ABSTRACT
Pulsed electron-electron double resonance (PELDOR or DEER) in combination with site-directed spin labelling has emerged as an important method for measuring nanometer distance constraints that are used to obtain coarse-grained structures of biomolecules or to follow their conformational changes. Translating measured spin-spin distances between spin labels into structural information requires taking the conformational flexibility of spin label side chains into account. Here, we present an analysis of orientation selective PELDOR data recorded on six singly MTSSL-labelled azurin mutants. The analysis yielded conformational MTSSL ensembles, which are considerably narrower than those predicted using in silico spin labeling methods but match well with spin label conformations found in the corresponding crystal structures. The possible reasons and consequences for predicting spin label conformers in the fold of biomolecules are discussed.
Subject(s)
Nitrogen Oxides/chemistry , Spin Labels , Azurin/chemistry , Crystallography, X-Ray , Electron Spin Resonance SpectroscopyABSTRACT
Pulsed electron electron double resonance (PELDOR) is a well-established method for measuring nanometer distances between paramagnetic centres. Here, we demonstrate on three rigid and conjugated biradicals how the presence of an exchange coupling constant J and its distribtion DeltaJ influences PELDOR data and its analysis. In principle two combinations of J and D fulfill the experimental data in each case. The correct one, including the sign of J, can be determined via simulations in case the two halves of the Pake pattern are separated enough.
ABSTRACT
Long range distance measurement on RNA allow the determination of RNA folds. Here we report the site specific incorporation of nitroxide spin labels at U,C and A by "on column synthesis". PELDOR (Pulsed Electron Double Resonance) measurements of several RNAs in the range of 2-6 nm were successful.
Subject(s)
Cyclic N-Oxides/chemistry , Oligonucleotides/chemical synthesis , RNA/chemistry , Spin Labels , DNA/chemical synthesis , Electron Spin Resonance Spectroscopy/methods , Iodine/chemistry , Oligonucleotides/chemistry , RNA/chemical synthesisABSTRACT
The radical pair recombination of an intramolecular electron-transfer system containing a transition metal moiety has been addressed by femtosecond spectroscopy. The radical pair is formed by ultrafast electron transfer (90 fs) from a ferrocene residue to a photoexcited Nile blue moiety. Its recombination proceeds on the picosecond time scale in a multiexponential fashion. The kinetic pattern is a manifestation of spin processes competing with electron transfer. Magnetic field effects on these kinetics allow one to disentangle the two contributions. Their temperature dependencies yield the activation parameters of the two processes. The discussion focuses on the mechanism of electron spin relaxation. Strong evidence for the Orbach/Kivelson mechanism will be given.
ABSTRACT
A new facile method for spin-labeling suitable for DNA and RNA oligonucleotides is presented. The nitroxide 3-ethenyl-2,2,5,5-tetramethyl-pyrrolin-1-yloxy was directly introduced during automated solid-phase synthesis by a Pd(0) cross coupling reaction. The main advantages of this procedure are the small amount of spin-label needed for the derivatisation of the oligonucleotide and the high coupling efficiency on the solid phase.
Subject(s)
Oligonucleotides/chemical synthesis , Spin Labels/chemical synthesis , DNA/chemical synthesis , DNA/chemistry , Nucleic Acid Conformation , Oligonucleotides/chemistry , RNA/chemical synthesis , RNA/chemistryABSTRACT
Charge transfer in supramolecular assemblies of DNA is unique because of the notion that the pi-stacked bases within the duplex may mediate the transport, possibly leading to damage and/or repair. The phenomenon of transport through pi-stacked arrays over a long distance has an analogy to conduction in molecular electronics, but the mechanism still needs to be determined. To decipher the elementary steps and the mechanism, one has to directly measure the dynamics in real time and in suitably designed, structurally well characterized DNA assemblies. Here, we report our first observation of the femtosecond dynamics of charge transport processes occurring between bases within duplex DNA. By monitoring the population of an initially excited 2-aminopurine, an isomer of adenine, we can follow the charge transfer process and measure its rate. We then study the effect of different bases next to the donor (acceptor), the base sequence, and the distance dependence between the donor and acceptor. We find that the charge injection to a nearest neighbor base is crucial and the time scale is vastly different: 10 ps for guanine and up to 512 ps for inosine. Depending on the base sequence the transfer can be slowed down or inhibited, and the distance dependence is dramatic over the range of 14 A. These observations provide the time scale, and the range and efficiency of the transfer. The results suggest the invalidity of an efficient wire-type behavior and indicate that long-range transport is a slow process of a different mechanism.
Subject(s)
2-Aminopurine/chemistry , Adenine/analogs & derivatives , Base Pairing , DNA/chemistry , Adenine/chemistry , Electron Transport , Guanine/chemistry , Inosine/chemistry , Models, Molecular , Time FactorsABSTRACT
Studies of the Clinical Pharmacokinetics of Allopurinol/3rd Communication: Allopurinol/oxipurinol bioavailability and pharmacokinetics following the administration of a controlled release allopurinol formulation. Regarding the results of our studies on the localization of the absorption of allopurinol and the kinetic behavior of allopurinol/oxipurinol following multiple administration the bioavailability and kinetic properties of the drug delivered from controlled release tablets were studied in healthy volunteers. Allopurinol controlled release tablets (Sigapurol CR), containing 200 mg of the drug characterized by rapid absorption and 100 mg characterized by pH-dependent delivery, were identified as a formulation with advantages pharmacokinetic properties.
Subject(s)
Allopurinol/metabolism , Oxypurinol/metabolism , Pyrimidines/metabolism , Adult , Allopurinol/administration & dosage , Allopurinol/blood , Chromatography, High Pressure Liquid , Delayed-Action Preparations , Female , Humans , Kinetics , Male , Middle Aged , Oxypurinol/administration & dosage , Oxypurinol/blood , Suspensions , Therapeutic EquivalencyABSTRACT
The rate and extent of allopurinol absorption was studied following its oral ingestion in a "high frequency capsule" which allows the evaluation of the sites of drug absorption. If allopurinol is liberated in the duodenum or upper jejunum its absorption is fast and complete while its liberation in the lower jejunum results in a slow and incomplete absorption. A capacity limited absorption process for allopurinol, suggested from the results of a study on the allopurinol bioavailability from different formulations, could not be proved in the range of single doses between 200 and 600 mg resp. 2.2 to 12.8 mg/kg. AUC- and Cp-values of allopurinol and oxipurinol correspond to the calculated figures in relation to the different doses/kg.
Subject(s)
Allopurinol/metabolism , Oxypurinol/metabolism , Pyrimidines/metabolism , Adult , Allopurinol/administration & dosage , Biological Availability , Humans , Intestinal Absorption , Kinetics , MaleABSTRACT
In a pharmacokinetic study with 6 healthy volunteers the parameters for allopurinol and oxipurinol were compared following a single dose of allopurinol and multiple application of the drug. Pharmacokinetic data for allopurinol and oxipurinol are different after single doses and under steady state conditions. The oxipurinol half-life of 17 +/- 5.1 h is prolonged under steady state conditions to 19.7 +/- 5.8 h. Based on the results of this study and on data from different authors the range of 17-21 h is discussed as the most frequent oxipurinol half-life.