| dc.description.abstract | This thesis describes spectroscopic studies on two interesting biological amphiphiles, melittin and bilirubin. The former is a 26?residue polypeptide which comprises ~50% of the dry venom of honeybees. Melittin is cytotoxic and, being an amphiphilic peptide, is extremely membrane?active. It therefore provides a very good model for the study of protein–lipid interactions. Bilirubin is a tetrapyrrole, produced as a result of heme degradation in humans and many animals. Apart from being structurally interesting, disorders of bilirubin metabolism are physiologically important, particularly due to their implication in neonatal hyperbilirubinemia.
Difference spectroscopy, circular dichroism (CD), fluorescence, and nuclear magnetic resonance (NMR) techniques have been used in this investigation to probe the conformational properties, aggregation behaviour, and interactions of melittin and bilirubin. This thesis is divided into two parts. Section I deals with the studies on melittin, while Section II details the studies on bilirubin.
Section I — Melittin
Chapter I briefly summarizes the extant literature on melittin: its isolation and characterization, conformational studies in both aqueous and solid states, and its biological effects. This is followed by a summary of the vast literature on melittin–lipid interactions, including studies on the perturbing effect of melittin on lipid bilayers and its conformation when lipid?bound. Finally, the models proposed for lipid?bound melittin are discussed.
Chapters II, III, and IV describe the present work on melittin.
Chapter II focuses on the conformation of melittin in nonaqueous solutions as delineated from CD studies. Evidence is presented for an extended ??sheet?like conformation for melittin in an apolar solvent, dioxane. Transitions between extended and helical forms are observed in solvent mixtures. The solvent?dependent conformational change in melittin, especially in relation to conformational variability in membrane?active peptides, is discussed in detail.
Chapter III describes the utility of edge?excitation fluorescence measurements (the red?edge effect) in the study of melittin in both aqueous and lipid phases, beginning with an introduction to the red?edge effect. The red?edge effect, in conjunction with isotope effects, has been found to be a useful parameter to probe the aggregation state of melittin in both water and lipid bilayers. The studies permitted monitoring of lipid?phase aggregation of melittin molecules induced by changes in the phosphate ion concentration of the medium. The interaction of melittin with synthetic bilayers of differing thickness has been probed by this method. In addition, acrylamide quenching of fluorescence has been employed to determine the depth of insertion of melittin within bilayers of synthetic lipids.
Chapter IV presents studies on the ionophoretic ability of melittin. Using a difference spectrophotometric assay with a liposome?bound metallochromic dye, melittin has been shown to form channels across membranes that alter Ca²? fluxes. Furthermore, this assay is sufficiently sensitive to monitor the voltage?dependent insertion of melittin into bilayers.
Section II — Bilirubin
The studies on bilirubin constitute Chapters VI and VII, and, along with an introductory Chapter V, form Section II of this thesis.
Chapter V briefly reviews the available literature on bilirubin formation and the pathological implications of its faulty metabolism, focusing on this neurotoxic pigment. Special attention is given to the structure and conformation of bilirubin in solution. Various aspects of the interaction of bilirubin with human serum albumin (HSA) are discussed. The origin of bilirubin CD and its variability are briefly mentioned.
Chapter VI describes attempts to mimic the binding site of bilirubin on HSA using model amine receptors, including symmetrical alkyl diamines, chiral amines, and basic polypeptides. Enhanced fluorescence and induced optical activity have been used as probes to monitor such interactions. The dependence of bilirubin CD on the secondary structure of the binding site has been examined using polypeptides of various secondary structures. Covalent conjugation of bilirubin to chiral amino acids has been shown to result in induced circular dichroism of bilirubin. These studies suggest that the diverse CD spectra observed upon bilirubin interaction with serum albumins can, in fact, be mimicked by appropriate choices of model receptors.
Chapter VII presents extensive investigations on the interactions of bilirubin with bile salts using gel filtration, fluorescence, CD, and NMR methods. Gel filtration provides a particularly simple means of fractionating bilirubin–cholate complexes of defined stoichiometry. A detailed ¹H NMR study of the bilirubin–cholate system, using nuclear Overhauser effect (NOE) measurements, led to the delineation of the conformation of micelle?packaged bilirubin. In addition, intra? and intermolecular NOEs have been shown to be particularly useful probes for monitoring micellar aggregation and solubilization of hydrophobic solutes like bilirubin.
Chapter VIII summarizes the major findings of the present investigation. | |
