Comparative Evaluation of UV-Vis Spectroscopy-Based Approaches for Hemoglobin Quantification: Method Selection and Practical Insights

Abstract

The growing demand for effective alternatives to red blood cells (RBCs) has spurred significant research into hemoglobin (Hb)-based oxygen carriers (HBOCs). Accurate characterization of HBOCs-including Hb content, encapsulation efficiency, and yield-is crucial for ensuring effective oxygen delivery, economic viability, and the prevention of adverse effects caused by free Hb. However, the choice of quantification methods for HBOCs is often driven more by tradition than by a thorough assessment of available options. This study meticulously compares various UV-vis spectroscopy-based methods for Hb quantification, focusing on their efficacy in measuring Hb extracted from bovine RBCs across different concentration levels. The findings identify the sodium lauryl sulfate Hb method as the preferred choice due to its specificity, ease of use, cost-effectiveness, and safety, particularly when compared to cyanmethemoglobin-based methods. Additionally, the study discusses the suitability of these methods for HBOC characterization, emphasizing the importance of considering carrier components and potential interferences by analyzing the absorbance spectrum before selecting a method. Overall, this study provides valuable insights into the selection of accurate and reliable Hb quantification methods, which are essential for rigorous HBOC characterization and advancements in medical research.

Keywords: absorbance-based assays; blood substitutes; hemoglobin; hemoglobin-based oxygen carriers; protein quantification.

Scheme 1

Representation of the different hemoglobin (Hb) quantification methods: (A) The bicinchoninic acid (BCA) assay, showing the biuret reaction, the BCA–cuprous ion (Cu⁺) complex formed, and its characteristic absorbance (Abs) spectrum. (B) The Bradford Plus assay (Coomassie blue method), showing the interaction of the Coomassie blue G-250 dye and a protein and its characteristic Abs spectrum. (C) The method of Abs at 280 nm (Abs280), showing a fragment of the Hb beta chain amino acid (AA) sequence (i.e., residues 34–38, VYPWT), with two representative aromatic AAs that contribute to the Abs signal at 280 nm. The characteristic Abs spectrum in that region is also shown. (D) The cyan (CN)–Hb method, which starts with the oxidation of oxygenated Hb (oxyHb) into methemoglobin (metHb) by potassium hexacyanoferrate (III) K₃[Fe(CN)₆]. The addition of potassium cyanide (KCN) results in the formation of the CN-metHb complex. The characteristic CN-metHb Abs spectrum is also shown. (E) The sodium lauryl sulfate (SLS)–Hb method, showing the interactions between the SLS head group and the positively charged peptide chains of Hb, as well as the hemichrome structure. The Abs spectrum of the SLS-Hb complex is also shown.

Scheme 2

Schematic illustration of the experimental setup for the quantification of hemoglobin (Hb). For simplicity, only the high-concentration Hb stock is shown, but the same process was repeated for the low- and medium-concentration Hb stocks. Briefly, different Hb standards and dilutions of the Hb stocks were prepared. Then, both non-specific (i.e., bicinchoninic acid (BCA) assay, Coomassie blue (CB), and absorbance (Abs) at 280 nm (Abs280)) and Hb-specific quantification methods (i.e., cyan–Hb (CN-Hb) and sodium lauryl sulfate (SLS)–Hb) were conducted by measuring the Abs signal at the specified wavelength for each method.

Figure 1

(A) Reaction mechanism of the bicinchoninic acid (BCA) assay: (i) schematic of the biuret reaction showing the protein’s ability to reduce the cupric ion (Cu²⁺) to the cuprous one (Cu⁺) and (ii) formation of the BCA-Cu⁺ complex, which has the characteristic absorbance (Abs) signal at 562 nm. (B) UV-vis spectra of the bovine hemoglobin (Hb) standards after incubation with the BCA reagents. (C) Standard curve correlating the concentrations of the Hb standards (i.e., 0–1.5 mg mL⁻¹) with their Abs signal at 562 nm.

Figure 2

(A) Reaction mechanism of the Coomassie blue (CB) assay: electrostatic interactions between the Coomassie G-250 dye and the basic amino acids of a protein. (B) UV-vis spectra of the bovine hemoglobin (Hb) standards after incubation with the CB reagent. (C) Standard curve correlating the concentrations of the Hb standards (i.e., 0–1 mg mL⁻¹) with their absorbance (Abs) signal at 595 nm.

Figure 3

(A) Fragment of the hemoglobin (Hb) beta chain amino acid (AA) sequence (i.e., residues 34–38, VYPWT) containing two aromatic AAs that contribute to the absorbance (Abs) signal at 280 nm. (B) UV-vis spectra of bovine hemoglobin (Hb) standards in the 240–320 nm range. (C) Standard curve correlating the concentrations of the Hb standards (i.e., 0–2 mg mL⁻¹) with their Abs signal at 280 nm.

Figure 4

(A) Reaction mechanism of the cyan (CN)–hemoglobin (Hb) method: potassium hexacyanoferrate (III) (K₃[Fe(CN)₆]) oxidizes Hb into methemoglobin (metHb), which is finally converted into CN-metHb by potassium cyanide (KCN). (B) UV-vis spectra of the bovine Hb standards after incubation with K₃[Fe(CN)₆] and KCN. (C) Standard curve correlating the concentrations of the Hb standard solutions (i.e., 0–5 mg mL⁻¹) with their absorbance (Abs) signal at 540 nm.

Figure 5

(A) Reaction mechanism of the sodium lauryl sulfate (SLS) assay: following SLS addition, hemoglobin (Hb) is transformed into methemoglobin (metHb) rendering a hemichrome-like complex. (B) UV-vis spectra of the bovine hemoglobin (Hb) standards after incubation with SLS. (C) Standard curve correlating the concentrations of the Hb standards (i.e., 0–20 mg mL⁻¹) with their absorbance (Abs) signal at 539 nm.

Figure 6

Concentrations of the three hemoglobin (Hb) stocks (low, medium, and high Hb concentrations) assessed by the different assays. (A) Boxplots with data points, showing the quartile distribution and the mean value (red square). (B) Mean values with standard deviation for n = 3 data analysis. The dotted gray lines represent the average Hb concentration of each stock, calculated from the results of all the methods (i.e., 66.0 ± 1.8 mg mL⁻¹, 136.0 ± 4.3 mg mL⁻¹, and 179.3 ± 5.0 mg mL⁻¹ for the low-, medium-, and high-concentration Hb stocks, respectively). BCA: bicinchoninic acid assay; CB: Coomassie blue assay; Abs₂₈₀: absorbance at 280 nm method; CN: cyan–Hb method; SLS: sodium lauryl sulfate–Hb method.