PubMed · 7634081
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
N O Artemyev, H E Hamm. 1994. Probing G-protein function.. https://doi.org/10.1038/nsb1194-752
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
There is a need for rapid methods to detect pathogenic bacteria in food products as alternatives to the current laborious and time-consuming culture procedures. We report a microbial detection technique that combines the selectivity of antibody-coated superparamagnetic beads with the rapidity and sensitivity of electrochemical detection in a format termed enzyme-linked immunomagnetic electrochemistry. In it, Salmonella typhimurium were sandwiched between antibody-coated magnetic beads and an enzyme-conjugated antibody. With the aid of a magnet, the beads (with or without bound bacteria) were localized onto the surface of disposable graphite ink electrodes in a multi-well plate format. Enzyme substrate was added and conversion of substrate to an electroactive product was measured using electrochemical detection. The electrochemical response was directly proportional to the number of captured bacteria. Using this technique, a minimum detectable level of 8 x 10(3) cells/ml of Salmonella typhimurium in buffer was achieved in ca. 80 min.
Knowledge of instrumental complications is vital both in interpreting experimental observations and in achieving true results. In a study of palladium electrodeposition on highly ordered pyrolytic graphite by in situ electrochemical scanning tunnelling microscopy, some unexpected experimental artefacts caused by instrumental design and tip-surface interactions have been recognised. The electrodeposition system employed in our in situ studies has been found to be very sensitive to an initial cathodic current surge when the potentiostat control was applied to the electrodes at the measured open circuit potential. As a result, palladium and/or hydrogen were immediately deposited on the graphite surface within a small radius under the imaging tip. The cause of this current surge was investigated and found to be related to the performance of the potentiostat. The deposit was removed either by anodic potential sweeps or under positive local electric fields of the imaging tip. Further cathodic deposition was possible on the surge-induced deposit. Characterisation, by ex situ x-ray diffraction, atomic force microscopy, scanning transmission electron microscopy and high resolution electron microscopy, showed that the deposits were Pd. Use of the various techniques demonstrated their complementarity in studies of the structure and dimensions of surface deposits.
The release of fibrinopeptides A and B by the slow and fast forms of thrombin was studied over the temperature range from 5 to 45 degrees C and the salt concentration range from 100 to 800 mM. The sequential mechanism for the release of fibrinopeptides originally proposed by Shafer was found to be obeyed under all conditions examined. The origin of preferential binding of fibrinogen and fibrin I to the fast form of thrombin in the transition state is in the second-order rate constant for association, k(l). In the case of fibrinogen, the values of k(l) for interaction with the fast and slow forms at 25 degrees C are 19 +/- 4 and 2.5 +/- 0.3 microM(-1) s(-1), with an activation energy of about 10 kcal/mol in both forms. In the case of fibrin I, the analogous values of k(l) are 9.1 +/- 0.7 and 2.5 +/- 0.2 microM(-1) s(-1), and the activation energy is about 4.5 kcal/mol in both forms. The mechanism of recognition of fibrinogen and fibrin I by thrombin entails a diffusion-controlled step with a small energy barrier. Analysis of the temperature dependence of the coupling free energy for allosteric switching indicates that the preferential interaction of fibrinogen and fibrin I with the fast form of thrombin in the transition state is entropy-driven, signaling a contribution of the hydrophobic effect to the slow-->fast transition. The salt dependence of the release of fibrinopeptides shows a constant coefficient Gamma(salt) = d ln(k(cat)/K(m))/d ln [salt] in the concentration range examined. Interestingly, the value of Gamma(salt) is independent of the salt used (NaCl, ChCl, or NaF) and is -1.5 +/- 0.1 for fibrinopeptide A and -2.5 +/- 0.1 for fibrinopeptide B. Hence, Gamma(salt) reflects predominantly the electrostatic contribution to the formation of the transition state, with a larger contribution seen in the interaction of thrombin with fibrin I. It is concluded that the interaction of thrombin with fibrinogen and fibrin I, leading to the release of fibrinopeptides A and B, is driven by electrostatic forces that presumably favor the correct preorientation of the enzyme and the substrate to form a productive complex in the transition state. This electrostatic-steering effect, also reported for thrombin-hirudin interaction, leads to a diffusion-controlled encounter with a very small energy barrier. Once the complex is formed, the enzyme switches to the fast form as a result of entropic factors presumably linked to water release from a more extended surface of recognition. While the release of fibrinopeptides as a function of salt concentration was being studied, an important observation was made on the role of Cl- in the formation of the fibrin clot. This anion drastically and specifically reduces the thickness of fibrin fibers, as judged by the 10-fold decrease in the equilibrium turbidity of clots developed in NaCl as compared to the turbidity of clots developed in NaF. Hence, the transition from a "coarse" to a "fine" clot induced by an increase in ionic strength as first described by Ferry is, instead, due to the specific binding of Cl- to intermediates in the ensuing polymerization. In fact, no change in the clotting curve is observed when the ionic strength is changed with NaF.