Lamba 25 spectrophotometer

The continuous ODC enzyme activity was measured by the reactions that were coupled with the phosphoenolpyruvate carboxylase and malate dehydrogenase, and the ODC enzyme (0.38 μM) was inhibited with various quantities of AZ proteins [17 (link)]. The assay mixture in a final volume of 0.5 mL contained 30 mM Tris-HCl at pH 7.4, 10 mM ornithine, 0.02 mM pyridoxal 5′-pyrophosphate, and 0.4 mL of the CO2-L3K assay kit solution (DCL, Charlottetown, Canada), which had 12.5 mM PEP, >0.4 U/mL phosphoenolpyruvate carboxylase (microbial), >4.1 U/mL malate dehydrogenase (mammalian), and 0.6 mM NADH analog.
The reaction was traced at the absorbance decrease at 405 nm using a Perkin–Elmer Lamba-25 spectrophotometer, and the production of 1 mmol of CO₂ was accompanied by the oxidation of 1 mmol of NADH analog in this coupled reaction. For the NADH analog, an extinction coefficient of 2410 cm⁻¹ mM⁻¹ was used in the calculations. The IC₅₀ value of each inhibition plot was calculated with the following equation: where A and B are the minimum and maximum ODC enzyme activity, respectively, and the Hill slope provides the largest slope of the curve. The IC₅₀ value denotes the AZ concentration that is required for inhibiting 50% of the ODC enzyme activity. All calculations were carried out with the SigmaPlot 10.0 software program (Jandel, San Rafael, CA, USA).

The total antioxidant potential of tomato peel extracts was determined using the FRAP assay reported by Benzie and Strain [67 (link)]. This is based on the reduction in Fe³⁺-2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ) to a blue-colored Fe²⁺-TPTZ. Briefly, FRAP reagent was freshly prepared and the solution was heated at 37 °C for one hour. The absorbance was read at 593 nm using an UV-Vis spectrophotometer (Perkin Elmer, Lamba 25 spectrophotometer, Waltham, MA, USA). The FRAP values of the samples, which were expressed as µmol of Fe²⁺ per g of fresh weight (FW), were determined from a standards curve built up using ferrous sulphate.