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Chemical Permeabilization of Cells for Intracellular Product Release
Published in Juan A. Asenjo, Separation Processes in Biotechnology, 2020
Thomas J. Naglak, David J. Hettwer, Henry Y. Wang
Chaotropic agents, such as guanidine and urea, are capable of bringing some normally hydrophobic compounds into aqueous solution. They accomplish this by disrupting the structure of water, making it a less hydrophilic environment and weakening the hydrophobic interactions among solute molecules (Hammes and Swann, 1967; Hatefi and Hanstein, 1974). Ingram (1981) has studied the effect of the chaotropes ethanol, trichloracetate, Perchlorate, thiosulfate, iodide, nitrate, and urea on E. coli. He determined that all these chaotropes can induce lysis of growing but not of stationary cells. He also showed that ethanol inhibits the assembly of cross-linked peptidoglycan, possibly explaining the growth dependence of lysis. Ingram concluded that the effects of the chaotropic agent ethanol were due to its weakening of hydrophobic interactions.
Approaches to the Measurement of Biological Pollutants
Published in Somenath Mitra, Pradyot Patnaik, Barbara B. Kebbekus, Environmental Chemical Analysis, 2018
Somenath Mitra, Pradyot Patnaik, Barbara B. Kebbekus
The methods of RNA isolation depends upon the tissue and the type of RNA to be extracted. Procedures to isolate total cellular RNA include chemical extractions and centrifugation. Phenol extraction was one of the first techniques to successfully isolate RNA from many sources. However, guanidinium salts have been found to be better options, even for those tissues that are rich in RNA degrading enzymes. Guanidinium hydrochloride and guanidinium thiocyanates are very powerful chaotropic agents. Guanidinium thiocyanate-based methods are quite popular for the isolation of good-quality RNA from a variety of tissues. Cells (or tissues) are homogenized directly in a solution containing guanidinium salt and reducing agents such as 2-mercaptoethanol (2-ME) or dithiothreitol (DTT) to break intramolecular protein disulfide bonds. These conditions rapidly inactivate RNases by distorting the secondary and tertiary folding of the enzymes when the cells are disrupted. Using these reagents, it is possible to isolate intact RNA even from RNase-rich tissues and cells.
Protocols for Key Steps in the Development of an Immunoassay
Published in Richard O’Kennedy, Caroline Murphy, Immunoassays, 2017
Caroline Murphy, Richard O’Kennedy
The addition of low concentrations of salts can help to stabilise proteins [31]. Kosmotropic agents (their name derives from the word ‘kosmotrope’ (order-making)) function to stabilise proteins and hydrophobic aggregates in solution. They form strong hydrogen bonds and act to exclude hydrophobic residues (usually forming the internal structure of the protein) from the solvent, thereby maintaining the structural integrity of the protein. Too high a concentration of kosmotropic reagents will cause the protein to ‘salt out’. Therefore, a balance must be achieved. Chaotropic agents get their name from the word ‘chaotrope’ (disorder-making) and function to destabilise proteins. They break up hydrogen bonds and increase the solubility of proteins in aqueous solutions. This encourages the protein to denature. Amino acids: During the production of recombinant proteins, arginine can be used to enhance the amount of appropriately refolded proteins. Arginine, used at high concentrations (2M) has the capacity to suppress aggregation of partially folded intermediates [32].
Increase in cysteine-mediated multimerization under attractive protein–protein interactions
Published in Preparative Biochemistry & Biotechnology, 2022
Leo A. Jakob, Tomás Mesurado, Alois Jungbauer, Nico Lingg
Since the dimeric species of the CASPON enzyme is most stable and active, we further investigated its multimerization behavior in the presence of kosmotropic, neutral and chaotropic salts (ammonium sulfate, sodium chloride and guanidium hydrochloride, respectively). We have selected a pH range of 6-8 to study multimerization behavior. At this practically relevant pH range, the protonation state of cysteines varies from partially protonated and fully protonated to partially deprotonated and fully deprotonated at pH 6 and 8, respectively[23]. Temperatures for incubation were set to either 4 °C or 25 °C to emulate conventional temperatures in downstream processing. During this experiment, minor peaks occurred at higher retention times than the dimeric species and due to their later retention time compared to the dimer in SEC, we identified the species as the CASPON enzyme monomer.
Microemulsion as Model to Predict Free Energy of Transfer of Electrolyte in Solvent Extraction
Published in Solvent Extraction and Ion Exchange, 2022
Simon Gourdin-Bertin, Jean-François Dufrêche, Magali Duvail, Thomas Zemb
It is well known in the field of selective extraction that “hydrophilic” simple electrolytes are less extracted than “hydrophobic”. This is qualitative and more pronounced for anions than for cations [76]. However, the term “hydrophilic” is unclear and is not quantified in the same way by different authors. In biophysical literature, the terms “chaotropic” and “kosmotropic” are used [77]. The combination of chaotropic anion and kosmotropic cation renders the situation more complex, with the concept of “antagonistic” salts as introduced by Onuki and co-workers [78]. The experimental observation is that “inversions” occur when ordering on either the Hofmeister or the lyotropic series, or even the HSAB (Hard and Soft Acids and Bases) series, which are correlated to the observed extraction. To the best of our knowledge, only the theory of Schwierz et al. is compatible with the experimentally observed inversions [79], using the central assumption that any surface can be described by hydrophilic domains patches of the order of 1 nm2 or more and by patches that are mainly hydrophobic.
Dynamic properties of aqueous electrolyte solutions from non-polarisable, polarisable, and scaled-charge models
Published in Molecular Physics, 2019
Shuwen Yue, Athanassios Z. Panagiotopoulos
We obtain mobility results similar to that of Rasaiah, Lynden-Bell, and Koneshan, although with more rigorous sampling and error analysis. Figure 5 displays the mobility calculations for each single ion species following the Hofmeister order. As we move from chaotropic toward kosmotropic ions, the mobility predictably decreased. The maximum found in Rasaiah, Lynden-Bell, and Koneshan's work was not seen, however this may be because the range of ions studied in this work did not include the larger ions found in the earlier work. Notably, the results of the ionic mobility followed closely to the B-coefficient calculations. Li showed its anomalous behaviour, and the F− ion displayed a sharp jump from the rest of the anions in both the mobility and B-coefficient calculations. The agreement with experiment for the anion series was slightly worse than in the cation series. One explanation could be that the ion-water model parameters were obtained from equilibrium properties of small clusters where larger anion effects were not fully captured. Furthermore, deviations in diffusion and mobilities may be at least partly attributed to the failure of the SPC/E water + JC ion model combination to predict accurate solution densities, as shown in Supporting Information.