Physical and Biological Studies on Some Enzymes

Abstract Not Provided

Studies on Some Biological Reactions of Agricultural Interest

The general introduction out lines briefly, with particular reference to ribonucleases, the character of the enzymes which depolymerise nucleic acids. The publication by H. S. Kaplan and L. A. Heppel in J. Biol. Chem. 222 907 (1956) that a heat stable ribonuclease M. W. 2,000 - 5,000 could be isolated from calf spleen led to the research work presented in this thesis. These workers reported the purification of a ribonuclease similar to pancreatic ribonuclease in heat stability and specificity. Publications by other workers revealed that several ribonuclease activities could be extracted from calf spleen. The work reported here describes the procedures taken to purify the low molecular weight ribonuclease. Since several ribonucleases were reported, the original purification scheme of Kaplan and Heppel was adhered to initially. Section I outlines this procedure and the salient points are highlighted. In addition to a heat treatment there were four fractionations by conventional precipitation techniques, a lengthy dialysis and an Amberlite resin treatment. Section II, in addition to drafting the criteria for enzyme isolation and purification, reports the investigation of the techniques used originally by these workers. The unfavourable results obtained are presented in detail. Although an equivalent purification was achieved the yield of heat stable ribonuclease activity was poor. Each of the steps rejected ~50% of the activity. With the exception of the heat treatment which was considered essential, this original system was abandoned in favour of preliminary fractionation end concentration by precipitation with ammonium sulphate before and after the heat treatment. In this way ~75% of the heat stable activity was concentrated ready for molecular sieve and ion exchange chromatography. Section III reports the chromatography procedures undertaken to develop a purification scheme for the heat stable spleen ribonuclease. Some early experiments on the gel filtration behaviour of the active sample, particularly with respect to pancreatic ribonueleaee, are described. Gel filtration on a Sephadex G-75 column, 5cm x 75cm, developed to desalt the crude spleen sample. This measure eliminated the lengthy dialysis and achieved a complete recovery of activity with some purification. The desalting technique was followed by ion exchange chromatography. Chromatography on a Carboxymethyl-cellulose column fractionated the heat stable sample into two ribonuclease active peaks "A" and "B". A satisfactory adsorption of the crude sample on the C. M. cellulose was difficult to effect initially thus the sample was reduced by passing it through Diethylaminoethyl-cellulose as a pretreatment. All the ribonuclease activity passed through the column leaving ~37% of the contaminants adsorbed. The preliminary column work necessary to achieve the fractionation on C. M. cellulose using a gradient elution system is described. This column method was scaled up tenfold to cope with the large quantity of crude spleen preparation and prepare sufficient amounts of the active peaks "A" and "B" for rachromatography. Rechromatography on Carboxymethyl Sephadex revealed an elution irregularity at the chromatography on C.M. cellulose. Although ribonuclease active peak "B" was eluted as a single peak, the active peak "A" on rechromatography split into two peaks, one of which was eluted at a similar position to active peak "B". This indicated a distribution of activity for peak "A" similar to the chromatography of the crude preparation. On subsequent chromatography the rechromatographed peak "A" did not split again. These results indicated that calf spleen contains two heat stable ribonucleases. The activity "A" amounted to 16% of the total heat stable ribonuclease as determined by the general assay method. This activity was shown to be as heat stable as the bulk of the reparation. Attempts are made to explain the irregular chromatography effect. Section IV outlines the merit of disc electrophoresis on polyacrylamide gels as a technique for estimating the purity of protein samples. The spleen ribenuclease fractions "A" and "B" were examined by this technique. It was demonstrated that active fraction "B" had been extensively purified and had an electrophoretic mobility similar to pancreatic ribonuclease. Active fraction "A" though considerably purified contained at least three contaminants. An estimate of the molecular weights of the two ribonucleases is presented in Section V. A linear relationship exists between elution volume and log. (molecular weight) for globular proteins at gel filtration. To carry out the estimation, a Sephadex G-75 column was calibrated by protein standards of known molecular weight and gel filtration behaviour. After determining the elution volumes for the heat stable spleen ribonucleases, molecular weights of ~24,000 and 10,000 were attributed to activity "A" and activity "B" respectively. Ho evidence could be found to support the previous report that a heat stable ribonuclease M.W. 2,000-5,000 was present in calf spleen

Chromatographic Separation and Stability Analysis of Small Interfering RNA and Lipid Vehicles Using Ion-Pair Reversed Phase Liquid Chromatography

Chromatographic methods were developed capable of separating and quantitating siRNA, lipids, and their potential breakdown products due to oxidation or hydrolysis. Such methods are essential to developing lipid nanoparticles (LNPs) as a formulation delivery system for siRNAs. Separation of siRNAs was achieved using ion-pair reversed-phase liquid chromatography. Part 1 of the thesis describes the development of an ion-pair reversed-phase HPLC method for the separation of closely related stereoisomers of a chemically modified siRNA duplex. A systematic evaluation of key chromatographic parameters showed that a BEH C18 column with sub-2 [mu]-m particle size, coupled with the use of triethylammonium acetate as the ion-pair reagent and acetonitrile as the strong solvent of the hydro-organic mobile phase, achieved baseline resolution of siRNA stereoisomers and their desulfurization products. A high column temperature, creating a denaturing condition for siRNA, is critical to the separation of stereoisomers. An aprotic organic modifier, such as acetonitrile, can effectively disrupt the hydrogen bonding interaction between the duplex and enable the separation of stereoisomers by promoting hydrophobic interactions between the C18 stationary phase and the stereoisomers. Part 2 of the thesis expands the utility of the ion-pair reversed-phase liquid chromatography to include a simultaneous separation of the main lipid components of an LNP system. Ion-pair reversed-phase separation conditions were developed that can reduce the retention gap between siRNAs and lipids that have significant differences in their physical and chemical properties. Studies showed that a BEH phenyl column could significantly retain siRNA due to a combination of both hydrophobic and [pi] – [pi] interactions. In contrast, the lipids experienced a reduced retention with the phenyl column, a key advantage attributed to the presence of a short alkyl chain component in the stationary phase compared to octyl- or octadecyl-derivatized silica. While the ion-pair reagents had virtually no impact on the separation of the lipids, the retention times of the siRNAs showed a quantitative correlation with the structure of the ion-pair reagents in the mobile phase. The chromatographic separation conditions with a phenyl stationary phase, particularly with dibutylammonium acetate as the ion-pair reagent, markedly reduced the retention gap between the siRNAs and the lipids, achieving a baseline resolution of a complex matrix containing five siRNAs and six lipids in a 20-minute gradient elution method. Finally, the ion-pair reversed-phase method was applied to the degradation products of a model siRNA system. Stress testing showed that the model siRNA developed minimal hydrolysis products at neutral pH. This indicated the importance of chemical modification at the 2’-position in the ribose unit of siRNA molecules. In contrast, the siRNA was prone to oxidation by hydrogen peroxide, with or without trace levels of a transition metal, and to oxidation by a radical initiator. Desulfurization and phosphodiester strand scission were the likely main degradation pathways contributing to the observed oxidative reactivity.Ph.D., Chemistry -- Drexel University, 201

Studies on Nucleotides and Related Compounds in Plants

Abstract Not Provided

47th Rocky Mountain Conference on Analytical Chemistry

Final program, abstracts, and information about the 47th annual meeting of the Rocky Mountain Conference on Analytical Chemistry, co-endorsed by the Colorado Section of the American Chemical Society and the Rocky Mountain Section of the Society for Applied Spectroscopy. Held in Denver, Colorado, July 31 - August 4, 2005

Engineering protein biosynthesis apparatus, advanced design and screening strategies for small and fluorinated substrates in orthogonal translation

Protein engineering is a comprehensive toolbox for the chemical modification of enzymes in particular, and for the expansion of molecular functional diversity in general. In recent decades, two different categories have become established for the engineering of proteins. These include the approach of directed evolution approaches on the one hand and the strategies of rational protein design on the other hand. In particular, the use of noncanonical amino acids to introduce new functionalities has gained importance in the engineers’ toolbox. These include isostructural analogues of canonical amino acids as well as molecules with reactivities that can provide sites for further protein modifications. In this study, we have presented a strategy for manipulating the protein biosynthesis machinery towards the incorporation of noncognate fluorinated substrates. In general, fluorinated amino acids are not genetically encoded. These mainly synthetic building block are valuable for the design of particularly stable protein folds and for targeting highly specific protein-protein interactions. Fluorine is small and has a very low polarizability and the strongest inductive effect among the chemical elements found on earth. Due to these unique stereoelectronic properties, fluorine substitution is advantageously used in protein and peptide design. In this context, the strategy of directed evolution was applied to construct isoleucyl-transfer ribonucleic acid synthetase libraries for the isoleucine AUA rare codon reassignment with small aliphatic fluorinated amino acids, such as L-trifluoroethylglycine, by random mutagenesis. A suitable screening plasmid containing a mutant of superfolder green fluorescent protein (sfGFP) as reporter protein and a modified isoleucine transfer ribonucleic acid (tRNA_UAU) from Escherichia coli was produced to create an enhanced molecular adaptor level for gene expression. However, the required selection strain could not be constructed by genome editing due to the complexity of essential gene modification. In the second part of the study, different reporter proteins were used in advanced design with noncanonical amino acids for improvement of their biophysical, chemical, and biological properties. A robust alkene-tagged sfGFP variant was obtained, which is a valuable target in medicinal chemistry. In addition, the residue-specific incorporation of proline analogues into green fluorescent protein (GFP) derivates ─ enhanced green fluorescent protein (EGFP), NowGFP, and KillerOrange ─ enables the study of the role of prolines in the typical β-barrel structure organization

PURIFICATION AND INTERACTION STUDIES OF HISTONE, HMGB, AND PPI PROTEINS, FACILITATED BY THE DIPOLAR NATURE OF HMGBS

High mobility group box (HMGB) proteins are the most abundant non-histone proteins in the nuclei of eukaryotic cells and are highly conserved in animals. They bind dynamically to chromosomal DNA, interact with other proteins including transcription regulators, and have key roles in gene transcription and DNA repair. These fundamental activities require HMGB proteins to interact with the histone proteins found in chromatin, but the precise mechanisms remain unclear - the work here provides steps towards their clarification. Acid extraction of histones from chicken erythrocytes was explored, as this provides high yields without the use of ultracentrifugation. Histone protein markers (MW range 11.4 – 22.5kDa) suitable for use with SDS-PAGE were prepared using sulphuric acid extraction. For future research, almost pure histone octamers were also prepared, using phosphoric acid extraction combined with potassium chloride to ensure the octamers remained intact and in a nuclear-like environment. The remaining work was based on proteins extracted by mild methods from chicken erythrocytes to retain post-translational modifications. A nuclear protein set was isolated containing almost entirely histones and HMGB proteins, and was subject to cation-exchange chromatography, with phosphate buffers chosen to partially simulate nuclear conditions. HMGB molecules have a C-terminal acidic tail which in free solution is folded back onto the remaining basic part of the protein. Analysis of the chromatogram peaks suggested the HMGB proteins had unfolded into a dipolar configuration, with their basic parts binding to the cation-exchange column and their acidic tails binding to the histones. Gel filtration chromatography applied to fractions eluted from the cation-exchange column suggested the presence of one or more unidentified complexes. Native HMGB proteins where isolated using a novel method based on their dipolar nature. At high concentration, HMGB1 proteins were used to pull out a potential HMGB1/FKBP3 complex from a pool of nuclear proteins. Technology to significantly enhance the concentration of HMGB1 in chicken plasma was also developed, as the basis for prompt, low cost measurement of HMGB1 in biofluids (this has medical applications, such as in cancer diagnosis and prognosis). Further exploiting the dipolar nature of HMGB proteins, a method was developed for isolating the peptidyl-prolyl isomerases FKBP3 and Cyp B

The Mutarotation of Some Aldoses

The kinetics of the mutarotation of a series of nine 5-substituted aldoses of the D-glucose configuration and five 2-substituted glucoses, catalysed by a wide range of catalysts has been studied. It was found that substituents in the 5-position of the pyranose ring had a much greater effect on the kinetics of mutarotation than substituents in the 2-position of the ring. Electron withdrawing substituents (in the 5-position) were found to enhance the rate of the base catalysed mutarotation but to decrease the rate of acid and water catalysed mutarotation. Reasons for this are discussed with reference to the possible mechanisms of mutarotation. Steric hindrance of general base catalysis of mutarotation has been observed with the bases 2,6-lutidine, diethanolamine and tris-hydroxymethyl methylamine as catalysts. Attempts have been made to establish conclusively the reasons for this steric hindrance and to use it as a tool in distinguishing between kinetically indistinguishable mechanisms of mutarotation. The kinetic data of the acid and base catalysed mutarotation of the 5-substituted aldoses have been fitted to linear free-energy relationships (Taft and Hammett Equations) and found to give, at best, only a moderately good correlation. Reasons for this have been suggested. The spontaneous mutarotation of the anions of 6-deoxy-alpha-D-glucohepturonic acid and 6-O-(o-hydroxyphenyl)-alpha-D-glucose have been found to be faster than anticipated on the basis of the inductive effects of the substituent on the 5-position of the ring. It is proposed that these rapid mutarotations result from intramolecular catalysis by the carboxylate and phenolate groups respectively. The possible mechanisms are discussed and a preference expressed for that mechanism which involves intramolecular general acid catalysis of the sugar anion in the rate determining step, rather than the kinetically equivalent true intramolecular general base catalysis. A brief comment is made on the possibility of testing the hypothesis of "tautomeric catalysis". Finally, the appendix describes the attempted preparation of suitable substrates for a kinetic investigation of the enzyme ribonuclease A. Although a substrate for ribonuclease was prepared in situ, the difficulty in preparation and isolation of such substrates proved too great