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Reactivities of Amino Acids and Proteins with Iodine
Published in Erwin Regoeczi, Iodine-Labeled Plasma Proteins, 2019
The stadium-shaped line represents the surface of a folded protein molecule with an exposed and a buried tyrosyl (hexagons). At neutral pH (left), both tyrosyls are protonated (dark dots). At the moment of alkali addition, the exposed tyrosyl ionizes instantaneously (center) while the buried one remains unchanged. However, as the protein unfolds, the buried residue becomes freely accessible to the alkali and loses its proton (right). (During the conformational change there is also a blue shift.) Because of the very rapid mixing achieved by the stopped- flow technique, distinction between the states depicted above in the center and on the right can be made. The conformational transition of α-lactalbumin is a first-order process lasting for approximately 20 sec. Changes that take place in absorbance during the transition are recorded on a high-speed recording device and extrapolated to zero time to find the number of those tyrosyl residues which are ionizable in the native state of the protein.184
Activation Techniques
Published in Frank Helus, Lelio G. Colombetti, Radionuclides Production, 2019
Stopped flow system — By this procedure the target box is filled with the target gas. In our arrangement the target volume by 11 bar is 1430 cm3. When the irradiation is completed, in comparison with the flow system it produced the same amount of activity, 1.5Ci, the gas is removed by the flow rate of 600 cm3/min. In this target volume the amount of nonactive carbon atoms is about 20 times lower than it was in the first procedure. The using of the first or the second method of producing radioactive gases depends more or less on the application and further chemical processing of the product. If the 11C in the form of 11CO2 or another simple form is applicated, the open flow system is used. For the complicated synthetic procedure the stopped flow system is preferred. The third possibility of removing of the activity from the gas targets is the closed circuit system.
Instrumentation
Published in Clive R. Bagshaw, Biomolecular Kinetics, 2017
Although stopped-flow methods evolved from the continuous-flow method (discussed further in this chapter), it is appropriate to consider this method next because it is the most widely used of the rapid-reaction techniques [45,385, 406–409]. It is a continuous assay procedure usually used with optical detection, but the technique can be applied to a number of other detection methods. The principle is straightforward. Solutions containing the reactants are expelled from two drive syringes through a mixing jet, into a detection chambe r, and finally into a third syringe whose plunger hits a back stop (Figure 7.2). A microswitch, usually located at the back stop, triggers the recording device so that the reaction can be monitored in real time after the detection chamber has been filled with newly mixed reactants and the flow is brought to a halt.
Exploration of the residues modulating the catalytic features of human carbonic anhydrase XIII by a site-specific mutagenesis approach
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2019
Giuseppina De Simone, Anna Di Fiore, Emanuela Truppo, Emma Langella, Daniela Vullo, Claudiu T. Supuran, Simona Maria Monti
All measurements were done according to Khalifah’s stopped flow method43. An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed CO2 hydration activity. Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes as buffer (pH 7.4). About 20 mM NaClO4 were also added to the assay system for maintaining constant ionic strength. The initial rates of the CA-catalysed CO2 hydration reaction were followed for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. The uncatalysed rates were determined in the same manner and subtracted from the total observed rates, as reported earlier44–46.
The RFK catalytic cycle of the pathogen Streptococcus pneumoniae shows species-specific features in prokaryotic FMN synthesis
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2018
María Sebastián, Adrián Velázquez-Campoy, Milagros Medina
Kinetic experiments in the pre-steady state were registered as previously described23, using stopped-flow spectroscopy on an Applied Photophysics SX17. MV spectrophotometer, using the Xscan software (Applied Photophysics Ltd., Leatherhead, UK). Fast kinetic measurements were carried out as previously described23, at 25 °C in PIPES 20 mM pH 7.0, 0.8 mM MgCl2. About 0.2 µM SpnFADS was mixed with reaction samples that contained increasing concentrations of the flavin ligands (FLV, herein indicating RF or FMN), in the presence and in the absence of ADP or ATP (herein referred as ANP). Controls were measured in the same buffer but without MgCl2. All concentrations indicated here are the final ones in the reaction cell. The kinetic traces were registered until obtaining three reproducible traces.
Strategies for targeting the cardiac sarcomere: avenues for novel drug discovery
Published in Expert Opinion on Drug Discovery, 2020
Joshua B. Holmes, Chang Yoon Doh, Ranganath Mamidi, Jiayang Li, Julian E. Stelzer
In contrast to in vitro motility and solution-based assays, skinned fiber preparations retain a well-organized sarcomeric lattice with essential regulatory and structural protein constituents and thus preserve near-normal structure-function relationships. Thus, testing newly identified drug candidates on skinned cardiac preparations may yield additional insights that might differ from findings obtained using lattice-free systems. For instance, initial solution-based stopped-flow and transient-state kinetic experiments with OM showed an increased rate of actin-dependent Pi release from cardiac myosin-S1 leading to the idea that the sarcomeric modulator OM accelerates myosin XB transition rate from weakly- to a strongly-bound force-generating state [53]. This would predict faster rates of force generation in cardiac muscle. However, contrary to this notion, recent skinned cardiac fiber experiments revealed that OM acts to slow overall force generation by slowing the rate of XB detachment (krel), rate of cooperative XB recruitment (kdf), and rate of tension redevelopment (ktr) [76,94]. Data from skinned fiber studies suggest that the OM-induced slowing of krel acts to extend the amount of time that OM-bound myosin XBs spend in their actin-bound state [76]. The prolonged binding-times of OM-bound myosins enhance cooperative thin filament activation via nearest-neighbor effects, allowing non-OM-bound myosins to bind to actin, and result in an OM-induced increase in net force production [69]. Additionally, OM-induced slowing of kdf and ktr also suggests that the drug prolongs the total time course of OM-bound XB transitions to force-bearing states, an effect that potentially spreads cooperative activation along the thin filaments during OM-induced force enhancement [93].