Interaction of Antimalarial Drugs (Pyrimethamine and Sulphadoxine) with Normal and Sickle Haemoglobins.
ABSTRACT
Crude haemoglobins were extracted from blood samples of identified individuals of normal (AA), sickle trait carrier (AS), and sickle (SS) by employing centrifugation techniques. The crude haemoglobins were dialysed at 4oC for 12hr against 50mM Tris-HCl buffer of pH 7.2.
The effects of pyrimethamine and sulphadoxine on the haemoglobins in the presence and absence of sodium dodecyl sulphate (SDS) were studied at pH 5.0 and 7.2 with uv-visible titration spectrophotometry.
The study showed that sodium dodecyl sulphate at pH 5.0 unfolded the studied proteins. These can be related to destabilization of haemoglobin structure by proteases such as plasmepsins and falcipains in the acidic environment of malaria parasite food vacuole due to malaria parasite infection.
Pyrimethamine and sulphadoxine at pH 5.0 and 7.2 decreased the concentration of oxyhaemoglobin and increased the concentrations of methaemoglobin and deoxyhaemoglobin of the studied proteins. The results also show how haemoglobins are deoxygenated due to interaction with sodium dodecyl sulphate.
Deoxygenation of haemoglobin as a result of their interaction with SDS can be likened to pathological condition whereby malaria parasites infection reduced the oxygen tension of erythrocytes of their host. HbS had the highest interaction with sulphadoxine and pyrimethamine followed by HbAS while HbA had the least interaction.
Formation of methaemoglobin is associated with lipid oxidation. Increase in absorbance at 275 nm observed in this study refers to dynamic motion of the studied proteins and their deviation from normal structure and function.
The interaction of haemoglobins with sulphadoxine-pyrimethamine combination at pH 5.0 caused a large perturbation of the protein conformation that was reflected in modest spectral shift of the soret band.
TABLE OF CONTENT
Title – – – – – – – – – – – i
Certification – – – – – – – – – – ii
Dedication – – – – – – – – – – iii
Acknowledgements – – – – – – – – – iv
Abstract – – – – – – – – – – v
Table of Content – – – – – – – – – vi
List of Figures – – – – – – – – – x
List of Tables – – – – – – – – – xiii
CHAPTER ONE: INTRODUCTION
1.1 Malaria – – – – – – – – – – 2
1.1.1 A health problem – – – – – – – – 2
1.1.2 History of malaria – – – – – – – – 2
1.1.3 The life cycle of the malaria parasite – – – – – – 2
1.1.4 Signs and symptoms of malaria – – – – – – 4
1.1.5 Diagnosis and treatment of malaria – – – – – – 5
1.2 Antifolates – – – – – – – – – 6
1.2.1 Combination of DHPS and DHFR inhibitors – – – – – 7
1.2.2 Sulphadoxine-pyrimethamine combination therapy – – – – – 8
1.2.3 Sulphadoxine and its pharmacological classification – – – – 8
1.2.4 The mechanism of action of sulphonamides – – – – – 9
1.2.5 Physico-chemical properties of sulphadoxine – – – – – 9
1.2.6 Pyrimethamine and its pharmacological classification – – – – 10
1.2.7 The mechanism of action of diaminopyrimidines – – – – 10
1.2.8: Binding mechanism of DHFR inhibitors – – – – – 10
1.2.9 Physico-chemical properties of pyrimethamine – – – – 12
1.2.10 Clinical uses and adverse effects of the combination – – – – 12
1.3 Chloroquine – – – – – – – – – 13
1.4 Lumefantrine – – – – – – – – – 14
1.5 Artemisinins and synthetic peroxides – – – – – – 15
1.5.1 Artemether – – – – – – – – – 15
1.6 Oxygen-carrying protein in the blood: Haemoglobin – – – – 17
1.6.1 Haemoglobin variants – – – – – – – – 18
1.6.2 Haemoglobin digestion by malaria parasite: Role of multiple proteases – 18
1.6.3 Oxidative stress generated by haemoglobin degradation – – – 19
1.7 Haem detoxification pathways of the malaria parasite – – – – 20
1.8 Haemozoin:The antimalarial drug target – – – – – – 21
1.8.1 Mechanism of haemozoin formation in the malaria parasite – – – 22
1.9 Spectral properties of haemoglobin – – – – – – 23
1.10 Aim and Objectives of Study – – – – – – – 25
1.10.1 Aim of the Study – – – – – – – – 25
1.10.2 Specific Objectives of the study – – – – – – 25
CHAPTER TWO: MATERIALS AND METHODS
2.1 Materials – – – – – – – – – – 26
2.1.1 Chemicals – – – – – – – – – 26
2.1.2 Equipment – – – – – – – – – 26
2.2 Methods – – – – – – – – – – 26
2.2.7 Collection of blood samples – – – – – – – 26
2.2.8 Isolation and purification of haemoglobin – – – – – 27
2.2.9 UV – Visible titration – – – – – – – – 27
2.2.10 Data analysis – – – – – – – – 28
CHAPTER THREE: RESULTS
3.1 Absorption spectra of haemoglobin A – – – – – 29
3.1.1 Absorption spectra of haemoglobin A in varying concentrations of pyrimethamine- 29
3.1.2 Absorption spectra of haemoglobin A in varying concentrations of pyrimethamine, in the presence of SDS- 29
3.1.3 Absorption spectra of haemoglobin A in varying concentrations of pyrimethamine and constant concentration of sulphadoxine – – – 29
3.1.4 Absorption spectra of haemoglobin A in varying concentrations of pyrimethamine and constant concentration of sulphadoxine in the presence of SDS- 33
3.1.5 Absorption spectra of haemoglobin A in varying concentrations of sulphadoxine- 33
3.1.6 Absorption spectra of haemoglobin A in varying concentrations of sulphadoxine in the presence of SDS -33
3.1.7 Absorption spectra of haemoglobin A in varying concentrations of sulphadoxine and constant concentration of pyrimethamine – – – 33
3.1.8 Absorption spectra of haemoglobin A in varying concentrations of sulphadoxine and constant concentration of pyrimethamine – – – 38
3.2 Absorption spectra of haemoglobin AS – – – – – – 40
3.2.1 Absorption spectra of haemoglobin AS in varying concentrations of pyrimethamine- – – – – – 40
3.2.2 Absorption spectra of haemoglobin AS in varying concentrations of pyrimethamine in the presence of SDS 40
3.2.3 Absorption spectra of haemoglobin AS in varying concentrations of pyrimethamine and constant concentration of sulphadoxine – – – 40
3.2.4 Absorption spectra of haemoglobin AS in varying concentrations of pyrimethamine and constant concentration of sulphadoxine in the presence of SDS- 40
3.2.5 Absorption spectra of haemoglobin AS in varying concentrations of sulphadoxine- 45
3.2.6 Absorption spectra of haemoglobin AS in varying concentration of sulphadoxine in the presence of SDS- 45
3.2.7 Absorption spectra of haemoglobin AS in varying concentrations of sulphadoxine and constant concentration of pyrimethamine – – – 45
3.2.8 Absorption spectra of haemoglobin AS in varying concentrations of sulphadoxine and constant concentration of pyrimethamine in the presence of SDS- 45
3.3 Absorption spectra of haemoglobin S – – – – – – 50
3.3.1 Absorption spectra of haemoglobin S in varying concentrations of pyrimethamine- 50
3.3.2 Absorption spectra of haemoglobin S in varying concentrations of pyrimethamine in the presence of SDS- 50
3.3.3 Absorption spectra of haemoglobin S in varying concentrations of pyrimethamine and constant concentration of sulphadoxine – – – 50
3.3.4 Absorption spectra of haemoglobin S in varying concentrations of pyrimethamine and constant concentration of sulphadoxine in the presence of SDS- 50
3.3.5 Absorption spectra of haemoglobin S in varying concentration of sulphadoxine- 55
3.3.6 Absorption spectra of haemoglobin S in varying concentrations of sulphadoxine in the presence of SDS – 55
3.3.7 Absorption spectra of haemoglobin S in varying concentration of sulphadoxine and constant concentration of pyrimethamine – – – 55
3.3.8 Absorption spectra of haemoglobin S in varying concentrations of sulphadoxine and constant concentration of pyrimethamine in the presence of SDS – – – – – – – – 55
CHAPTER FOUR: DISCUSSION
Discussion – – – – – – – – – – 60
Conclusion – – – – – – – – – – 62
References – – – – – – – – – – 63
Appendices – – – – – – – – – – 73
INTRODUCTION
One of the main causes of death today is malaria, especially in numerous parts of Asia, Sub-Saharan Africa and the America (Esparza, 2005). Of the four Plasmodia that cause malaria, Plasmodium falciparum is responsible for the majority of illness and death in mankind (Duraisingh and Refour, 2005; Idro et al., 2005; Okie, 2005; Worrall et al., 2005).
In Sub- Saharan Africa, this disease has a profound impact on children and infants, whilst millions have
already died from AIDS (Acquired Immunodeficiency Syndrome) (Esparza, 2005; Harms and Feldmeier, 2005).
In addition to this, malaria adds in mortality while the spread of chloroquine resistant strains of the plasmodium parasites across Africa increases (Farooq and Mahajan, 2004; Mahajan et al., 2005).
Approximately, three million people, of whom more than half are children, die of malaria caused by P. falciparum annually (Duraisingh and Refour, 2005).
Mortality and morbidity increase every year with over 500 million people infected with P. falciparum, presenting clinical symptoms of mild to severe malaria.
There exist several reasons for the increase in the occurrence of malaria including: i An increase of the protozoan parasite’s resistance to anti-malarial drugs, ii
The development of the anopheles mosquito vectors’ resistance to numerous insecticides iii The growth and the widespread migration of vulnerable populations to vastly endemic areas (Abdel-Hameed, 2003; Gregson and Plowe, 2005).
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