ABSTRACT
On the premise that the resistance of malaria is related to the structure of the sickle haemoglobin molecule, a comparative denaturation of haemoglobin A (HbA), haemoglobin AS (HbAS) and haemoglobin S (HbS) in their relaxed states using sodium dodecyl sulphate (SDS) was carried out at pH 5.0 and 7.2 in the presence of two antimalarial drugs artemether and lumefantrine in both singly and in combination forms and monitored by UV-Visible spectrophotometer in the range of 250 nm-700 nm. The results were analysed by convolution of the obtained absorption spectra. Artemether and lumefantrine have similar effects on all haemoglobins but lumefantrine show more pronounced effects when compared to artemether at pH 5.0. Both antimalarial drugs converted haemoglobin from the R-state to predominantly the deoxy structure as well as increasing the aromaticity and unfolding of haemoglobin. The effects of artemether and lumefantrine were more destabilizing for HbS when compared to HbA and HbAS. The concentration-dependent decrease of the Soret band was observed, a condition that is achieved as a consequence of heme alkylation and subsequent disruption of the p electron delocalized system. Also, artemether and lumefantrine in combination led to the progressive, slow decay and to eventual loss of the Soret band, as a consequence of direct alkylation of the porphyrin ring and of its subsequent degradation. The decay in the Soret band was highest in HbS when compared to other haemoglobins; suggesting it has a higher drug response which agrees with the apparent resistance of sickle cell carriers to malaria. HbS showed the lowest decrease in Soret band when compared the HbA. The difference spectra of all haemoglobins in the presence of SDS, pH 5.0 showed concentration-dependent positive peak at 275 nm and positive trough at 415 nm. The peak absorbance of the difference spectra at 275 nm suggest that the protein is unfolding and the unfolding predisposes the aromatic amino acid group to solvent in its environment and this causes the protein to expand while the positive trough at 415mm suggest exposure of the haem moiety of the studied proteins. Lumefantrine interracts more with haemoglobin when compared to arthemeter. This may suggest why arthemeter has high rapid onset but short term cure rate while lumefantrine has a long term cure rate with short treatment course. Artemether and lumefantrine combination shows a synergistic effect on reaction with haemoglobin and is effective in malaria treatment but cannot be used in prevention due to the generation of free radicals.
CHAPTER ONE
INTRODUCTION
Haemoglobin (Hb) is an iron- containing oxygen transport metalloprotein in the red blood cells of vertebrates (Maton et al., 1993) and tissue of some invertebrates. Adult haemoglobin (HbA) commonly referred to as normal haemoglobin is a globular protein made up of four polypeptide chains, 2α and 2β. A mutation which results in the replacement of glutamate with valine at position six of the two β-chains of HbA gives rise to mutant form of haemoglobin called sickle haemoglobin (HbS) (Nelson and Cox., 2005). It is well known that the main physiological function of haemoglobin is transport of oxygen. Malaria is an infectious disease caused by a parasite, Plasmodium, which infects red blood cells. Chloroquinne and its derivatives, are antimalarial drugs that have been effective in treating most types of malaria. However, widespread resistance of Plasmodium falciparum to quinoline-based drugs has made the disease situation difficult to manage in endemic malaria areas. Coartem is a combination product of arthaemter and lumefantrine which is indicated for treating acute and uncomplicated types of malaria due to Plasmodium falciparum (Sullivan, 2002). Since the parasite utilizes the haemoglobin molecule for its growth, and Coartem has been shown to represses malaria symptoms by inhibiting the growth and reproduction of the parasite, ittherefore interacts with haemoglobin. Artemether interacts with ferriprotoporphyrin IX (haem), or ferrous ions, in the acidic parasite’s food vacuole, which results in the generation of cytotoxic radical species. The generally accepted mechanism of action of peroxide antimalarials involves interaction of the peroxide-containing drug with haem, a haemoglobin degradation byproduct, derived from proteolysis of haemoglobin. This interaction is believed to result in the formation of a range of potentially toxic oxygen and carbon-centered radicals (Ibrahim et al, 2008). Lumefantrine is a blood schizonticide active against erythrocytic stages of Plasmodium falciparum. It is thought that administration of lumefantrine with artemether results in cooperate antimalarial clearing effects. Artemether has a rapid onset of action and is rapidly cleared from the body. It is thus thought to provide rapid symptomatic relief by reducing the number of malarial parasites. Lumefantrine has a much longer half life and is believed to clear residual parasites (Efferth, 2007).
1.1 Haemoglobin
The word haemoglobin is derived from two words; haem and globin. Globin is its protein part while haem is an iron containing compound (Garret and Grisham, 1999). Each subunit of haemoglobin is a globular protein with an embedded haem group. Each haem group of haemoglobin contains one iron atom that can bind one oxygen molecule through ion-induced dipole forces. The most common type of haemoglobin in mammals contains four such subunits. Also, haemoglobin can be seen to consist of four myoglobin units joined together. The structure of haemoglobin and myoglobin are shown in Fig. 1. Haemoglobin transports oxygen from the lungs or gills to the rest of the body where it releases the oxygen for cell use. It also has a variety of other roles of gas transport and effect modulation which vary from species to species, and are quite diverse in some invertebrates. The oxygen carrying protein has an oxygen binding capacity of between 1.36 and 1.37ml oxygen per gram haemoglobin (Ruiz et al., 1981) which increases the total blood oxygen capacity seventy fold (Costanzo, 2007).
Haemoglobin is synthesized in a complex series of steps. The haem part is synthesized in a series of steps in the mitochondria and cytosol of immature red blood cells, while the globin
protein parts are synthesized by ribosomes in the cytosol.
A B
Fig.1: Structures of haemoglobin and myoglobin;
(A)A ribbon diagram of the structure of haemoglobin. (B) Structure of the myoglobin protein with the position of the haem group highlighted.
1.1.1 Haem
In most humans, the haemoglobin molecule is assembly of four globular protein subunits. Each subunit is composed of a protein chain tightly associated with a non-protein haem group. A haem group consists of an iron (Fe) ion held in a heterocyclic ring known as a porphyrin as illustrated in Figure 2. The iron ion, which is the site of oxygen binding, coordinates with the four nitrogens in the center of the ring, which all lie in one plane. The iron is also bound strongly to the globular protein via the imidazole ring of the F8 histidine residue below the porphyrin ring. A sixth position can reversibly bind oxygen by a coordinate covalent bond (Nelson and Cox, 2005), completing the octahedral group of six ligands. Oxygen binds in an “end on bent” geometry where one oxygen atom binds Fe and the other protrudes at an angle. When oxygen is not bound, a very weakly bonded water molecule will fill the site, forming a distorted octahedron.
Figure 2: Structure of Haem;
The non-protein active site within myoglobin and haemoglobin. The side-groups which have been added to porphine are highlighted in magenta, and the central iron atom is shown in red. Source: (Nelson and Cox, 2005)
The iron ion may either be in the Fe2+ or Fe3+ state, but ferrihemogblobin (methaemoglobin) cannot bind oxygen (Linberg et al., 1998). In binding, oxygen temporarily oxidizes Fe2+ to Fe3+ so iron must exist in the +2 oxidation state inorder to bind oxygen. The enzyme methaemoglobin reductase reactivates haemoglobin found in the inactive (Fe+3) state by reducing the iron center. Oxyhaemoglobin – a form of haemoglobin with bound oxygen is formed during respiration when oxygen binds to the haem component of the protein haemoglobin in red blood cells. In vivo, the process occurs in the pulmonary capillaries adjacent
to the alveoli of the lungs. The oxygen then travels through the blood stream to be dropped off at cells where it is utilized in aerobic glycolysis and in the production of ATP by the process of oxidative phosphoxylation. Haemoglobin is always isolated in the form of oxyhaemoglobin (Antonin and Brunori, 1971). Deoxyheoglobin is the form of haemoglobin without the bound oxygen (Zhao et al., 2008).
1.2 Variants of Haemoglobin
In adult humans, the principal haemoglobin is a tetramer called haemoglobin A (HbA), consisting of two alpha (α) and two beta (β) subunits non-covantently bound, each made up of 141 and 146 amino acid residues, respectively (Stryer, 1989, Nelson and Cox 2005). This is denoted as α2β2. The subunits are structurally similar and about the same size. Each subunit has a molecular weight of about 68,000 daltons (64.458g/mol) (Van Beekuelt et al., 2001). Adults also have a minor haemoglobin (2% of the total haemoglobin) called haemoglobin, A2, which
contains delta ( ) chains in place of the β chains of haemoglobin A (Stryer, 1989). Thus the subunit composition of haemoglobin A2 is22 .
Mutations in the genes for the haemoglobin protein result to haemoglobin variants, some of which cause a group of hereditary diseases, termed the haemoglobinopathies in humans. The best known is sickle-cell diseases, which was the first human disease whose mechanism was understood at the molecular level. A separate set of diseases called thalassemias involves underproduction of normal and sometimes abnormal haemoglobin through problems and mutations in globin gene regulation (Warnick, 1986).
Several hundred mutant haemoglobin (Stryer., 1989) have been found by studies of the haemoglobin of patients with haematologic symptoms. One of most important mutant form of the principal haemoglobin is haemoglobin S (HbS). It results from a single amino acid substitution, a Val instead of a Glu residue at position 6 in the two β Chains (Nelson and Cox,
2005).The R group of Valine has no electric charge, whereas glutamate has a negative charge at pH 7.4. Haemoglobin S therefore have two fewer negative charges than HbA, one for each of the two β chains.
Other important mutant forms of the principal haemoglobin are those that result to altered active site of the mutant haemoglobin. Consequently, the defective subunit(s) cannot bind oxygen because of a structural change near the haem that directly affects oxygen binding (Stryer,
1989). For example, substitution of tyrosine for the histidine proximal or distal to haem stablizes the haem in the ferric form, which can no longer bind oxygen as illustrated in Figure 3. The tyrosine side chain is ionized in this complex with the ferric ion of the haem. Mutant haemoglobins characterized by a permanent ferric state of two of the haems are called haemoglobin M. The letter M signifies that the altered chains are in the methaemoglobin (ferrihaemoglobin) form (Remacher et al., 2000).
C CH2
Haem
N Fe2+ O2
Haemglobin A
C CH2
O-
Haemglobin M
Haem
H Fe2+
Figure 3: Structure of haemoglobin showing proximal histidine;
Substitution of tyrosine for the proximal His results in the formation of haemoglobin M. The negatively charged oxygen atom of tyrosine is coordinated to the iron atom, which is the ferric state. Water molecule instead of O2 is bound at the sixth coordination position. Source: (Shenai et al., 2000).
1.2 Haemoglobin and Malaria: The Relationship
The plasmodium parasite that causes malaria is transmitted from female anopheles mosquito to human. The parasites spend part of their life cycle in the mosquito and the rest in the human host. The complex nature of the malaria parasite life cycle in the human host presents several points at which the organism could be targeted for destruction. These points of destruction range from host immunity to red cell invasion and multiplication. A mutation that somehow destroys both the infected red cell and the parasite could therefore eliminate the malaria parasite. The destroyed infected cells would be replaced by new healthy cells (Woodrow et al., 2005).
Sickle haemoglobin provides the best example of a change in the haemoglobin molecule that impairs malaria growth and development. The initial hints of a relationship, between the two
came with the realization that the geographical distribution of the gene for haemoglobin S and the distribution of malaria in Africa virtually overlap. A further hint came with the observation that peoples indigenous to the highland regions of the continent did not display the high expression of the sickle Hb genes like their lowland neighbors in the malaria belt (Ringelhann et al., 1976). Sickle trail provides a survival advantage over people with normal haemoglobin in regions where malaria is endemic. Sickle cell trait provides neither absolute protection nor invulnerability to the disease (Ringelhann et al., 1976). Rather, people (and particularly children) infected with Plasmodium falciparumare more likely to survive the acute illness if they have sickle cell trait. When these people with sickle cell trait procreate, both the gene for normal haemoglobin and that for sickle haemoglobin are transmitted into the next generation (Ringelhann, et al., 1976).
The precise mechanism by which sickle cell trait imparts resistance to malaria is unknown. A number of factors are likely involved and contribute in varying degrees to the defense against malaria. Red cells from people with sickle trait do not sickle to any significant degree at normal venous oxygen tension. Very low oxygen tensions will cause the cells to sickle for example; extreme exercise at high altitude increases the number of sickled erythrocytes in venous blood samples from people with sickle cell trait (Martin et al., 1989). Sickle trait red cells infected with Plasmodium falciparum parasite deform, presumably because the parasite reduces the oxygen tension within the erythrocytes to very low levels as it carries out its metabolism. Deformation of sickle trait erythrocytes would mark these cells as abnormal and target them for destruction by phagocytes (Luzzatto, et al., 1970).
Studies have revealed that oxygen radical formation in sickle trait erythrocytes retards growth and even kills the plasmodium falciparum parasite (Woerdenbag et al., 1993). Sickle trait red cells produce higher levels of the superoxide anion (O2-) and hydrogen peroxide (H2O2) than do normal erythrocytes. Each compound is toxic to a number of pathogens including malaria parasites. Homozygous haemoglobin S red cells produce membrane associated hemin secondary to repeated formation of sickle haemoglobin polymers. This membrane-associated hemin can oxidize membrane lipids and proteins (Rank et al., 1985). Sickle trait red cells normally produce little in the way of such products. If the infected sickle trait red cell form sickle polymer due to the low oxygen tension produced by parasite metabolism, the cells might generate enough hemin to damage the parasites (Zijlstraet al., 1991).
1.2.1 Haemoglobin Denaturation and Peptide Digest
The malaria parasite like all organisms must acquire nutrients from the environment and convert these nutrients to other molecules or energy and the energy is used to maintain its homeostasis, growth and reproduction. They can synthesize protein using amino acids from three sources:
1) Denovo synthesis
2) Import from host plasma and
3) Degradation or digestion of host haemoglobin.
In the early stage of the parasite, it takes up the host stroma by pinocytosis into its vacuole; the food is acidic and contains protease activities. They include plasmepsins and falcipains. The digestion takes place by a semi ordered process involving the sequential action of different proteases (Goldberg, 2005). These proteases are effective in cleaving undenatured haemoglobin between phenylalanine and leucine residue located at position 33 and 34 on the alpha globin chains called the hinge region that is crucial to the stability of haemoglobin and cleavage at this region cause the dissociation of the subunits and exposes additional protease sites within the globins peptide chains. It’s been suggested that falcipain 2 (Shenai et al., 2000) and possibly falcipain 3 (Sijwali et al., 2001) are capable of digesting native haemoglobin and therefore may also participate in the cleavage of haemoglobin which can lead to liberation of the haem moiety of the haemoglobin (Tshefu et al.,2010).
1.2.2 Fate of Haem Liberation
Haem is a prosthetic group that consists of an iron atom contained in the center of a heterocyclic prophyrin ring. Free haem is toxic to cells so the parasite converts it into an insoluble crystalline form called haemozoin. Haemozoin is often called malaria pigment, and since the formation is essential to the existence of the parasite it is an essential point of attack of malaria drug. Haemozoins are produced by the parasites as they multiply within the red blood cells (Sullivan, 2002) and were used to identify the stage of malaria parasite infection. It is a prooxidant and catalyzes the production of reactive oxygen species. Oxidative stress is believed to be due to the conversion of of haem (ferroprotoporphyrin) to hematin (ferriprotoporphyrin). Free haematin can also bind and disrupt cell membrane, damaging cell structure and causing lysis of the host erythrocytes. A malaria parasite detoxifies hematin by biocrystallization,
converting it into insoluble and chemically inert beta hematin crystals called haemozoin (Hempelmann, 2007). Plasmodium has food vacuole filled with haemozoin crystals which are about 100-200nm long and each contains about 80,000 haem molecules (Sullivan, 2002). Detoxification through biocrystallization is distinct from the detoxification in mammals, where an enzyme called oxygenase instead breaks excess haem into biliverdin, iron and carbon monoxide (Kikuchi et al., 2005).
1.3 Generation of Reactive Oxygen Species and Membrane Lipid Peroxidation: the Role of
Iron.
Organic and inorganic iron compounds are involved in the catalysis of various stages of lipid peroxidation. Haem pigments are more powerful catalysts of lipid oxidation than inorganic iron compounds. The work of Halliwell and Gutteridge (1990) indicates that all simple iron complexes are capable of decomposing hydrogen peroxide (H2O2) to form hydroxyl radicals
(.OH). Studies on the other hand showed that iron chelated to iron binders or storage protein had
very weak or no catalytic effect (Valentine et al., 1995) on lipid peroxidation reaction. Transferin, an iron carrier protein that binds ferric iron tightly does not participate in .OH generation at physiological concentrations. This means that partly saturated transferrin protects cells from damage by binding iron that might catalyze .OH formation from superoxide (.O2-) and H2O2. Transferrin however, can release iron at low pH (below pH 5.6) which can accelerate lipid peroxidation. Ferritin is regarded as a safe iron storage protein, but has been shown to be involved in the formation of .OH if the iron ions are released from ferritin by ascorbate, dithionite, or .O2- radicals. Therefore the presence of ascorbate, H2O2, and xanthine oxidase system (XOS) can increase the release of iron from iron proteins such as ferritin and haemoglobin (Reeder and Wilson, 2005).
In vitro, Fe of Hb undergoes slow autooxidation to give Fe3+ Hb (methaemoglobin met
Hb) and superoxide. The fraction of met Hb, which cannot transport oxygen, in normal blood cells does not exceed 3% but it can increase in the presence of certain drugs (Gebicka and Banasiak, 2009). The superoxide formed during Hb autooxidation dismutases to hydrogen peroxide (Traylor et al., 1989). Hydrogen peroxide is reactive oxygen species involved in the propagation of cellular injury in various pathophysiological conditions. The reaction of hydrogen peroxide with Fe2+ Hb (deoxy Hb and oxy Hb) and Fe3+ Hb ( met Hb) results in the formation of ferryl haemoglobin (ferryl Hb) and ferryl Hb with a globin-based radical, respectively (Giulivi
and Davies, 1994). In vivo, these reactions are of physiological relevance under ischemic conditions (Patel et al., 1996). Ferryl haemoglobin is able to oxidize proteins, nucleic acids and lipids (Kowalczyk et al., 2007).
Decreased antioxidant defenses in sickle cell disease patients are accompanied by activation of enzymatic (NADPH Oxidase, Xanthine Oxidase) and non-enzymatic (Sickle haemoglobin auto-oxidation) sources of reactive oxygen species (Wood and Granger, 2007). These makes sickle cell to generate approximately two times more amount of reactive oxygen species (Hundekar et al., 2010). The reactive oxygen species can attack the cell membrane causing damage in the membranous lipid and protein structure that may ultimately result in hemolysis. This indicates that lipid peroxidation plays a major role in the pathophysiology of sickle cell anemia (Hundekar, et al., 2010).
1.4 Protein Folding and Unfolding
This is a biophysical process by which polypeptides fold into their characteristic and functional three dimensional structures from random coil (Albert et al., 2002). Each protein exists as an unfolded polypeptide that is translated from its mRNA to a linear chain of amino acids. These polypeptides spontaneously fold into their native conformations under physiological conditions. This implies that a protein’s primary structure dictates its three-dimensional structure (Warnick, 1986).
Proteins have stabilizing forces that exist within their structure. This means that a protein’s stability is produced from that protein’s structure. These stabilizing forces include intermolecular Vander weal force, hydrophobic interaction, hydrophilic interaction, hydrogen bond, promoter- promoter bonds; disulfide bond etc. studies have revealed that proteins have two- transition states: native and denatured states. External influences (i.e. physical/chemical agent) can affect the force of interaction and unfold protein slightly until a certain point is reached where the protein is denatured. At unfolded state, only breakage of disulfide bond is felt while covalent structure remains unchanged (Rastall, 1999). Native protein can be unfolded by physical agent such as temperature and mechanical force or by chemical agent (denaturant) such as urea, guanidinium chloride or SDS. Protein unfolding transitions can be measured by following the absorbance changes at 282-292 nm as a function of temperature or denaturant concentration. When denaturants are added to a protein solution, the absorbances of tyrosine and
tryptophan at 287 nm and at 291 nm, respectively, increase substantially even in the absence of structural transition. The observed increases in absorbance at 287 nm and 291 nm reflect a slight red shift of the spectra, which originates from the change in refractive index (i.e the polarity) of the solvent with the concentration of denaturant (Schmid, 1990).
UV-Vis spectroscopy is a very helpful technique to study the conformational changes of haem proteins, since the soret band of the haem provides very useful information on the secondary structure of haem proteins (Zhao et al., 2008). The position of the soret band will shift or the absorption will decrease if the structure of the haem protein is transformed. Matsui et al. (2008) revealed that the absorbance spectrum in the soret region results mainly from the interaction between the haem moiety and well- defined tertiary structure and hence can be used to monitor protein unfolding.
1.4.1 Effect of pH on Proteins
Proteins have pH where they function effectively and a slight change in pH may cause total destabilization of protein. A change in pH that may unfold protein results to ionization of amino acid side chain buried deeply into the protein molecule. Most of the histidine and tyrosine residues are not ionized in the hydrophobic interaction of the protein except in high pH (Rastall,
1999). These amino acids are present in haemoglobin and their ionization may drive the unfolding of the protein and also minimize charge repulsion at the protein’s surface.
1.4.2 Effect of SDS on Proteins
The hydrophobic tail of SDS interacts strongly with polypeptide chains. the number of SDS molecules bound by a polypeptide is proportional to the length (number of amino acid residues) of the polypeptide. Each dodecylsulphate contributes two negative chargers. Collectively, these chargers overwhelm any intrinsic charge that the protein might have. SDS is also a detergent that disrupts protein folding (Garret and Grisham, 1999). Sodium dodecyl sulptiate (SDS) is used more often than any other detergent as an excellent denaturing or “unfolding” detergent (Parker and Song, 1992). Studies have revealed that higher concentrations of SDS unfolds most proteins (Moodsavi- Movahedi et al., 1997) while few protein are compacted in the presence of SDS at low concentrations (Reza et al., 2002). Denaturation or unfolding of haem proteins such as haemoglobin or peroxidase leads to exposure or release of the haem moiety of the protein. The exposed haem group can form aggregates with other protein molecules (Eze and Chilaka, 2002). Its iron can oxidize to Fe+3 to forming methaemoglobin (MetHb) (Samir, 2006) and formation of metmyoglobin (and/or methaemoglobin) is highly correlated with lipid oxidation. The degree of conversion of haemoglobin to methaemoglobin depends on the degree of unfolding and can lead to existence of low and high spin states as it appears from a shift towards shorter wavelength of the soret band.(Ibrahim et al., 2008).
However, formation of ordered structure (- helix or β-Sheet) in certain peptides is known to be induced by interaction with SDS micelles. The SDS- induced structures formed by these peptides are amphiphilic, having both a hydrophobic and a hydrophilic face.
Most proteins in the body maintain their native conformations or if they become partially denatured, are either renatured through the auspices of molecular chaperons or are prototypically degraded. Chaperons have been labeled as “protein detergents” and are widespread and abundant in biological systems. As a chaperon interacts with a denatured molecule that have been caught in a dead end folding path or potential energy well, various amphiphilic sequences may be docked (hydrophobic collapse) unto a chaperon in a structurally ordered and thus stable manner. This docking might involve the same nonnative but ordered secondary structures that are involved in SDS micelle- protein interactions (Singh and Panwar, 2006).
SDS has also been revealed to have effect on the conformations of haemoglobin. Haemoglobin normally has three conformational structures. These are the Oxy-, deoxy- and met- conformations. In the oxy- conformation, haemoglobins haem center is in the reduced form and has the relaxed (R) conformation. However, the haem group has the tense (T) conformation in the deoxy form. Finally in the met-conformation, the haem center is in an oxidized state and has a relaxed- like conformation (Traylor and Traylor, 1982). These findings have been related to inhibition of haemoglobin authoxidatin by SDS, in the mechanisms for myoglobin and haemoglobin autoxidation, the starting species are Fe (II) O2, where hydrogen bound between oxygen and like N atom of distal hitidine are present. In the first step of autoxidation mechanism, the disruption of the hydrogen bond and dissociation of haemoglobin and O2 occur. In the second step, a water molecule associates with haemoglobin (Fe2+) to occupy the sixth coordination site.
In the third step, an electron is transferred from haemoglobin (Fe2+) to molecular oxygen via an outer spHere mechanism. In this meachanism, if the entrance of water is blocked by any factor (like SDS), the autoxidation of haemoglobin could be prevented. This is presumably the
mechanism by which low SDS can prevent haemoglobin autoxidation. Reza et al., (2002) concluded that low SDS concentrations induce the folded compact state of haemoglobin as a stabilized form of the protein, which prevents met formation, perhaps by water exclusions from the folded protein.
1.5 Spectral Properties of Protein
The peptide groups of the protein main chain absorb light in the “far-Uv” range (180-
230nm). The aromatic side chains of Tyrosine (Tyr) TryptopHan (Trp) light in this region and in addition, they absorb in the 240-300nm region. This region is called the “near-Uv” or the “aromatic” region. Disulfide bond that form between two cysteine residues also shown an absorbance band near 250nm (Schmid, 1990).
The absorbance properties of the aromatic amino acids are shown in Table 1. In the near- Uv the molar absorbance of phe ( max 258 nm) is much smaller than that of Tyr and Trp and the spectrum of a protein between 240 and 300nm is therefore dominated by the contributions from the Tyr and Trp side- chains. Phenylalanine residues contribute fine structure (wiggles) to the spectrum between 250 and 260nm. The aromatic amino acids do not absorb above 310nm and therefore absorbance of solutions containing only protein should be zero at wavelengths greater than 310nm (Schimid, 1990) and solutions containing only protein without Trp residues do not absorb above 300nm.
Table 1: Absorbance of the aromatic amino acid
| Compound | max (nm) | -1 -1 max (Lmrl cm ) | -1 280 (lmsl cm ) |
| TryptopHan | 280 | 5600 | 5500 |
| Tyrosine | 275 | 1400 | 1490 |
| PHenylalanine | 258 | 200 |
(Source; Roberts et al., 1981)
Absorption coefficient at max
in water and neutral pH
The absorption spectra of the aromatic amino acids depend on the nature of the molecular neighborhood of the respective chromophores. This environmental sensitivity can result in broadening of bands, shifts in wavelength of maximal absorption and over- all changes in intensity. In general, the shift in wavelength maximal absorption predominates. A red- shift of protein spectrum is observed when the polarity of the solvent increases (Schmid, 1990). For example, the maximum of the absorption of tyrosine is blue- shifted by about 3nm from 277 nm to 274nm, when the solvent is changed from carbon tetrachloride to water. This spectra shift combined with minor changes in the strength of absorbance and in the fine structure of the spectrum, leads to maxima (prominent peak) in the difference spectra in the descending slope of the original spectrum, which is in the 285-288nm region for tyrosine and 291-294nm for tryptophan.
In folded native proteins, some of the aromatic amino acid residues are buried within the hydrophobic core of the molecule. They become exposed to the aqueous solvent during unfolding, giving rise to a decrease in absorbance in the 288 to 295nm region. The difference in the absorption spectra between the native and the unfolded state of a protein is generally small; nevertheless the difference spectrum can be determined with good accuracy by difference spectroscopy (Ahn et al., 1993).
The size and shape of the difference spectrum depends on the kind and number of aromatic amino acids, as well as on the degree of burial of their side chains in interior of the native protein. The contribution of phe residues to the difference spectrum is very small. It is sometimes apparent as a ripple structure in the 250 – 260nm region. Proteins that lack Trp display a Tyr difference spectrum with a prominent positive peak at 287nm and a minor peak at around 278nm. Proteins that contain both Tyr and Trp show an additional prominent peak around 292nm that originates from the buried Trp residues in the folded protein. The difference spectra are usually too complex to sort out the contributions of individual amino acids; they are extremely useful as convenient means to monitor conformational changes of a protein. Also, measurement of spectral changes that accompany unfolding transitions provides a very powerful technique to determine the stability of proteins and to follow the kinetics of conformational changes.
1.5.1 Spectral Properties of Haemoglobin
The haem group consists of a porphyrin ring with a ferrous or ferric iron co-ordinated centrally. the conjugated double bond system of the porphyrin ring causes a strong absorption in haemprotein termed α, β, γ-bands. Typically, α-bands occur at the longest wavelengths (550- 650), γ bands at shortest wavelengths (also called Soret bands, after the Swiss scientist who first examined the near UV region of cytochromes) and the β bands lie between. Solution of oxyhaemogloin is bright red, and tends to a yellowish colour at high dilutions. A dark red colour indicates the presence of some ferric haemoglobin. Oxyhaemoglobin shows two visible absorption bands with maxima at 577 nm and 541 nm fro mammalian haemoglobin; these bands are shifted towards the red (591 and 542 nm) in myoglobin. The Soret band of oxyhaemoglobin lies near 415nm (Antonin and Brunori, 1971). Solutions of deoxygenated haemoglobin and myoglobin have a typical red violet colour which becomes greenish at high dilutions. The absorption spectrum of deoxygenated is characterized by a single, broad and asymmetrical band in the visible, with a maximum at about 555 nm; the soret band, in the near ultraviolet shows a maximum at 430nm in deoxygenated haemoglobin and 435 nm in myoglobin. The spectrum of ferric haemoglobin or myoglobin is pH sensitive. Acidic ferric haemoglobin and myoglobin appear dark brown in concentrated solution and yellow-green in dilute solution. The spectrum shows two bands in the visible region with maxima at about 500 and 625 nm; the soret band lies at about 405 nm. Alkaline ferric haemoglobin has two bands in the visible region with maxima at about 540 and 580 nm. The soret band of the alkaline form has a maximum at 412 nm. Ferric haemoglobin usually has two forms (states) known as low and high-spin forms (states). The spectroscopic difference between high and low-spin derivatives of ferric haemoglobin is shown in Table 2.
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THE INTERACTION OF THE ANTIMALARIAL DRUGS (ARTEMETHER AND LUMEFANTRINE) WITH HAEMOGLOBIN A AND S: A UV- VISIBLE STUDY
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