Interaction of Normal and Sickle Hemoglobins for Sodium Dodecylsulphate and Hydrogen Peroxide at pH 5.0 and 7.2

Clinical manifestations of malaria primarily result from proliferation of the parasite within the hosts' erythrocytes. The malaria parasite digests hemoglobin within its digestive vacuole through a sequential metabolic process involving multiple proteases. The activities of these proteases could lead to the production of ROS which could lead to the death of the parasites due to the destruction of their membrane. The action of SDS on hemoglobins can be likened to the way malarial proteases destabilizes host hemoglobin. Hence, the study was designed to determine the binding parameters of SDS and H2O2 for normal, sickle trait carrier and sickle hemoglobins at pH 5.0 and 7.2 using UV-VIS Titration Spectrophotometry. Hb-SDS interactions were significantly different at pH 5.0 but were not at pH 7.2. Also, Hb-H2O2 interactions were statistically different at pH 5.0 and 7.2. The interactions suggest that HbA and HbS are easily destabilized than HbAS and that HbAS has more affinity for H2O2. These suggest a production of more ferryl intermediates or hydroxyl radicals. All these interactions may hinder the development of the malaria parasite at the intraerythrocytic stage and could likely account for a significant proportion of the mechanism that favours the resistance to malaria by individuals with HbAS.


Introduction
Malaria is one of the most important infectious disease problems of humans, particularly in developing countries. Plasmodium falciparum, the most virulent human malaria parasite, is responsible for hundreds of millions of illnesses and more than one million deaths each year [1]. Clinical manifestations of malaria primarily result from proliferation of the parasite within the hosts' erythrocytes. During this process, hemoglobin is utilized as the predominant source of nutrition. This is because during the intraerythrocytic development and proliferation, the parasites ingest more than 75% of the hosts' hemoglobin and digest them within the digestive vacuole-an acidic organelles with estimated pH of 5.0-5.4through a sequential metabolic process involving multiple proteases [2,3], and action of sodium dodecylsulphate (SDS) on hemoglobins can be likened to the way proteases secreted by malaria parasites destabilizes host hemoglobin for their homeostasis. Hydrogen peroxide is a major reactive oxygen species in living organisms and can produce reactive hydroxyl radicals or ferryl intermediate [Fe(IV)=O] 2+ by Fenton or Fenton like reaction [4]. Hanspal et al. [5] reported that Plasmodium falciparum-derived cysteine protease, falcipain-2, cleaves host erythrocyte hemoglobin at acidic pH and specific components of the membrane skeleton at neutral pH. Invasion of erythrocytes by plasmodium merozoites is a complex multistep process which is mediated by specific molecular interactions between host receptors and parasite ligands. A clear understanding of the molecular mechanisms involved in erythrocyte invasion and proliferation of the parasite could lead to the development of novel approaches to inhibit invasion, limit blood-stage parasite growth, and protect against malaria [6].
Molecular recognition lies at the heart of biological processes, and much effort is being made by biological chemist to understand the molecular details of macromolecule-ligand interactions of which hemoglobin-SDS and hemoglobin-H 2 O 2 are not exception. Also, medical chemists are trying to exploit this understanding in developing useful pharmaceutics [7]. Dissociation constant ( ) is a reciprocal of binding constant ( ), and it is a useful way to present the affinity of a ligand for a macromolecule. This is because its value quickly tells us the concentration of ligand that is required to yield a significant amount of interaction with the macromolecule. The precise mechanism by which sickle cell trait imparts resistance to malaria is unknown. Hence, investigation was carried out on affinities of hemoglobins for SDS and H 2 O 2 , which could provide insight to mechanism that favours the resistance to malaria by individuals with HbAS variant.

Materials and Methods
2.1. Materials. Sodium dodecylsulphate (SDS) and other chemicals used in this work obtained from BDH, England and Sigma, Germany are of analytical grade. All reagents were freshly prepared unless otherwise stated.

Methods.
Four milliliters (4 mL) of blood samples were collected from each of the identified individuals of genotype AA, AS, and SS after informed consent. In each case, the blood sample was collected with an ethylene di-amine tetra acetic acid (EDTA) vial.

Isolation and Purification of Hemoglobin.
Each of the blood samples was combined with normal saline in 50 mM Tris-HCl (pH 7.2) in the ratio of 2 : 3 and centrifuged at 4 ∘ C for 10 min at 4000 rpm [8]. Thereafter, the supernatants were removed by aspiration. The centrifugation was repeated for 2-4 times until a clear supernatant is gotten in each case. The clear supernatants were removed and the resulting pellets in the case were made up to 5 mL with 50 mM Tris-HCl (pH 7.2). The red cells were lysed and 5% NaCl was added to the resulting volume and centrifuged for 10 min at 4000 rpm to remove inorganic phosphates and other ions. The crude hemoglobins were collected with separate vials and labeled appropriately. Each of the crude hemoglobins (HbA, HbAS, or HbS) was dialyzed at 4 ∘ C for 24 hr against 50 mM Tris-HCl buffer, pH 7.2. The dialyzed hemoglobins were collected and stored at -20 ∘ C for further experiments.

Calculation of Dissociation
where Hb represents HbA, HbAS, or HbS; L represents SDS or H 2 O 2 ; also known as ℎ represents number of binding sites of SDS or H 2 O 2 for hemoglobin. , the dissociation constant is defined as .
Dividing both numerator and denominator of (4) by [Hb] and multiplying by gives From (5) The value of was determined from where Δ max is the maximum change in the absorption intensity at 415 nm when SDS or H 2 O 2 is saturated with Hb. It represents a fraction of a limiting compound of the system (in this case Hb) that binds to a compound with surplus concentration (in this case SDS or H 2 O 2 ): ISRN Hematology  (7) can be modified to A plot of Δ versus [L] o was used to estimate Δ max . Then, Δ max was used for calculating values according to (6). The actual concentration of L in each titration can be calculated from from (3) [11]. The direct plot of versus [L] according to (7) was used for the determination of using nonlinear least-square regression analysis ( ≤ 0.05), a statistical package in GraphPad Prism version 5.04.

Results and Discussion
Titration of 0.1 mL of 0.01 mM of hemoglobins with SDS or H 2 O 2 (0-0.748 mM) was monitored by absorption spectroscopy as shown in Figure 1 (Figure 2). The increase in the absorbance of the aromatic band refers to dynamic motion of the molecule and its deviation from normal structure and function [12,13], or unfolding of the hemoglobins [14,15]. These can be likened to destabilization of hemoglobin structure by proteases such as plasmepsins and falcipains in the acidic environment of malaria parasite food vacuole as a result of malaria parasite infection. This unfolding exposes the heme moiety and buried aromatic amino acids of the proteins which explains the increase in absorbance observed at the soret and aromatic bands of the hemoglobins by SDS (pH 5.0). The decrease in absorbance on these bands by SDS at pH 7.2 suggests that the hemoglobins are folding while that by H 2 O 2 (pH 5.0 and 7.2) suggests depletion of their heme content.
The value of , Δ max , and ℎ were determined from the plot of versus the concentration of free ligand, L, ([SDS] or [H 2 O 2 ]) as shown in Figure 3. The data were analyzed at ≤ 0.05 according to (7) using nonlinear least-square regression, a statistical package in GraphPad. The calculated values of , Δ max , and ℎ are shown in Table 1. At pH 5.0, the of HbAS-SDS interaction was higher than that of HbA-SDS and HbS-SDS interactions. Action of SDS on hemoglobins can be likened to the way proteases secreted by malaria parasites destabilizes host hemoglobin for their homeostasis, therefore the result suggests that malaria parasite proteases easily destabilize HbA and HbS than HbAS at acidic pH. At pH 7.2, the values calculated for HbA-SDS, HbAS-SDS and HbS-SDS were apparently the same ( Table 1)

Conclusion
Action of SDS on hemoglobins can be likened to the way proteases secreted by malaria parasites destabilizes host hemoglobin for their homeostasis. The higher for HbAS-SDS interaction (pH 5.0) suggest that HbA and HbS are easily destabilize more than HbAS while the higher affinity of HbAS for H 2 O 2 (pH 5.0) suggests the production of more ferryl intermediates or hydroxyl radicals. All these interactions may hinder the development of the malaria parasite at the intraerythrocytic stage and could likely account for a significant proportion of the mechanism that favours the resistance to malaria by individuals with the HbAS hemoglobin variant.