**2.3 Hemoglobin oxidation studies**

Prepare stocks of iron, glutathione, deferoxamine, and deferiprone solution and dilute them with distilled water to reach the exact quantities and concentrations required for the experiment. The absorbance spectra of hemoglobin were measured using a UV-VIS spectrophotometer, either in hemolysate or pure form.

"There is no discernible difference in the visible area between the absorption spectra obtained from hemolysate and pure hemoglobin," writes Horecker (1943). To view the distinctive spectrum activity of oxy-Hb, the oxy-Hb must be scanned in *Perspective Chapter: Iron Chelation Inhibits Reduced Glutathione (GSH) as a Prooxidant… DOI: http://dx.doi.org/10.5772/intechopen.109462*

(500–700 nm) wavelength before adding the iron or chelators to analyze their influence on oxyhemoglobin spectra, oxy-Hb has two distinct peaks at 541 and 577 nm. The oxidation of hemoglobin was evaluated by spectrophotometric analysis (500–700 nm); the concentrations of oxy-, met-, and hemichrome hemoglobin were estimated using the Winterbourn technique.

After assessing the effect of iron on oxy-Hb in both models, hemolysate and pure Hb, the iron concentration that causes the most oxidative damage will be chosen for the next tests. Because hemolysate contains its own glutathione, we shall solely evaluate the impact of reduced glutathione on purified Hb. The prepared glutathione solution, as well as precise iron concentrations, will be added. The impact of the iron chelator will then be investigated.

Microsoft Excel was used for statistical analysis. The concentration of oxyhemoglobin was determined using the Winterbourn technique from the absorption spectra. Experiments were carried out in triplicate at each time point, and the scanning data displayed are representative of the outcomes of the experiments.

### **3. Results and data analysis**

**Figures 1** and **2** show the findings of spectrum measurements in iron-mediated oxyhemoglobin oxidation on hemolysate with regard to time in minutes. The UV-visible spectra in **Figure 1A** and **B** indicate the effect of dose-dependent ironmediated oxidative damage on oxyhemoglobin in hemolysate during the first 10 minutes of iron addition. **Figure 2** is visibly expressing the effect of iron by causing oxidative damage to the oxyhemoglobin content of hemolysate. The hemolysate is losing the normal red pigmentation of the hemoglobin into brown color due to iron effect with different concentrations concerning time (5 minutes).

The results of the spectral measurements in iron-mediated oxyhemoglobin oxidation on purified hemoglobin only with respect to the time that was measured

#### **Figure 1.**

*UV-visible spectra of dose-dependent iron-mediated oxidative damage on oxyhemoglobin in hemolysate after 10 minutes of iron addition are shown in A. The % oxyhemoglobin derived from the spectral shift with dosedependent iron-induced hemoglobin oxidation in hemolysate is shown in B.*

#### **Figure 2.**

*Figure demonstrating the effect of iron on hemolysate oxyhemoglobin concentration, which causes oxidative damage. Because of the iron activity in variable levels throughout time, the hemolysate loses its normal red color and turns brown (10 minutes). (1) represents the control, (2) represents 100 μM Fe3+, (3) represents 175 μM Fe3+, and (4) represents 250 μM.*

in minutes are shown in **Figure 3**. The UV-visible spectra shows the effect of dosedependent iron-mediated oxidative damage on oxyhemoglobin in purified hemoglobin during the first minute of iron addition, where there was no visible change. **Figure 4** visibly expresses the effect of iron in causing oxidative damage to the oxyhemoglobin content of purified hemoglobin. There is no visible change in the normal red pigmentation of the hemoglobin of different iron concentrations concerning time (5 minutes). **Figure 5** is a graph showing the binding of iron chelators with the iron. As the iron

#### **Figure 3.**

*A: UV-visible spectra showing the effect of dose-dependent iron-mediated oxidative damage on oxyhemoglobin in purified hemoglobin after 5 minutes of iron addition. B shows there is no change in spectra after 10 minutes.*

*Perspective Chapter: Iron Chelation Inhibits Reduced Glutathione (GSH) as a Prooxidant… DOI: http://dx.doi.org/10.5772/intechopen.109462*

#### **Figure 4.**

*Figure visibly expressing the effect of iron in causing oxidative damage to the oxyhemoglobin content of purified hemoglobin. There is no visible change in the normal red pigmentation of the hemoglobin different iron concentrations with respect to time (5 minutes). (1): the control, (2): 100 μM Fe3+, (3): 175 μM Fe3+, and (4): 250 μM Fe3+.*

chelators are added to the iron in the cuvette, the color changed. Also, the absorbance is changed indicating a reaction (peaks) between iron and the chelating agent. As shown, the red and black lines, where the iron is bound with chelators are showing noticeable peaks that are indicative of a binding reaction. The maximum absorbance of L1-Fe and DFO-Fe complexes is achieved at 450 to 470 nm and 430 to 460 nm, respectively [4].

**Figure 6** depicts iron-mediated hemoglobin oxidation and the inhibitory impact of DFO and L1 in hemolysate. In A, the addition of 250 µM results in a significant drop in the percentage of oxyhemoglobin, and when the iron chelator DFO is applied, the percentage of oxyhemoglobin is recovered to above 90%. Similarly, in B, a bidentate iron chelator L1 has a similar response, inhibiting iron-mediated hemoglobin oxidation [5].

**Figure 7** depicts the iron-mediated oxidation of pure hemoglobin, with no substantial loss of % oxyhemoglobin in purified hemoglobin solution as compared to hemolysate. However, the mixing of iron and glutathione in pure hemoglobin produced results comparable to hemolysate. Iron chelator DFO inhibits iron and glutathione-mediated hemoglobin oxidation in A, whereas iron chelator L1 inhibits comparable findings in B.

### **4. Discussions**

Excess bio-reactive iron contributes to the formation of free radicals, which can lead to oxidative damage to the hemoglobin in our red blood cells. This oxidative

#### **Figure 5.**

*The graph depicts the binding of iron chelators to iron; as the iron chelators are added to the iron in the cuvette, the color changes, suggesting a reaction (peaks) between iron and the chelating agent. The red and black lines, where the iron is bonded with chelators, display prominent peaks, indicating a binding response. L1-Fe and DFO-Fe complexes had maximal absorbance at 450 to 470 nm and 430 to 460 nm, respectively [1].*

#### **Figure 6.**

*A: Fe3+-mediated hemoglobin oxidation in hemolysate. Shown is the oxyhemoglobin concentration following the addition of exogenous 250 μM Fe3+ and DFO in hemolysate at pH 7.4. Hemoglobin concentration was adjusted to ~ 40 μM heme. B: shows the inclusion of 250 μM Fe3+ and deferiprone L1 a bidentate iron chelator.*

#### **Figure 7.**

*Reduced glutathione (GSH) has been found to be a mediator of Fe3+-mediated hemoglobin oxidation in crude purified HbA (α2 ß2). The oxyhemoglobin content in HbA solution prepared of crude purified hemoglobin after the addition of exogenous 100 μM Fe3+ and 1 mM GSH is shown. In contrast to μ100 M Fe3+ alone, no appreciable hemoglobin oxidation occurs. A and B shows DFO and L1 at 100 μM or 1 mM reduced Fe3+ GSH-driven hemoglobin oxidation. The content of hemoglobin was adjusted to 40 M heme.*

#### *Perspective Chapter: Iron Chelation Inhibits Reduced Glutathione (GSH) as a Prooxidant… DOI: http://dx.doi.org/10.5772/intechopen.109462*

damage happens as a result of conditions, such as thalassemia and other hemoglobinopathies [6]. In these illnesses, the hemoglobin moiety's qualitative or quantitative deficiency causes iron to be ejected from the red cell into circulation [2]. Patients with thalassemia or sickle cell anemia receive blood transfusions on a regular basis as a treatment technique, which contributes to the same problem of having excess circulatory iron. This fact was examined in this study. The effect of iron on hemoglobin was investigated *in vitro* using two study models: hemolysate and pure hemoglobin solution. Winterbourn has previously demonstrated in high-throughput investigations that switching from oxyhemoglobin to methemoglobin leads to the loss of the peaks of typical normal hemoglobin spectra [7] and that is similar to what we have shown here.

From research findings, it is clear that iron has a great impact on the hemoglobin oxidation rate in hemolysate [8]. Upon adding iron in dose-dependent manner (100, 175, and 250 μM) the hemolysate constitution of oxyhemoglobin is reduced. It is also visibly seen that the hemolysate is losing the normal red pigmentation of the hemoglobin into brown color due to the iron effect of different concentrations to time (5 minutes). RBCs have been the most ferruginous cells in the human body, where the single circulating RBC contains ~ 20 mM iron, [9]. With mild hemolysis, loose iron in the body may cause the observed type of oxidative events in our body.

In contrast to hemolysate, iron does not affect purified hemoglobin. Even after 5 minutes, no change has been observed for the oxy-Hb absorbance spectra. Iron, being an oxidizing agent, requires a catalyst to produce free radicals, which are responsible for oxidative damage to hemoglobin. Free radicals are formed by enzymes, such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, nitric oxide synthase (NOS), xanthine oxidase (XO), cytochrome P450, cyclooxygenase (COX), and lipoxygenase [10]. As evidence, **Figure 4** shows that there is no visible change in the normal red pigmentation of the hemoglobin with different iron concentrations with respect to time (5 minutes). However, when glutathione was added, there was a steep decrease in the percentage of oxyhemoglobin as a result of the pro-oxidation effect of glutathione. Similarly, it was proved from Atamna's research that reduced glutathione (GSH) can degrade heme in solution with a pH of 7 [11].
