Ram P. Singhal

New ligands for boronte affinity chromatography : Synthesis and propertiesa☆

Abstract In order for a boronate ligand to be useful in affinity chromatography for the purification of biomolecules, it must be able to form a stable complex in an environment (pH) in which the affinity molecule is stable. A major limitation of the widely used ligand, 3-aminophenylboronate, is its high ionization constant (pKa 8375). to make this complex under more faborable pH conditions, different methods have been explored here in order to introduce an electron-withdrawing (nitro) group in the phenyl ring. Reagents and procedures for the preparation of ortho-, meta- and para-nitro derivatives of succinamidophenhylboronic acid using nitronium trifluromethanesulfonate are described. Preferential substitution of the nitro functinality into the ortho position of the boronic acid is exploited by selective use of acetic anhydrided for the reaction medium. This method yields mostly an ortho-nitro derivative (;Ka 7.4) under selected reaction conditions. The ionization and solute—ligand interaction of several phenylboronates are studied in solution by using 11B NMR and spectrophotometric methods. The results indicate the presence of specific chemical shifts for the neutral (δ 30), the boronate anions (δ 30, and the cis-diol-complex boronate species (δ 7.5). In the presence of a cis-diol derivative, the complex formation is favored over anionization of neutral species. Moreover, the complex is formed approximately on pH unit below the ionization constant of the ligand and is stable, i.e. fails to break down in boronate anion, even when the solution pH is raised appreciably. Two boronate affinity column matrices were examined for their binding capacity and apparent dissociation constant. The results clearly indicate that the formation and also the breakdown of the complex are greatly enhanced because of the presence of the elecgtron-withdrawing group in the boronate ligand. The results further demonstrate that small structural differences in affinity moleculew have significant differences on their binding capacities. A comparison of binding betwen alkyl-cis-diols and arylcis-diols to different boronate matrices indicates taht the aryl affinity molecules not only form a complex but do so very effectively. The significance of this work lies in the demonstration that the best environment for the ligand—solute interaction can be established by carrying out studies in solution, without prior immobilization of the ligand. The results from in-solution studies and those from the affinity columns are in very good agreement. The new nitrophenylboronate matrix offers enhanced binding of most affinity molecules over those examined with the phenylboronate matrix. In addition, the new matrix offers chromatographic separations of alkali-unstable biomolecules.

Structure, function and evolution of 5-S ribosomal RNAs.

Publisher Summary This chapter discusses the structure, function, and evolution of 5-S ribosomal RNAs. The recent culmination of new and corrected nucleotide sequences of 5-S RNAs derived from the eubacteria, archaebacteria, eukaryotes, and the organelles reveals that the 5-S ribosomal RNA has been extremely conserved in nucleotide sequence and chain-length during the course of evolution. Eubacterial and eukaryotic 5-S RNAs differ primarily by the presence of a small number of insertions or deletions that are scattered throughout the molecule. Certain regions of the molecule tend to sustain chain-length changes more readily than other regions, e.g., helix V. The nucleotide sequences from a wide range of organisms conform to a generalized secondary structural model. Probes of the 5-S RNA structure by partial nuclease digestions largely confirm this model. In E. coli 5-S RNA, two double-helical regions (helices I and V) have been detected by nuclear magnetic resonance (NMR) spectroscopy. There is both phylogenetic and experimental evidence for the presence of non-Watson-Crick A–G base-pairing in the stem encompassing helices IV and V of the 5-S RNA. There is phylogenetic evidence also for the presence of A–A and A–C pairing, but as yet no experimental data reveal the presence of these base-pairs in the 5-S RNA.

Structure, Function, and Evolution of Transfer RNAs (with Appendix Giving Complete Sequences of 178 tRNAs)

Publisher Summary The chapter discusses the structure, function, and evolution of transfer RNAs (tRNA). A comparison of the sequences of tRNAs has provided new and supportive information concerning: (a) secondary and tertiary structures; (b) minor modified nucleotides and their possible roles; and (c) binding sites for specific and nonspecific interactions between tRNA and the aminoacyl-tRNA synthetase. The statistical data reveal that invariant or conserved nucleotides are present in each prokaryotic and eukaryotic tRNA. These invariant positions, determined for >60% and >90% of the tRNAs, suggest evolutionary or “primordial” structures that may have differentiated into the highly specific structures required for the aminoacylation process. The study of variant residues indicate that the prokaryotic and eukaryotic tRNAs, although stemming from a common “proto” tRNA, have evolved in two directions. Interestingly, tRNA structure has not changed drastically during this evolution. A statistical analysis of 129 tRNAs from eukaryotic, prokaryotic, and viral sources is carried out in an attempt to answer the following questions, which are: (a) what are the invariant (conserved) nucleoloids in tRNAs, (b) is the tertiary structure of yeast tRNA Phe representative of the tertiary bonds in other tRNAs, (c) what is the nature of the modified nucleotides in tRNAs, (d) what, if any, are the differences between prokaryotic and eukaryotic modifications, (e) how does the T-ψ-C sequence, a feature of most tRNAs, vary in certain tRNAs, (f) can the chapter predict one or more specific interaction sites between tRNA and the cognate synthetase after determining the variant and the invariant residues between the isoacceptor sequences.

Separation of DNA restriction fragments by polymer-solution capillary zone electrophoresis: Influence of polymer concentration and ion-pairing reagents

Abstract To achieve sensitive and fast separations of the DNA fragments, polymer-solution capillary zone electrophoresis (PS-CZE) is examined. A systematic study of the influence of the linear polymer concentration on the separation of restriction fragments indicates that the larger DNA fragrments are resolved with greater efficiency by using a lower concentration of the polymer, while the smaller ones are better resolved with a higher polymer concentration, when added to the buffer. A specific concentration of the polymer provides a certain limit of dynamic “porosity” (sieving), which is appropriate for a given range of the DNA fragments. Several ion-pairing reagents are examined here to improve resolutions of both small and large DNA fragments in PS-CZE. These reagents cause no interactions between the ammonium cations (reagent) and the silanol groups (capillary surface). However, they interact with the linear polymer, perhaps by hydrophobic functions, and change its physical properties, such as dynamic viscosity and sieving ability. Enhanced retention (interactions) of the DNA fragments is observed by increasing the ion-pairing contents in the polymer solution. The increased peak retention strongly suggests presence of interactions between the reagent and the DNA fragments. Different ion-pairing reagents produce different degrees of peak retentions. The interactions results from relatively greater hydrophobic interactions and smaller ionic interactions between the reagent and the polynucleotides. The study of ethidium bromide (EdBr) additon in PS-CZE indicates that a small concentration of EdBr reduces peak widths and decreases retention of all the fragments, but has little influence on the electroosmotic flow. However, a larger EdBr concentration results in broader peak widths and causes little change in their retention. Ion-pairing and intercalating reagents interact with the DNA fragments by different mechanisms. An anion-pairing agent appears to interact predominantly by hydrophobic interactions with the purine and pyrimidine bases, while EdBr intercalates with G:C base pairs and alter the chain length. Satisfactory separations of both larger and smaller fragments can be achieved by adding an appropriate ion-pairing reagent along with the linear polymer to the buffer. Critical concentrations of the linear polymer and the ion-pairing reagent must be determined in order to achieve satisfactory separations for a given range (size) of the DNA fragments.

Separation and Analysis of Nucleic Acids and Their Constituents by Ion-Exclusion and Ion-Exchange Column Chromatography

Abstract The recent commercial development of high pressure liquid chromatographic instruments, and the availability of small and uniformly sized resin beads has enhanced the role of column chromatography for the rapid assay of nucleic acid components. A major advancement in the purification and analysis has been due to the recent demonstration that the ion exclusion is more or equally effective than the ion exchange as a separation principle for the resolution of purine and pyrimidine bases, nucleosides, deoxynucleosides and nucleotides1–3. This article deals with the recent applications of separation principles that have been employed for the separation of nucleic acid constituents. The reader is referred to two review articles of Cohn for the earlier works on this subject4, 5. Separations by chromatography and electrophoresis on paper and thin layers are not described here; several monographs and review articles are available in the literature6–12.The reader is also referred to a recent methodological ...

Modification of guanine to queuine in transfer RNAs during development and aging

Abstract The degree of modification of guanine to queuine in the four queuine-containing tRNAs (Q-tRNAs) has been studied from rats of various age groups, and bacterial cells in different growth phases by measuring the amount of G-tRNA present in these tRNA preparations by tRNA-guanine transferase. In very young (one-week old) animals, only a small amount of G to Q modification was observed. However, this modification was essentially complete in the tRNAs of nine-month old animals, thereafter, the amount of Q decreased steadily. Studies of tRNAs from leukemic lymphocytes and bacterial cells indicated that the degree of G to Q modification was related to the metabolic state of the cell. The possible role of the Q-deficient isoacceptors in translation control is discussed.

Reversed-phase boronate chromatography for the separation of O-methylribose nucleosides and aminoacyl-tRNAs

Abstract Boronate forms an anionic complex with the cis -2′,3′ hydroxyls of unsubstituted ribonucleosides and the 3′-terminal adenosine of unacylated tRNAs, but not with ribosesubstituted nucleosides such as 2′- O -methylnucleosides and aminoacyl-tRNAs. We have synthesized phenyl boronates with hydrophobic side chains of about 1-nm-long and coated inert 10-μm solid beads of polychlorotrifluoroethylene with this material. This matrix complexes easily with compounds containing free cis -hydroxyls, but not with their O -alkyl or O -acyl derivatives. This permits the separation of mammalian and bacterial amino-acyl-tRNAs from uncharged tRNAs and O -methyl nucleosides from ribose-unsubstituted nucleosides in one chromatographic step, as the substituted members of each group do not undergo boronate complex formation and are thus not as much retarded in passing through the column. Complex formation between ribofuranoses and the boronate matrix appears to be enhanced by the hydrophobic “tail” of the boronate compound, by the high ionic environment of the solvent, and by the hydrophobic nature of the inert support. This method of one-step purification of tRNAs on reversed-phase boronate columns has been tested for several tRNAs specific for amino acids of different hydrophobicity and ionic character. The results indicate that each tRNA tested can be purified with appreciable purity (70–95%) and high yield (80%). However, recovery of the queuine base containing aminoacyl-tRNAs is only about 6% of the applied material. Several other boronate matrices have also been synthesized using cellulose, agarose. Sepharose, or porous glass beads as the inert support with different lengths of the spacer arm. Cellulose with a 1-nm-long spacer arm is satisfactory not only for the separation of aminoacyl-tRNAs and O -methylribose nucleosides, but also for the separation as a group of tRNAs containing the base of Q, queuine. However, other inert supports are unsatisfactory because of a non-specific binding of the tRNAs.

High-performance liquid chromatographic analysis of DNA composition and DNA modification by chloroacetaldehyde

The separation of common and modified deoxyribonucleosides derived from DNA hydrolyzates was examined under different chromatographic conditions on silica-based octadecyl (C18) columns, involving hydrophobic interactions with the matrix. A novel method for the analysis of the DNA composition is described. It involves the removal of RNA contaminants and enzymatic hydrolysis of DNA, first to deoxyribonucleoside monophosphates and then dephosphorylation of the latter to deoxyribonucleosides. Hydrolysis conditions were sought to avoid deamination of dA and dC residues to dI and dU contaminants, respectively. Elution of these contaminants and the artifacts (ribonucleosides derived from RNA) is described in relation to the elution of deoxyribonucleosides. Chromatographic separation of the hydrolyzate derived from a 15-micrograms sample of DNA under selected separation conditions and on one high-performance liquid chromatographic column is achieved in 18 min at room temperature. Detection of modified components (and contaminants) present in minute amounts is enhanced with the use of a diode-array detector. The power of this technique lies in its ability to characterize and quantitate accurately the amount of modified species present in the DNA structure (less than 2% of all the other residues). Examples of the composition analysis of DNA derived from a prokaryote (Escherichia coli B) and a eukaryote (salmon sperm) are described. Details of quantitation (calibration graphs) of different nucleosides are furnished for peak-area integration by commercially available software, and spectral properties of the nucleoside in the elution buffer are described for quantitation by other means. Application of the composition analysis is shown here for probing the DNA conformation in solution by chemical means, while using chloroacetaldehyde as the modifying agent.

Structure of Transfer RNAs: Listing of 150 Additional Sequences

Publisher Summary This chapter discusses the structure of transfer RNAs: listing of 150 additional sequences. The structure of various tRNAs can be organized in a manner to yield common nucleotide “positions” in the cloverleaf structure of the most tRNAs. To achieve this, “subpositions” of residues 17 (17: 1, 17 : 2) and 20 (20 : 1, 20 : 2, 20 : 3) in the dihydrouridine loop and residue 47 (47 : 1, 47 : 2, etc.) in the variable loop are introduced. Using this numbering scheme, each tRNA yields a structure with common “invariable” or “conserved” nucleotide positions. The chapter discusses in addition to “sub positions,” “invariable” (A, C, G, or U), and “semi conserved” (R, which is A or G; Y, which is C or U; G or C) positions found in most tRNA sequences. tRNA sequences have been grouped according to their origins (sequences published in the earlier listing are indicated by sequence code numbers in italics). An Arabic number in parentheses after the sequence code indicates the number of different organisms having that common tRNA structure as referred to by the sequence code.

Hardware and software for microprocessor controlled HPLC: interfacing of converters (A/D and D/A) for data acquisition and display

Abstract To allow direct acquisition and “real-time” processing of chromatographic data, hardware interfaces and a software operating system are described here for a dual-processor Hplc controller. The interfaces consist of a multiplexed, analog-digital converter, and digital-analog converters with built-in event marker circuits. The analog amplifiers for the converters are specifically designed to permit interfacing of conventional Hplc detectors and recorders to increase the flexibility of the system. A digital logic design is developed that minimizes interface servicing during “real-time” program execution. The software operating system consists of machine-language “kernel” routines for communication between the two processors, and high-level language programs to simplify the oper.