Homocysteine and its cyclic ester homocysteine thiolactone (HTL) have been involved in the detrimental consequences associated to hyperhomocysteinemia, an independent risk factor for vascular diseases. HTL reacts with protein lysine residues in a process named N-homocysteinylation. The aim of our study was to evaluate the in vitro effects of HTL on the fibrinogen through electrophoretic methods. Fibrinogen was incubated with HTL at different molar ratios and structural changes of the protein were assessed by polyacrylamide gel electrophoresis (PAGE), capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF). Ellman´s reaction, CZE and proton nuclear magnetic resonance (1H NMR) were used to evaluate HTL hydrolyisis. On denaturing PAGE numerous bands were observed, being the three lower bands identical to those obtained by treatment with 2-mercaptoethanol. This effect was also detected by CZE. The results show a reducing action of HTL on the fibrinogen molecule, probably attributed to the sulfhydryl groups generated by N-homocysteinylation and/or by the ones present in the homocysteine molecule yielded by HTL hydrolysis. In order to distinguish between these two options, HTL stability was evaluated at different pH and incubation times. The results showed minimum HTL hydrolysis in our experimental conditions. We postulate that the reducing effect observed would be mainly associated to the new sulfhydryl groups generated by the N-homocysteinylation process. Moreover, a displacement of the HTL-treated fibrinogen isoforms towards more acidic pH values was detected. The structural changes of N-homocysteinylated fibrinogen could be involved in the pathological consequences of hyperhomocysteinemia. © 2017, Springer Science+Business Media, LLC.
|Título:||Structural changes of fibrinogen molecule mediated by the N-homocysteinylation reaction|
|Autor:||Genoud, V.; Quintana, P.G.; Gionco, S.; Baldessari, A.; Quintana, I.|
|Filiación:||Facultad de Ciencias Exactas y Naturales, Departamento de Química Biológica, Laboratorio de Hemostasia y Trombosis, Universidad de Buenos Aires, Ciudad Universitaria-Pabellón 2, Buenos Aires, C1428EHA, Argentina|
Facultad de Ciencias Exactas y Naturales, Departamento de Química Orgánica y UMYMFOR, Laboratorio de Biocatálisis, Universidad de Buenos Aires, Ciudad Universitaria-Pabellón 2, Buenos Aires, C1428EHA, Argentina
|Palabras clave:||Electrophoretic methods; Fibrinogen; Homocysteine-thiolactone hydrolysis; N-homocysteinylation; Proton nuclear magnetic resonance; fibrinogen; homocysteine; homocysteine thiolactone; mercaptoethanol; thiol group; fibrinogen; homocysteine thiolactone; Article; biochemical recurrence; capillary isoelectric focusing; capillary zone electrophoresis; controlled study; disulfide bond; homocysteinylation; hydrolysis; in vitro study; molecular stability; pH; polyacrylamide gel electrophoresis; priority journal; protein modification; protein structure; proton nuclear magnetic resonance; analogs and derivatives; capillary electrophoresis; chemistry; human; hyperhomocysteinemia; isoelectric focusing; metabolism; pathology; two dimensional gel electrophoresis; Electrophoresis, Capillary; Electrophoresis, Gel, Two-Dimensional; Fibrinogen; Homocysteine; Humans; Hydrogen-Ion Concentration; Hydrolysis; Hyperhomocysteinemia; Isoelectric Focusing|
|Página de inicio:||66|
|Página de fin:||76|
|Título revista:||Journal of Thrombosis and Thrombolysis|
|Título revista abreviado:||J. Thromb. Thrombolysis|
|CAS:||fibrinogen, 9001-32-5; homocysteine, 454-28-4, 6027-13-0; homocysteine thiolactone, 10593-85-8, 3622-59-1, 6038-19-3; mercaptoethanol, 60-24-2; Fibrinogen; Homocysteine; homocysteine thiolactone|
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---------- APA ----------Genoud, V., Quintana, P.G., Gionco, S., Baldessari, A. & Quintana, I.
. Structural changes of fibrinogen molecule mediated by the N-homocysteinylation reaction. Journal of Thrombosis and Thrombolysis, 45(1), 66-76.
---------- CHICAGO ----------Genoud, V., Quintana, P.G., Gionco, S., Baldessari, A., Quintana, I.
"Structural changes of fibrinogen molecule mediated by the N-homocysteinylation reaction"
. Journal of Thrombosis and Thrombolysis 45, no. 1
(2018) : 66-76.
---------- MLA ----------Genoud, V., Quintana, P.G., Gionco, S., Baldessari, A., Quintana, I.
"Structural changes of fibrinogen molecule mediated by the N-homocysteinylation reaction"
. Journal of Thrombosis and Thrombolysis, vol. 45, no. 1, 2018, pp. 66-76.
---------- VANCOUVER ----------Genoud, V., Quintana, P.G., Gionco, S., Baldessari, A., Quintana, I. Structural changes of fibrinogen molecule mediated by the N-homocysteinylation reaction. J. Thromb. Thrombolysis. 2018;45(1):66-76.