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Modified cupric reducing antioxidant capacity (CUPRAC) assay for measuring the antioxidant capacities of thiol-containing proteins in admixture with polyphenols.
Talanta. 2009 Jul 15; 79(2):344-51.T

Abstract

Proteins are not considered as true antioxidants but are known to protect antioxidants from oxidation in various antioxidant activity assays. This study aims to investigate the contribution of proteins, especially thiol-containing proteins, to the observed overall antioxidant capacity measured by known methods. To determine the antioxidant properties of thiol-containing proteins, the CUPRAC method of antioxidant assay using the oxidizing reagent Cu(II)-neocuproine previously used for simultaneous analysis of cystine and cysteine was adopted. While the CUPRAC method is capable of determining all antioxidant compounds including thiols in complex sample matrices, the Ellman method of thiol quantitation basically does not respond to other antioxidants. The antioxidant quantities in the selected samples were assayed with the ABTS and FRAP methods as well as with the CUPRAC method. In all applied methods, the dilutions were made with a standard pH 8 buffer used in the Ellman method by substituting the Na(2)EDTA component of the buffer with sodium citrate. On the other hand, the standard CUPRAC protocol was modified by substituting the pH 7 ammonium acetate buffer (at 1M concentration) with 8M urea buffer adjusted to pH 7 by neutralizing with 6M HCl. Urea helps to partly solubilize and denaturate proteins so that their buried thiols be oxidized more easily. All methods used in the estimation of antioxidant properties of proteins (i.e., CUPRAC, Ellman, ABTS, and FRAP) were first standardized with a simple thiol compound, cysteine, by constructing the calibration curves. The molar absorptivities of these methods for cysteine were: epsilon(CUPRAC)=7.71x10(3), epsilon(Ellman)=1.37x10(4), epsilon(ABTS)=2.06x10(4), and epsilon(FRAP)=2.98x10(3)L mol(-1)cm(-1). Then these methods were applied to various samples containing thiols, such as glutathione (reduced form:GSH), egg white, whey proteins, and gelatin. Additionally, known quantities of selected antioxidants were added to these samples to show the additivity of responses.

Authors+Show Affiliations

Istanbul University, Faculty of Engineering, Department of Chemistry, Avcilar 34320 Istanbul, Turkey.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Evaluation Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

19559889

Citation

Cekiç, Sema Demirci, et al. "Modified Cupric Reducing Antioxidant Capacity (CUPRAC) Assay for Measuring the Antioxidant Capacities of Thiol-containing Proteins in Admixture With Polyphenols." Talanta, vol. 79, no. 2, 2009, pp. 344-51.
Cekiç SD, Başkan KS, Tütem E, et al. Modified cupric reducing antioxidant capacity (CUPRAC) assay for measuring the antioxidant capacities of thiol-containing proteins in admixture with polyphenols. Talanta. 2009;79(2):344-51.
Cekiç, S. D., Başkan, K. S., Tütem, E., & Apak, R. (2009). Modified cupric reducing antioxidant capacity (CUPRAC) assay for measuring the antioxidant capacities of thiol-containing proteins in admixture with polyphenols. Talanta, 79(2), 344-51. https://doi.org/10.1016/j.talanta.2009.03.061
Cekiç SD, et al. Modified Cupric Reducing Antioxidant Capacity (CUPRAC) Assay for Measuring the Antioxidant Capacities of Thiol-containing Proteins in Admixture With Polyphenols. Talanta. 2009 Jul 15;79(2):344-51. PubMed PMID: 19559889.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Modified cupric reducing antioxidant capacity (CUPRAC) assay for measuring the antioxidant capacities of thiol-containing proteins in admixture with polyphenols. AU - Cekiç,Sema Demirci, AU - Başkan,Kevser Sözgen, AU - Tütem,Esma, AU - Apak,Reşat, Y1 - 2009/04/05/ PY - 2008/10/28/received PY - 2009/03/23/revised PY - 2009/03/27/accepted PY - 2009/6/30/entrez PY - 2009/6/30/pubmed PY - 2009/9/10/medline SP - 344 EP - 51 JF - Talanta JO - Talanta VL - 79 IS - 2 N2 - Proteins are not considered as true antioxidants but are known to protect antioxidants from oxidation in various antioxidant activity assays. This study aims to investigate the contribution of proteins, especially thiol-containing proteins, to the observed overall antioxidant capacity measured by known methods. To determine the antioxidant properties of thiol-containing proteins, the CUPRAC method of antioxidant assay using the oxidizing reagent Cu(II)-neocuproine previously used for simultaneous analysis of cystine and cysteine was adopted. While the CUPRAC method is capable of determining all antioxidant compounds including thiols in complex sample matrices, the Ellman method of thiol quantitation basically does not respond to other antioxidants. The antioxidant quantities in the selected samples were assayed with the ABTS and FRAP methods as well as with the CUPRAC method. In all applied methods, the dilutions were made with a standard pH 8 buffer used in the Ellman method by substituting the Na(2)EDTA component of the buffer with sodium citrate. On the other hand, the standard CUPRAC protocol was modified by substituting the pH 7 ammonium acetate buffer (at 1M concentration) with 8M urea buffer adjusted to pH 7 by neutralizing with 6M HCl. Urea helps to partly solubilize and denaturate proteins so that their buried thiols be oxidized more easily. All methods used in the estimation of antioxidant properties of proteins (i.e., CUPRAC, Ellman, ABTS, and FRAP) were first standardized with a simple thiol compound, cysteine, by constructing the calibration curves. The molar absorptivities of these methods for cysteine were: epsilon(CUPRAC)=7.71x10(3), epsilon(Ellman)=1.37x10(4), epsilon(ABTS)=2.06x10(4), and epsilon(FRAP)=2.98x10(3)L mol(-1)cm(-1). Then these methods were applied to various samples containing thiols, such as glutathione (reduced form:GSH), egg white, whey proteins, and gelatin. Additionally, known quantities of selected antioxidants were added to these samples to show the additivity of responses. SN - 1873-3573 UR - https://www.unboundmedicine.com/medline/citation/19559889/Modified_cupric_reducing_antioxidant_capacity__CUPRAC__assay_for_measuring_the_antioxidant_capacities_of_thiol_containing_proteins_in_admixture_with_polyphenols_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0039-9140(09)00264-1 DB - PRIME DP - Unbound Medicine ER -