Chaotropic reagents

For precipitated protein, buffered solutions containing chaotropic reagents such as 0.1% SDS, 8 M urea, or 6 M guanidine or proteolytic enzymes such as pepsin may be used. However, an extended washing with buffer is required to remove SDS and guanidine. Unexpected elution behavior can occur if these reagents are not removed completely. 

The cells are lysed in a buffer containing strong chaotropic reagents such as guanidine thiocyanate and 2-mercaptoethanol, which completely denatures any ribonuclease present. The supernatant is then placed on a cushion of CsCl (5.7 mol l-1) and centrifuged at 100000 g for 18 h. The RNA passes through the CsCl and is pelleted, while the DNA and protein remain in the aqueous solution. The RNA pellet is dissolved in buffer and concentrated by precipitation in cold ethanol. 

Integral proteins are dissolved into the lipid bilayer of the membrane through interactions of the hydrophobic amino acid side chains and fatty acyl groups of phospholipids. In order to remove integral membrane proteins, the membrane must be disrupted by addition of detergents or other chaotropic reagents to solubilize the protein and to prevent aggregation and precipitation of the hydrophobic proteins upon their removal from the membrane. 

Furthermore, the necessity to expose the probe tip to chaotropic reagents makes the measurement both cumbersome and discontinuous. 

However, Kazakevich and co-workers demonstrated the importance of the adsorption of chaotropic ions onto the reversed stationary phase [106]. The rank of an ion in the Hofmeister series is another measure of its tendency to accumulate at the stationary phase in RP-HPLC and be quantified via its adsorption isotherm [88]. Clearly a specific surface excess of the chaotropic reagent results in the development of a potential difference between the stationary phase and the bulk eluent, modulating retention of analytes [107]. We discussed in Chapter 3 the way a comprehensive theory can take this experimental evidence into account. 

Equation 10.12 is algebraically correspondent [15] to the final relationship that describes analyte retention under IPC conditions (Equation 3.21). It upgrades the parallel stoichiometric equation of the model by Kazakevich and co-workers [16] that is inherently inadequate because it cannot predict the decrease of retention for analytes similarly charged to the chaotropic reagent and the electrostatic tuning of the retention of the unpaired analyte in the presence of the electrified stationary phase. It also upgrades electrostatic models [17,18] that disregard the role played by the ion-pair complex (final term in Equation 10.12). 

SR-proteins can be expressed as recombinant proteins in Escherichia coli, but they lack the post-translational phosphorylation of the serine residues and are poorly soluble in the absence of chaotropic reagents. It is possible to phosphorylate bacterially produced protein by preincubation in nuclear extract or, even more efficiently, by the addition of purified recombinant SR-protein specific kinases Clk/Sty8 or SRPK.9 Soluble and phosporylated SR-proteins can also be produced in insect cells using the baculovirus system10 although it is not resolved whether these proteins behave exactly as those produced in human cells. 

Narayanasami R, Nishimura JS, McMillan K, Roman LJ, SheaTM, RobidaAM, Horowitz PM, Masters BS (1997) The influence of chaotropic reagents on neuronal nitric oxide synthase and its flavoprotein module. Urea and guanidine hydrochloride stimulate NADPH-cytochrome c reductase activity of both proteins. Nitric oxide Biol Chem 1 39-49... 

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