D5941

A stock solution was prepared by adding 15 g of bovine hemoglobin (BH) or human hemoglobin (HH) in 100 mL of ultrapure water. The supernatant was recovered after centrifugation at 4000 min⁻¹ for 30 min (Eppendorf AG, 22,331 Hamburg, Germany; Centrifuge 5804 R, Brinkmann Instruments, Westbury, NY, USA). The Drabkin method established by Crosby, Munn, and Furth (1954) was used to determine the real concentrations of BH (C_BH) and HH (C_HH) (1954). This is a spectrophotometric method used to quantitatively determine the hemoglobin concentration in whole blood. For this purpose, 20 µL of the sample was added to 10 mL of Drabkin’s reagent D5941, Sigma-Aldrich, for 15 min at room temperature and it was protected from light. Then, the absorbance was measured using a UV spectrophotometer (ChemStation UV–VIS spectrophotometer 8453A, Agilent Technologies, Santa Clara, CA, USA) at a wavelength of 540 nm. The measurements were reported on the calibration curve. From this concentration C of the bovine or human stock solution, several hemoglobin solutions were prepared by dilution to the following precise concentrations C_BH and C_HH: 1%, 2%, 8% and 10% (w/v).

Drabkin’s reagent is a spectrophotometric method used for the quantitative determination of hemoglobin concentration in whole blood. Briefly, 40 µL of the sample were added to 10 mL of the Drabkin’s reagent (D5941, Sigma-Aldrich, Oakville, ON, Canada) for 15 min at room temperature. Then, the absorbance was read at a wavelength of 540 nm by a UV spectrophotometer (ChemStation 8453A UV-Vis Spectrophotometer, Agilent Technologies, Santa Clara, CA, USA) and reported into the calibration curve. From the concentration C of the stock solution, solution of purified bovine hemoglobin was prepared by dilution to precise concentrations of hemoglobin C_BH: 1% (w/v).