on the ideXlab platform.
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binding of enterolactone and enterodiol to human serum albumin increase of cysteine 34 Thiol Group reactivity
Food & Function, 2016Co-Authors: Marija Takic, Ivan D Pavicevic, Vesna Jovanovic, Jelena M Acimovic, Tamara N Uzelac, Danijela Risticmedic, Ljuba M MandicAbstract:The interaction of polyphenolic molecules with human serum albumin (HSA) could lead to changes in the reactivity of the HSA Cys34 Thiol Group (HSA-SH). The influences of enterolactone (EL) and enterodiol (ED) binding on HSA-SH reactivity in fatty acid (FA)-free HSA, and in HSA with bound stearic acid (S) in S/HSA molar ratios of 1:1 and 4:1, were investigated by the determination of the pseudo first order rate constants (k') for the Thiol reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). The binding affinities and binding sites of EL and ED were also determined, using fluorescence measurements of the intrinsic fluorescence of Trp214 and diazepam (binding site marker). EL and ED binding to HSA increased the reactivity of HSA-SH in all assayed HSA-enterolignan complexes by 9.1-33.1%. The strongest effects were obtained for FA-free HSA-enterolignan complexes. S modulated/reduced the effect of EL on HSA-SH reactivity, while its influence on the effect of ED was negligible. The binding of enterolignans to HSA was investigated: the binding constants were the highest for FA-free HSA (EL: 11.64 × 10(4) M(-1) and ED: 5.59 × 10(4) M(-1) at 37 °C) and the lowest for S/HSA 4:1-enterolignan complexes (EL: 2.43 × 10(4) M(-1) and ED: 1.92 × 10(4) M(-1)). When the S/HSA ratio was increased, the binding affinities and number of binding sites for EL and ED were decreased. At the same time, a high correlation between binding constants and increased Cys34 reactivity was found (r = 0.974). Competitive experiments using diazepam indicated that the binding of ED and of EL was located in the hydrophobic pocket of site II in HSA. Overall, it is evident that stearic acid could modulate the enterolignan effects on HSA-SH reactivity as well as their binding to HSA. This finding could be important for pharmacokinetics and the expression of enterolignan antioxidant effects in vivo after an intake of lignan rich food.
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fatty acids binding to human serum albumin changes of reactivity and glycation level of cysteine 34 free Thiol Group with methylglyoxal
Chemico-Biological Interactions, 2014Co-Authors: Ivan D Pavicevic, Vesna Jovanovic, Marija Takic, Ana Penezic, Jelena M Acimovic, Ljuba M MandicAbstract:Fatty acids (FAs) binding to human serum albumin (HSA) could lead to the changes of Cys-34 Thiol Group accessibility and reactivity, i.e. its scavenger capacity and antioxidant property. The influence of saturated, mono and poly unsaturated, and fish oil FAs binding to HSA on the carbonylation level and the reactivity of HSA-SH and HSA modified with methylglyoxal (MG-HSA-SH) was investigated. Changes of Thiol Group reactivity were followed by determination of pseudo first order rate constant (k') for Thiols reaction with 5,5'-dithiobis(2-nitrobenzoic acid). HSA changes were monitored using native PAG electrophoresis and fluorescence spectroscopy. For FA/HSA molar ratios screening, qTLC and GC were used. FAs increase Thiol Group carbonylation levels from 8% to 20%. The k' values obtained for FAs-free HSA-SH and FAs-free MG-HSA-SH are almost equal (7.5×10(-3) and 7.7×10(-3)s(-1), resp.). Binding of all FAs amplify the reactivity (k' values from 14.6×10(-3) to 26.0×10(-3)s(-1)) of HSA-SH Group for 2-3.5times in the order: palmitic, docosahexaenoic, fish oil extract, stearic, oleic, myristic and eicosapentaenoic acid, due to HSA conformational changes. FAs-bound MG-HSA-SH samples follow that pattern, but their k' values (from 9.8×10(-3) to 14.3×10(-3)s(-1)) were lower compared to unmodified HSA due to additional conformation changes of HSA molecules during carbonylation. Carbonylation level and reactivity of Cys34 Thiol Group of unmodified and carbonylated HSA depend on type of FAs bound to HSA, which implies the possibility for modulation of -SH reactivity (scavenger capacity and antioxidant property) by FAs as a supplement.
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the influence of fatty acids on determination of human serum albumin Thiol Group
Analytical Biochemistry, 2014Co-Authors: Vesna Jovanovic, Ivan D Pavicevic, Marija Takic, Jelena M Acimovic, Ana Z Penezicromanjuk, Ljuba M MandicAbstract:During investigation of the changes of the Cys34 Thiol Group of human serum albumin (HSA) (isolated by affinity chromatography with Cibacron Blue (CB)) in diabetes, we found that the HSA-SH content was higher (11-33%) than the total serum Thiol content. The influence of fatty acids (FA) binding to HSA on this discrepancy was investigated in vitro (using fluorescence and CD spectroscopy and GC) and with HSA samples from diabetic (n=20) and control Groups (n=17). HSA-bound FA determine the selection of HSA molecules by CB and enhance reactivity and/or accessibility of the SH Group. A high content of polyunsaturated FA (35.6%) leads to weaker binding of HSA molecules to CB. Rate constants of DTNB reaction with the SH Group of HSA applied to a CB column, bound-HSA and unbound-HSA fractions, were 4.8×10(-3), 21.6×10(-3), and 11.2×10(-3) s(-1), respectively. The HSA-SH Group of diabetics is more reactive compared with control individuals (rate constants 20.9×10(-3)±4.4×10(-3) vs 12.9×10(-3)±2.6×10(-3) s(-1), P<0.05). Recovery values of the SH Group obtained after chromatography of HSA with bound stearic acid ranged from 110 to 140%, while those for defatted HSA were from 98.5 to 101.7%. Thus, HSA-bound FA leads to an increase of HSA-SH content and a contribution to total serum Thiols, which make the determination of the Thiol Group unreliable.
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improving the reliability of human serum albumin Thiol Group determination
Analytical Biochemistry, 2013Co-Authors: Vesna Jovanovic, Ivan D Pavicevic, Jelena M Acimovic, Ana Z Penezicromanjuk, Ljuba M MandicAbstract:Abstract The Thiol (Cys34) content of human serum albumin (HSA-SH) decreases during oxidative and carbonyl stress and, therefore, could represent a useful parameter in clinical practice. Nevertheless, the reliability of HSA-Thiol determination with Ellman’s method depends on the purity of isolated HSA. Determination of total serum Thiols (mmol/L) and HSA-SH content (mmol -SH/mmol HSA) after HSA isolation from diabetic patient and control sera by a two-step precipitation with ammonium sulfate (AS), as well as HSA-SH contribution (%) to total serum Thiols, was assessed. Purity and yield of isolated HSA were monitored spectrophotometrically and by native polyacrylamide gel electrophoresis. Precipitation of HSA from serum via a two-step method with AS produced HSA with 91.9 ± 3.6% purity and 69.7 ± 4.4% yield, allowing for precise (relative standard deviation of 3.2%) and reliable (comparing with total serum Thiols) measurement of HSA-SH content with DTNB [5,5′-dithiobis-(2-nitrobenzoic acid)]. The content of the HSA-SH Group in patients with type 2 diabetes was significantly ( P
Ivan D Pavicevic - One of the best experts on this subject based on the ideXlab platform.
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binding of enterolactone and enterodiol to human serum albumin increase of cysteine 34 Thiol Group reactivity
Food & Function, 2016Co-Authors: Marija Takic, Ivan D Pavicevic, Vesna Jovanovic, Jelena M Acimovic, Tamara N Uzelac, Danijela Risticmedic, Ljuba M MandicAbstract:The interaction of polyphenolic molecules with human serum albumin (HSA) could lead to changes in the reactivity of the HSA Cys34 Thiol Group (HSA-SH). The influences of enterolactone (EL) and enterodiol (ED) binding on HSA-SH reactivity in fatty acid (FA)-free HSA, and in HSA with bound stearic acid (S) in S/HSA molar ratios of 1:1 and 4:1, were investigated by the determination of the pseudo first order rate constants (k') for the Thiol reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). The binding affinities and binding sites of EL and ED were also determined, using fluorescence measurements of the intrinsic fluorescence of Trp214 and diazepam (binding site marker). EL and ED binding to HSA increased the reactivity of HSA-SH in all assayed HSA-enterolignan complexes by 9.1-33.1%. The strongest effects were obtained for FA-free HSA-enterolignan complexes. S modulated/reduced the effect of EL on HSA-SH reactivity, while its influence on the effect of ED was negligible. The binding of enterolignans to HSA was investigated: the binding constants were the highest for FA-free HSA (EL: 11.64 × 10(4) M(-1) and ED: 5.59 × 10(4) M(-1) at 37 °C) and the lowest for S/HSA 4:1-enterolignan complexes (EL: 2.43 × 10(4) M(-1) and ED: 1.92 × 10(4) M(-1)). When the S/HSA ratio was increased, the binding affinities and number of binding sites for EL and ED were decreased. At the same time, a high correlation between binding constants and increased Cys34 reactivity was found (r = 0.974). Competitive experiments using diazepam indicated that the binding of ED and of EL was located in the hydrophobic pocket of site II in HSA. Overall, it is evident that stearic acid could modulate the enterolignan effects on HSA-SH reactivity as well as their binding to HSA. This finding could be important for pharmacokinetics and the expression of enterolignan antioxidant effects in vivo after an intake of lignan rich food.
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fatty acids binding to human serum albumin changes of reactivity and glycation level of cysteine 34 free Thiol Group with methylglyoxal
Chemico-Biological Interactions, 2014Co-Authors: Ivan D Pavicevic, Vesna Jovanovic, Marija Takic, Ana Penezic, Jelena M Acimovic, Ljuba M MandicAbstract:Fatty acids (FAs) binding to human serum albumin (HSA) could lead to the changes of Cys-34 Thiol Group accessibility and reactivity, i.e. its scavenger capacity and antioxidant property. The influence of saturated, mono and poly unsaturated, and fish oil FAs binding to HSA on the carbonylation level and the reactivity of HSA-SH and HSA modified with methylglyoxal (MG-HSA-SH) was investigated. Changes of Thiol Group reactivity were followed by determination of pseudo first order rate constant (k') for Thiols reaction with 5,5'-dithiobis(2-nitrobenzoic acid). HSA changes were monitored using native PAG electrophoresis and fluorescence spectroscopy. For FA/HSA molar ratios screening, qTLC and GC were used. FAs increase Thiol Group carbonylation levels from 8% to 20%. The k' values obtained for FAs-free HSA-SH and FAs-free MG-HSA-SH are almost equal (7.5×10(-3) and 7.7×10(-3)s(-1), resp.). Binding of all FAs amplify the reactivity (k' values from 14.6×10(-3) to 26.0×10(-3)s(-1)) of HSA-SH Group for 2-3.5times in the order: palmitic, docosahexaenoic, fish oil extract, stearic, oleic, myristic and eicosapentaenoic acid, due to HSA conformational changes. FAs-bound MG-HSA-SH samples follow that pattern, but their k' values (from 9.8×10(-3) to 14.3×10(-3)s(-1)) were lower compared to unmodified HSA due to additional conformation changes of HSA molecules during carbonylation. Carbonylation level and reactivity of Cys34 Thiol Group of unmodified and carbonylated HSA depend on type of FAs bound to HSA, which implies the possibility for modulation of -SH reactivity (scavenger capacity and antioxidant property) by FAs as a supplement.
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the influence of fatty acids on determination of human serum albumin Thiol Group
Analytical Biochemistry, 2014Co-Authors: Vesna Jovanovic, Ivan D Pavicevic, Marija Takic, Jelena M Acimovic, Ana Z Penezicromanjuk, Ljuba M MandicAbstract:During investigation of the changes of the Cys34 Thiol Group of human serum albumin (HSA) (isolated by affinity chromatography with Cibacron Blue (CB)) in diabetes, we found that the HSA-SH content was higher (11-33%) than the total serum Thiol content. The influence of fatty acids (FA) binding to HSA on this discrepancy was investigated in vitro (using fluorescence and CD spectroscopy and GC) and with HSA samples from diabetic (n=20) and control Groups (n=17). HSA-bound FA determine the selection of HSA molecules by CB and enhance reactivity and/or accessibility of the SH Group. A high content of polyunsaturated FA (35.6%) leads to weaker binding of HSA molecules to CB. Rate constants of DTNB reaction with the SH Group of HSA applied to a CB column, bound-HSA and unbound-HSA fractions, were 4.8×10(-3), 21.6×10(-3), and 11.2×10(-3) s(-1), respectively. The HSA-SH Group of diabetics is more reactive compared with control individuals (rate constants 20.9×10(-3)±4.4×10(-3) vs 12.9×10(-3)±2.6×10(-3) s(-1), P<0.05). Recovery values of the SH Group obtained after chromatography of HSA with bound stearic acid ranged from 110 to 140%, while those for defatted HSA were from 98.5 to 101.7%. Thus, HSA-bound FA leads to an increase of HSA-SH content and a contribution to total serum Thiols, which make the determination of the Thiol Group unreliable.
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improving the reliability of human serum albumin Thiol Group determination
Analytical Biochemistry, 2013Co-Authors: Vesna Jovanovic, Ivan D Pavicevic, Jelena M Acimovic, Ana Z Penezicromanjuk, Ljuba M MandicAbstract:Abstract The Thiol (Cys34) content of human serum albumin (HSA-SH) decreases during oxidative and carbonyl stress and, therefore, could represent a useful parameter in clinical practice. Nevertheless, the reliability of HSA-Thiol determination with Ellman’s method depends on the purity of isolated HSA. Determination of total serum Thiols (mmol/L) and HSA-SH content (mmol -SH/mmol HSA) after HSA isolation from diabetic patient and control sera by a two-step precipitation with ammonium sulfate (AS), as well as HSA-SH contribution (%) to total serum Thiols, was assessed. Purity and yield of isolated HSA were monitored spectrophotometrically and by native polyacrylamide gel electrophoresis. Precipitation of HSA from serum via a two-step method with AS produced HSA with 91.9 ± 3.6% purity and 69.7 ± 4.4% yield, allowing for precise (relative standard deviation of 3.2%) and reliable (comparing with total serum Thiols) measurement of HSA-SH content with DTNB [5,5′-dithiobis-(2-nitrobenzoic acid)]. The content of the HSA-SH Group in patients with type 2 diabetes was significantly ( P
Jelena M Acimovic - One of the best experts on this subject based on the ideXlab platform.
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binding of enterolactone and enterodiol to human serum albumin increase of cysteine 34 Thiol Group reactivity
Food & Function, 2016Co-Authors: Marija Takic, Ivan D Pavicevic, Vesna Jovanovic, Jelena M Acimovic, Tamara N Uzelac, Danijela Risticmedic, Ljuba M MandicAbstract:The interaction of polyphenolic molecules with human serum albumin (HSA) could lead to changes in the reactivity of the HSA Cys34 Thiol Group (HSA-SH). The influences of enterolactone (EL) and enterodiol (ED) binding on HSA-SH reactivity in fatty acid (FA)-free HSA, and in HSA with bound stearic acid (S) in S/HSA molar ratios of 1:1 and 4:1, were investigated by the determination of the pseudo first order rate constants (k') for the Thiol reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). The binding affinities and binding sites of EL and ED were also determined, using fluorescence measurements of the intrinsic fluorescence of Trp214 and diazepam (binding site marker). EL and ED binding to HSA increased the reactivity of HSA-SH in all assayed HSA-enterolignan complexes by 9.1-33.1%. The strongest effects were obtained for FA-free HSA-enterolignan complexes. S modulated/reduced the effect of EL on HSA-SH reactivity, while its influence on the effect of ED was negligible. The binding of enterolignans to HSA was investigated: the binding constants were the highest for FA-free HSA (EL: 11.64 × 10(4) M(-1) and ED: 5.59 × 10(4) M(-1) at 37 °C) and the lowest for S/HSA 4:1-enterolignan complexes (EL: 2.43 × 10(4) M(-1) and ED: 1.92 × 10(4) M(-1)). When the S/HSA ratio was increased, the binding affinities and number of binding sites for EL and ED were decreased. At the same time, a high correlation between binding constants and increased Cys34 reactivity was found (r = 0.974). Competitive experiments using diazepam indicated that the binding of ED and of EL was located in the hydrophobic pocket of site II in HSA. Overall, it is evident that stearic acid could modulate the enterolignan effects on HSA-SH reactivity as well as their binding to HSA. This finding could be important for pharmacokinetics and the expression of enterolignan antioxidant effects in vivo after an intake of lignan rich food.
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fatty acids binding to human serum albumin changes of reactivity and glycation level of cysteine 34 free Thiol Group with methylglyoxal
Chemico-Biological Interactions, 2014Co-Authors: Ivan D Pavicevic, Vesna Jovanovic, Marija Takic, Ana Penezic, Jelena M Acimovic, Ljuba M MandicAbstract:Fatty acids (FAs) binding to human serum albumin (HSA) could lead to the changes of Cys-34 Thiol Group accessibility and reactivity, i.e. its scavenger capacity and antioxidant property. The influence of saturated, mono and poly unsaturated, and fish oil FAs binding to HSA on the carbonylation level and the reactivity of HSA-SH and HSA modified with methylglyoxal (MG-HSA-SH) was investigated. Changes of Thiol Group reactivity were followed by determination of pseudo first order rate constant (k') for Thiols reaction with 5,5'-dithiobis(2-nitrobenzoic acid). HSA changes were monitored using native PAG electrophoresis and fluorescence spectroscopy. For FA/HSA molar ratios screening, qTLC and GC were used. FAs increase Thiol Group carbonylation levels from 8% to 20%. The k' values obtained for FAs-free HSA-SH and FAs-free MG-HSA-SH are almost equal (7.5×10(-3) and 7.7×10(-3)s(-1), resp.). Binding of all FAs amplify the reactivity (k' values from 14.6×10(-3) to 26.0×10(-3)s(-1)) of HSA-SH Group for 2-3.5times in the order: palmitic, docosahexaenoic, fish oil extract, stearic, oleic, myristic and eicosapentaenoic acid, due to HSA conformational changes. FAs-bound MG-HSA-SH samples follow that pattern, but their k' values (from 9.8×10(-3) to 14.3×10(-3)s(-1)) were lower compared to unmodified HSA due to additional conformation changes of HSA molecules during carbonylation. Carbonylation level and reactivity of Cys34 Thiol Group of unmodified and carbonylated HSA depend on type of FAs bound to HSA, which implies the possibility for modulation of -SH reactivity (scavenger capacity and antioxidant property) by FAs as a supplement.
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the influence of fatty acids on determination of human serum albumin Thiol Group
Analytical Biochemistry, 2014Co-Authors: Vesna Jovanovic, Ivan D Pavicevic, Marija Takic, Jelena M Acimovic, Ana Z Penezicromanjuk, Ljuba M MandicAbstract:During investigation of the changes of the Cys34 Thiol Group of human serum albumin (HSA) (isolated by affinity chromatography with Cibacron Blue (CB)) in diabetes, we found that the HSA-SH content was higher (11-33%) than the total serum Thiol content. The influence of fatty acids (FA) binding to HSA on this discrepancy was investigated in vitro (using fluorescence and CD spectroscopy and GC) and with HSA samples from diabetic (n=20) and control Groups (n=17). HSA-bound FA determine the selection of HSA molecules by CB and enhance reactivity and/or accessibility of the SH Group. A high content of polyunsaturated FA (35.6%) leads to weaker binding of HSA molecules to CB. Rate constants of DTNB reaction with the SH Group of HSA applied to a CB column, bound-HSA and unbound-HSA fractions, were 4.8×10(-3), 21.6×10(-3), and 11.2×10(-3) s(-1), respectively. The HSA-SH Group of diabetics is more reactive compared with control individuals (rate constants 20.9×10(-3)±4.4×10(-3) vs 12.9×10(-3)±2.6×10(-3) s(-1), P<0.05). Recovery values of the SH Group obtained after chromatography of HSA with bound stearic acid ranged from 110 to 140%, while those for defatted HSA were from 98.5 to 101.7%. Thus, HSA-bound FA leads to an increase of HSA-SH content and a contribution to total serum Thiols, which make the determination of the Thiol Group unreliable.
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improving the reliability of human serum albumin Thiol Group determination
Analytical Biochemistry, 2013Co-Authors: Vesna Jovanovic, Ivan D Pavicevic, Jelena M Acimovic, Ana Z Penezicromanjuk, Ljuba M MandicAbstract:Abstract The Thiol (Cys34) content of human serum albumin (HSA-SH) decreases during oxidative and carbonyl stress and, therefore, could represent a useful parameter in clinical practice. Nevertheless, the reliability of HSA-Thiol determination with Ellman’s method depends on the purity of isolated HSA. Determination of total serum Thiols (mmol/L) and HSA-SH content (mmol -SH/mmol HSA) after HSA isolation from diabetic patient and control sera by a two-step precipitation with ammonium sulfate (AS), as well as HSA-SH contribution (%) to total serum Thiols, was assessed. Purity and yield of isolated HSA were monitored spectrophotometrically and by native polyacrylamide gel electrophoresis. Precipitation of HSA from serum via a two-step method with AS produced HSA with 91.9 ± 3.6% purity and 69.7 ± 4.4% yield, allowing for precise (relative standard deviation of 3.2%) and reliable (comparing with total serum Thiols) measurement of HSA-SH content with DTNB [5,5′-dithiobis-(2-nitrobenzoic acid)]. The content of the HSA-SH Group in patients with type 2 diabetes was significantly ( P
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the role of the Thiol Group in protein modification with methylglyoxal
Journal of The Serbian Chemical Society, 2009Co-Authors: Jelena M Acimovic, Bojana Stanimirovic, Ljuba M MandicAbstract:Methylglyoxal is a highly reactive α-oxoaldehyde with elevated production in hyperglycemia. It reacts with nucleophilic Lys and Arg side-chains and N-terminal amino Groups causing protein modification. In the present study, the importance of the reaction of the Cys Thiol Group with methylglyoxal in protein modification, the competitiveness of this reaction with those of amino and guanidine Groups, the time course of these reactions and their role and contribution to protein cross-linking were investigated. Human and bovine serum albumins were used as model systems. It was found that despite the very low levels of Thiol Groups on the surface of the examined protein molecules (approx. 80 times lower than those of amino and guanidino Groups), a very high percentage of it reacts (25-85 %). The amount of reacted Thiol Groups and the rate of the reaction, the time for the reaction to reach equilibrium, the formation of a stable product and the contribution of Thiol Groups to protein cross-linking depend on the methylglyoxal concentration. The product formed in the reaction of Thiol and an insufficient quantity of methylglyoxal (compared to the concentrations of the Groups accessible for modification) participates to a significant extent (4 %) to protein cross-linking. Metformin applied in equimolar concentration with methylglyoxal prevents its reaction with amino and guanidino Groups but, however, not with Thiol Groups.