A quantitative method for the assessment of homocysteine thiolactonase enzyme activity.

This assay elucidates an accurate, simple, and precise protocol to quantify the activity of homocysteine thiolactonase (HTase). To establish HTase activity, the enzyme samples were incubated with a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, which contained suitable concentrations of the homocysteine thiolactone as a substrate. To stop the enzyme's reaction, the CUPRAC reagent (Cu(Nc)22+) was added after a suitable incubation time. The reduction of Cu(II)-neocuproine complex (Cu(Nc)22+) to highly coloured Cu(I)-neocuproine complex (Cu(Nc)2+) by the produced homocysteine was quantified spectrophotometrically at 450 nm (CUPRAC method). The increase in the absorbance of the coloured Cu(I)-neocuproine complex (Cu(Nc)2+) was correlated directly to the activity of HTase. ANOVA analysis was utilised to validate the new method against homocysteine thiolactonase activity using the H+ ions liberating method in matched samples. In conclusion, according to the obtained correlation coefficient (0.9995) from the comparison of the current method with the reference method, the current method is effective in assay HTase activity with high reliability.

Enzymatic determination of hypoxanthine in fish samples as a freshness indicator using the CUPRAC colorimetric sensor.

Fish consumption is essential for a healthy diet. However, all seafood including fish are susceptible to deterioration unless properly preserved. Controlling the freshness of fresh or packaged fish is a challenging issue for the food industry in terms of human health and shelf life determination. One of the main indicators showing the freshness of fish is undoubtedly the amount of hypoxanthine (Hx). As soon as the organism dies, Hx begins to be released with the cessation of ATP synthesis and shows a gradual increase over time. Therefore, Hx determination is an important indicator in the control of fish freshness. Based on this fact, a colorimetric method for the enzymatic determination of Hx using the CUPRAC (Cupric ion Reducing Antioxidant Capacity) sensor was developed. Uric acid (UA) and H2O2 are enzymatically produced by xanthine oxidase (XOD) from Hx, and both products respond to the CUPRAC reagent to produce the cuprous neocuproine (Cu(I)-Nc) chromophore chelate formed in situ on a Nafion anionic membrane on which the cationic Cu(II)-Nc complex was fixed. Hx was measured at different time intervals in the meat samples taken from sea bass (Dicentrarchus labrax), which was left to stand at room temperature for a time period between 0 and 24 h; the level of spoilage was determined from the coloration of the CUPRAC membrane sensor (via absorbance measurement at 450 nm). It was observed that there was a linear increase in the amount of Hx during the measurement period. The method was optimized for Hx determination, verified with interference analysis and standard additions to real samples, and validated against HPLC. The linear detection range of the developed method for Hx was 2.0-32.0 µM with an LOD of 0.79 µM, and early stages of fish degradation could be detected at several nanomoles of Hx per gram of fish meat. The proposed method was demonstrated to have distinct superiority over many recent colorimetric sensors of fish freshness in regard to its lower LOD for Hx, wider linear range, capability to cope with interferents (including biologically important antioxidants, such as cysteine, reduced glutathione, ascorbic acid, UA and α-tocopherol) and applicability to real samples.

Synthesis of New Regioisomers of 5-Nitro-1,4-Naphthoquinone, Evaluation of Antioxidant and Catalase Inhibition Activities.

The studies on nitronaphthoquinone derivatives are rare in the literature, and the nitro group associated with the aromatic ring in the quinone system is known to increase the biological activity of naphthoquinone due to its electron-withdrawing properties. In the course of quinone derivatives, the new N(H)-substituted-5-nitro-1,4-naphthoquinones (NQ) as regioisomers were synthesized by reactions of 2,3-dichloro-5-nitro-1,4-naphthoquinone with some heterocyclic ring substituted nucleophiles such as anilines, piperazines, or morpholines, according to a Michael 1,4-addition mechanism. Five NQ regioisomer couples having different functional group (2-chloro-isomers 3, 5, 7, 9 and 13; 3-chloro-isomers 2, 4, 6, 8 and 12) are reported here. All new synthesized compounds were characterized by spectroscopic methods and two-dimensional NMR techniques 1H-1H correlated spectroscopy (COSY).The synthesized NQ regioisomers were evaluated for catalase enzyme inhibitory activities and antioxidant efficiency. The synthesized regioisomers were screened for their antioxidant capacity using the cupric-reducing antioxidant capacity (CUPRAC) method. 2-Chloro-3-((2,4-dimethoxyphenyl)amino)-5-nitronaphthalene-1,4-dione (5) showed the highest antioxidant capacity with a 1.80±0.06 CUPRAC-trolox equivalent antioxidant capacity (TEAC) coefficient. Compound 5 also showed strongest catalase enzyme inhibitory activity. The antioxidant capacity results of all 2-chloro regioisomers are higher than the 3-chloro regioisomers. Likewise, also catalase enzyme inhibitory activities results were determined in the same way, except for one regioisomer pair. The catalase was effectively inhibited by the newly synthesized compounds, with % inhibition values in the range of 0.71-0.86%. Some of these NQ compounds also showed remarkable antioxidant capacities.

Determination of total antioxidant capacity of humic acids using CUPRAC, Folin-Ciocalteu, noble metal nanoparticle- and solid-liquid extraction-based methods.

Total antioxidant capacity (TAC) of humic acid (HA) samples was determined using CUPRAC (CUPric Reducing Antioxidant Capacity), FC (Folin-Ciocalteu), QUENCHER-CUPRAC, QUENCHER-FC, Ag-NP (Silver nanoparticle)‒ and Au-NP (Gold nanoparticle)‒based methods. Conventional FC and modified FC (MFC) methods were applied to solid samples. Because of decreased solubility of Folin-Ciocalteu's phenol reagent in organic solvents, solvent effect on TAC measurement was investigated using QUENCHER-CUPRAC assay by using ethanol:distilled water and dimethyl sulfoxide:distilled water with varying ratios. To see the combined effect of solubilization (leaching) and TAC measurement of humic acids simultaneously, QUENCHER experiments were performed at 25°C and 50°C; QUENCHER-CUPRAC and QUENCHER-FC methods agreed well and had similar precision in F-statistics. Although the Gibbs free energy change (ΔG°) of the oxidation of HA dihydroxy phenols with the test reagents were negative, the ΔG° was positive only for the reaction of CUPRAC reagent with isolated monohydric phenols, showing CUPRAC selectivity toward polyphenolic antioxidants. This is the first work on the antioxidant capacity measurement of HA having a sparingly soluble matrix where enhanced solubilization of bound phenolics is achieved with coupled oxidation by TAC reagents.

Novel pro-oxidant activity assay for polyphenols, vitamins C and E using a modified CUPRAC method.

In this study, a direct assay, a modified CUPRAC (Cupric Ion Reducing Antioxidant Capacity) method, is developed to determine transition metal ion (Cu(II))-catalyzed pro-oxidant activity of polyphenolic compounds, vitamins C and E, and herbal samples in the presence of proteins containing thiol groups. Since transition metal ion-catalyzed pro-oxidant activity of phenolics is usually initiated with the reduction of the metal to lower oxidation states (as a prerequisite of Fenton-type reactions), this method involves the reduction of copper(II) ions to copper(I) by polyphenolic compounds (simultaneously giving rise to reactive species), binding of the formed Cu(I) to egg white protein -SH groups, and liberation of copper(I)-neocuproine (Cu(I)-Nc) chelate (showing maximum absorbance at 450 nm) by treating the incubation product with a neocuproine-ammonium acetate mixture. The proposed method is validated against atomic absorption spectrometric (AAS) determination of protein-bound copper and protein carbonyl assay of oxidative stress. The proposed assay is faster and more specific than the carbonyl assay, and uses low-cost reagents and equipment. Pro-oxidant activity (i.e. proportional to absorbance) varies linearly over a relatively wide range with concentration, as opposed to the reciprocal correlations (i.e. linear regression of 1/(pro-oxidant activity) versus 1/concentration) of other similar assays. The pro-oxidant activity order of the tested antioxidant compounds in terms of 'Quercetin Equivalent Pro-oxidant Activity' (QREPA) coefficients is: gallic acid > epicatechin > quercetin ≈ catechin > α-tocopherol > rosmarinic acid > trolox > caffeic acid > ascorbic acid.