Study of Insulin Attached onto Magnetic Nanoparticles

Glucose regulation is compromised in diabetic patients and hence diabetes is characterized by accumulation of glucose in blood. As a standard practice diabetic patients usually self-administer subcutaneous insulin injections daily, which are usually associated with pain, tissue necrosis, microbial contamination and nerve damage to local areas. Glucose-responsive implantable devices have provided a hope for a brighter future of diabetes management. However, one of the limitations of such devices is their refill requirement, which often requires surgical procedures leading to lower patient compliance. To overcome this limitation led to the idea of reusing insulin after it has been in the body circulation system and later becomes residues. To make this idea work, the first step proposed in this thesis is to tag insulin with magnetic nanoparticles and then to use a magnetic guidance system to bring it back the residue insulin to the implanted device before it can go to the clearance sites. Obviously, the precondition for the foregoing idea to work is to make sure that insulin’s conformation is not affected by the attachment with magnetic nanoparticles. This thesis was designed to study this precondition. The hypothesis is that the insulin’s conformation will not be affected by the attachment with the magnetic nanoparticles. Two specific objectives are: (1) assessment of the feasibility of potential capturing techniques and analysis of the attachment of insulin onto the magnetic nanoparticles to confirm the attachment; (2) measurement of the insulin’s conformation before and after it is attached with the magnetic nanoparticles. The spectroscopy techniques, including Fourier transform infrared, circular dichroism, absorbance and fluorescence spectroscopy, were used to conduct data collection and analysis. All four of these spectroscopies provide important information concerning the research objectives of this thesis. The results from the fluorescence and absorbance spectroscopy confirm the attachment of insulin onto the magnetic nanoparticles, hence the achievement of Objective 1. The results from the CD and FTIR spectroscopy show that insulin’s conformation is unchanged before and after its attachment onto magnetic nanoparticles, hence the achievement of Objective 2. The general conclusion of the study is that the insulin’s conformation will not be affected by the attachment of it with magnetic nanoparticles

Magnetic properties of metallic fine particle systems

A study of the magnetic properties of metallic fine particle systems in the form of magnetic fluids has been made. The fluids were prepared utilising the organometallic decomposition route (detailed separately by N. Mason, Ph.D. thesis, Durham University 1986) and single metal systems containing Fe, Co and Ni were prepared from new precursors. The properties of the first hydrocarbon based mixed metal particle systems are also reported. For systems prepared with Fe precursors it is thought that the fine particles are not in the α-Fe phase but may be amorphous and/or consist of iron carbides. Co and Ni systems result in particles with bulk-metal like structures although Co usually forms in the f.c.c. phase. The h.c.p. is also observed. The mixed metal systems were of FeCo and Ni(_3)Fe and a tendency to form the superlattice or ordered structures was observed. In both cases Fe has been lost to the particles and this is thought to be due to the formation of volatile iron compounds during preparation and/or surfactant complexes. Narrow size distributions have been obtained in all cases with mean particle diameters in the range 4-10 nm and standard deviations of between 0.8 and 1.9. The form of the size distribution has been found to be Gaussian. A study of the anisotropy of the particles using torque and magnetisation measurements has found uniaxial anisotropy with the first anisotropy constant of the order lO(^5)Jmֿ(^3).The values observed are too large to be ascribed solely to shape anisotropy. Low temperature magnetisation measurements have revealed the existence of a paramagnetic component in the fluids. Loss of magnetisation has occurred in all systems and is thought to be due to oxidation of the metal

The biophysical and biochemical characterisation of recombinant AP1 and a structural determination of two DNA binding sites, the TRE and CRE

The experimental work of this thesis was carried out on the DNA binding domain of the Fos and Jun proteins to further characterise transcription factor function. The leucine coiled-coil and basic region (the DNA binding domain) were overexpressed and purified to homogeneity using metal chelation affinity. The DNA binding specificity was characterised by gel retardation studies. It was shown that the binding of the wbFos:wbJun heterodimer to an oligodeoxynucleotide containing the AP1 DNA binding site was specific. Reducing conditions (5 mM DTT) significantly improved the specific shift observed. The specific binding was demonstrated by competition assays using oligodeoxynucleotides containing the DNA binding sites of other transcription factors. 53 amino acid residues were helical in the wbFos:wbFos homodimer, 35 in the wbJun:wbJun homodimer and 36 amino acid residues in the wbFos:wbJun heterodimer. In a buffer containing 50 [percent] trifluoroethanol the helical content of the wbFos:wbFos homodimer increased by 20 amino acid residues, consistent with the folding of the basic region. The relative stability of the dimers, estimated from the van't Hoff enthalpy, showed that the heterodimer was significantly more stable than the wbFos:wbFos homodimer. The Gibb's free energy at 310 K for the wbFos:wbFos homodimer was 1.6 kJ/mol, with a measured Kd of 5-10 [mu]M. The wbJun:wbJun homodimer had a Gibb's free energy at 310 K of -5.3 kJ/mol and a about 0.75 [mu]M. The Gibb's free energy at 310 K for the the wbFos:wbJun heterodimer had an enthalpy of unfolding of -171 kJ/mol and a Kdmuch less than 0.10 [mu]M. This suggests that the preferential formation of the heterodimer was through the destabilisation of the wbFos:wbFos homodimer. Two oligodeoxynucleotides containing the AP1 and GCN4 DNA binding site were characterised by high resolution NMR studies. 114 out of 124 protons were assigned for the symmetric tetradecamer d(GCATGACGTCATGC)2 containing the consensus GCN4\DNA binding site. The pseudorotation phase angle of the nucleotides was 144[degrees] for pyrimidines and 162[degrees]for purines. The fraction in the south conformation of nucleotides G1 to G13 was greater than 80[percent]. The pseudorotation phase angle of the 3' terminal nucleotide (C14) was 54[degrees]. The conformation of the base relative to the nucleotide (X), was typical of B-DNA for all the nucleotides G1 through G13. The correlation time for the tetradecamer of 3.5 +/- 0.05 ns was measured from the cytosine (C7 and C10) H5-H6 NOE timecourse. Further characterisation of the symmetric tetradecamer compared the relaxation rate constants for number of the protons at different spectrometer frequencies. The intrinsic relaxation rate constants (p) showed little variation with spectrometer frequency, whilst the initial slopes determined from the inversion recovery timecourse significantly varied with spectrometer frequency. The initial slopes were also very different from the apparent R1 values determined from the inversion recovery timecourse. The recovery timecourses appeared monoexponential. Adenine H2 showed a pronounced lag in the recovery. The effect of a short recycle time diminished the cross peak intensity. However the saturation factors were similar and normalised cross peaks showed little dependence on the recycle time. The protons of the pentadecamer containing the consensus AP1 DNA binding site, 5'd(CAAGTGACTCAGCGC):d(GCGCTGAGTCACTTG), were incompletely assigned. CD demonstrated that the overall conformation of both oligodeoxynucleotides was B- DNA

Spectroscopic studies of proteins in reversed micelles

The spectroscopic characterization of some protein-containing reversed micellar systems has been investigated. The concept of protein solubilization in hydrocarbon media via reversed micelles was first introduced by Gitler and Montal. Their system consisted of proteolipids, solubilized in an apolar solvent, with phospholipids as surfactant. Some years later Wells introduced the term "enzymes in reversed micelles" based on a study of phospholipase in diethylether/methanol mixtures with phosphatidylcboline as the surfactant. In this case the enzyme was already soluble in some organic solvents and the surfactant acted also as the substrate of the enzyme.One of the first hydrophilic enzymes, incorporated into reversed micelles, was chymotrypsin. Detailed activity and spectroscopic data were reported by the groups of Martinek, Luisi and Menger.In the following years, numerous publications, dealing with reversed micellar enzymological subjects, appeared. Three partially overlapping fields of interest can be distinguished. The first one is the study of enzymatic activity in reversed micellar solutions. Among this belongs the work done in our laboratory, which is concentrated on the optimization of biocatalytic processes in reversed micellar systems. The second field of interest consists of structural and dynamic aspects of protein containing reversed micelles. During the last few years, several groups have reported on spectroscopic and ultracentrifugation studies. The work presented in this thesis belongs to this category. The third line of interest is the study of enzyme recovery using reversed micellar systems. This type of research has gained increasing attention during the last years. It is possible to selectively extract proteins from an aqueous phase into a hydrocarbon phase containing reversed micelles and to re-extract the protein again into a second aqueous phase. This process can be applied to the purification of proteins.In Chapter 2 a review is presented about spectroscopic studies performed with reversed micellar systems. In this chapter a number of techniques is described, which can be used in reversed micellar research. These techniques are both applied to "empty" and protein-containing systems.Chapter 3 provides a detailed description of the time-resolved fluorescence technique and analysis, that we have applied to protein- containing reversed micellar systems. Examples are given of the fluorescence properties of three well characterized proteins in a normal aqueous environment. These examples illustrate the kind of information obtainable by studying time-resolved fluorescence properties.In Chapters 4,5 and 6 investigations on Protein containing reversed micellar systems are presented. Chapters 4 and 5 describe micellar systems, containing the fluorescent cytochrome c derivatives porphyrin cytochrome c or Zn-porphyrin cytochrome c. Chapter 6 reports en studies of the same micellar systems but with alcohol dehydrogenase as the guest protein instead of cytochrome c.In the last chapter of this thesis (Chapter 7) a summarizing discussion of the results is presented

Analytical Applications of Circular Dichroism in Pharmaceutical Determinations

Chemistr

21st Rocky Mountain Conference on Analytical Chemistry

Abstracts and meeting program from the 21st annual meeting of the Rocky Mountain Conference on Analytical Chemistry, co-sponsored by the Rocky Mountain Section of the Society for Applied Spectroscopy and the Rocky Mountain Chromatography Discussion Group. Held in Denver, Colorado, July 30 - August 1, 1979