Effect of buffer,electric field,and separation time on detection of aptamer-ligand complexes for affinity probe capillary electrophoresis

The separation and detection of complexes of aptamers and protein targets by capillary electrophoresis (CE) with laser-induced fluorescence was examined. Aptamer-thrombin and aptamer-immunoglobulin E (IgE) were used as model systems. Phosphate, 3-(N-morpholino)propanesulfonic acid with phosphate, and tris(hydroxyamino)methane-glycine-potassium (TGK) buffer at pH 8.4 were tested as electrophoresis media. Buffer had a large effect with TGK providing the most stable complexes for both protein-aptamer complexes. Conditions that suppressed electroosmotic flow, such as addition of hydroxypropylmethylcellulose to the media or modification of the capillary inner wall with polyacrylamide, were found to prevent detection of complexes. The effect of separation time and electric field were evaluated by monitoring complexes with electric field varied from 150-2850 V/cm and effective column lengths of 3.5 and 7.0 cm. As expected, shorter times on the column greatly increased peak heights for the complexes due to a combination of less dilution by diffusion and less dissociation on the column. High fields were found to have a detrimental effect on detection of complexes. It is concluded that the best conditions for detection of noncovalent complexes involve use of the minimal column length and electric field necessary to achieve separation. The results will be of interest in developing affinity probe CE assays wherein aptamers are used as affinity ligands.     

APPLICATION OF TIME-RESOLVED FLUOROMETRY TO THE RESOLUTION OF PORPHYRIN-PHOTOPRODUCT MIXTURES

Abstract —The quantitation of porphyrin and its photoproducts in biological samples is important in diverse applications including, but not limited to, tumor phototherapy. Differences in fluorescence properties, e.g., emission spectra and lifetimes, of the various components can be utilized to resolve the individual contributions. In this report time-resolved measurements using multifrequency phase and modulation rluorometry are employed to study mixtures of protoporphyrin IX and photoprotopor-phyrin. Individual lifetimes of 14.85 ns and 5.95 ns were determined for dioxane solutions of protoporphyrin IX and photoprotoporphyrin, respectively. These lifetime values were utilized to resolve the individual emission spectrum of each component in the mixture using the phase-resolved Huorometry technique.     

Measuring chirality in NMR in the presence of a static electric field

The scalar Hamiltonian of nuclear spins in the presence of a static electric field supports chirality. However, the eigenvalues of the Hamiltonian are not chiral; hence, chirality is not manifested in the usual NMR experiment. In this work, we show that the magnetization response to certain radio frequency pulse sequences exhibits chirality as well as handedness.     

The role of particles in stabilising foams and emulsions

The use of particles as foam and emulsion stabilising species, with or without surfactants, has received great interest in recent years. The majority of work has studied the effects of particles as stabilisers in emulsion systems, but recent successes has widened consideration into foams, where industries such as flotation and food processing have encountered the effects of particle stabilisation for many years. This review seeks to clarify studies into emulsions, highlighting new research in this area, and relate similarities and differences to foam systems. Past research has focused on defining the interaction mechanisms of stability, such as principles of attachment energies, particle-particle forces at the interface and changes to the interfilm, with a view to ascertain conditions giving optimum stability. Studied conditions include effects of particle contact angle, aggregation formations, concentration, size and interactions of other species (i.e. surfactant). Mechanisms can be complex, but overall the principle of particles creating a steric barrier to coalescence, is a straitforward basis of interaction. Much research in emulsions can be applied to foam systems, however evidence would suggest foam systems are under a number of additional constraints, and the stability 'window' for particles is smaller, in terms of size and contact angle ranges. Also, because of increased density differences and interfilm perturbations in foam systems, retardation of drainage is often as important to stability as inhibiting coalescence.     

New insights into iron release from ferritin: direct observation of the neurotoxin 6-hydroxydopamine entering ferritin and reaching redox equilibrium with the iron core

Iron release from the iron storage protein ferritin has been studied extensively because of its important role in oxidative stress and its possible role in the progression of Parkinson's disease. For many years external indicators, notably strong iron(II) chelators, have been used to investigate this reaction. Such chelators can, however, drastically affect the electrochemical and thermodynamic properties of iron. The present study is unique in that it has been possible to follow a reaction taking place within the ferritin shell. This was made possible by our serendipitous discovery that, at physiological pHs, the oxidation product of 6-hydroxydopamine (a deprotonated quinone) acts as its own indicator (G. N. L. Jameson and W. Linert, J. Chem. Soc., Perkin Trans. 2, 2001, 563-568). The redox equilibrium data and the kinetics of the formation of this red-coloured species can only be explained on the basis that reduction of the iron(III) takes place within the ferritin shell. This is, in fact, the first time that a reaction actually taking place inside the ferritin shell has been followed. It has also been established that, at least in vitro, all eight hydrophilic channels are capable of being simultaneously involved in the reaction. It has also been possible to calculate the rate of oxidation of the 6-hydroxydopamine within the ferritin and it is demonstrated that a redox equilibrium is established within the protein. Finally, evidence is provided confirming that chelators are in fact intrinsically linked to iron removal from ferritin.     

Molecular reorientation of CD(4) in gas-phase mixtures

Spin-lattice relaxation times were measured for the deuterons in CD(4) in pure gas and in mixtures with the following buffer gases: Ar, Kr, Xe, HCl, N(2), CO, CO(2), CF(4), and SF(6). Effective collision cross sections sigma(theta, 2) for the molecular reorientation of CD(4) in collisions with these ten molecules are obtained as a function of temperature. These cross sections are compared with the corresponding cross sections sigma(J) obtained from (1)H spin-rotation relaxation in mixtures of CH(4) with the same set of buffer gases. Various classical reorientation models typically applied in liquids predict different ratios of the reduced correlation times for the reorientation of spherical tops. The Langevin model comes closest to predicting the magnitude of the sigma(theta, 2)/sigma(J) ratio that we obtain for CD(4).     

Rhenium(I) compounds bound by tripodal ligands of pyridine and N-methylimidazole

The reaction of Re(CO)(5)Br with tris(2-pyridyl)methanol (tpmOH) leads to unexpectedly complex chemistry with three new compounds forming instead of a single product. In compound 1, the tpmOH ligand binds to the metal in the N,N',N'-mode; 2 has tpmO(-) bound in the N,N',O-mode; while 3 is a dimer with the tpmO(-) ligand utilizing each of the four donor atoms to bridge the two metal centers. The analogous methyl ether ligands, tris(2-pyridyl)methoxymethane (tpmOMe) or tris[2-(l-methylimidazolyl)]methoxymethane (timmOMe), each yielded a single product, 4 and 5, respectively, bound in the N,N',N'-mode, and are new leads for potential radiotherapeutic agents. All compounds have been structurally characterized.