Hydrophobic variants

Lastly, it has been shown that in rare cases trisulfide bridges (Cys-SSS-Cys) can be found as hydrophobic variants of recombinant proteins. The experimental mass of such a trisulfide-bridged peptide would be increased by 32 amu. Andersson et al.125 have described this phenomenon for recombinant human growth hormone during expression in E. coli. However, the receptor binding properties were not affected by this trisulfide modification. 

The execution of this test with a 0.2 pm hydrophobic filter membrane is as follows. The entire filter case is filled with water and brought under a pressure of about 200 kPa using pressurised air. This pressure is not sufficient to fill the pores and cause a liquid flow through the filter this would require around 1,200 kPa. Transport of water due to evaporation does occur. The amount of water that evaporates per time unit is dependent on the pore size. As a result of this evaporation, the pressure in the filter case would decrease. The amount of air that is added to the filter case to maintain the air pressure is measured. This amount should not exceed a value provided by the manufacturer. The method strongly resembles the pressure hold test and can be regarded as the hydrophobic variant of this test. 

Hydrophobic silicas are aftertreated products. Aftertreatment processes can be carried out with the various Aerosll types as well as with the different precipitated silicas. Aerosil R 972 has been offered on the market since 1962. It is the oldest chemically aftertreated synthetic silica, i.e. the first hydrophobic product. In contrast to the silicas which are hydrophilic by nature, the hydrophobic variants are not wetted by water. Despite the higher density of these hydrophobic silicas in comparison with water, they float on the water surface. 

Other substitutes that have been advanced are derivatives of BIS-GMA obtained by selective reaction of the secondary hydroxyl groups, (e,g, esters, urethanes) as shown in Fig, 2C (11,12), Still more hydrophobic variants of BIS-GMA include the dimethacrylate ester of bisphenol A (BIS-MA) and 2,2-bis[p-(2 -methacryloxyethoxy) phenyl] propane (Fig, 2D), These solid monomers, which are relatively easy to purify by recrystallization, should permit the formulation of highly pure monomer systems based on these dimethacrylates (13,14),... 

AChE-R (in purple) Naturally rare, stress-induced variant, which lacks a hydrophobic domain and is incapable of binding to ColQ or PRiMA. Therefore, it remains soluble, and its secreted form shows greater mobility than AChE-S. AChE-R can intra-cellularly interact through its C-terminal tail with the Protein Kinase C Receptor RACK1, a scaffold protein which modifies multiple cellular processes. 

Hydrophobic interaction chromatography (HIC) can be considered to be a variant of reversed phase chromatography, in which the polarity of the mobile phase is modulated by adjusting the concentration of a salt such as ammonium sulfate. The analyte, which is initially adsorbed to a hydrophobic phase, desorbs as the ionic strength is decreased. One application demonstrating extraordinary selectivity was the separation of isoforms of a monoclonal antibody differing only in the inclusion of a particular aspartic acid residue in the normal, cyclic, or iso forms.27 The uses and limitations of hydrophobic interaction chromatography in process-scale purifications are discussed in Chapter 3. 

Fukuoka was found to be homozygous for the 1615 G to A (539 Asp to Asn) mutation. This mutation occurred at relatively conserved amino acid residues and caused an alteration in hydrophobicity. Recently, we examined the structure-function relationship of these variants using the recombinant protein (F14). Although all of the four variants were found to be heat labile, the residual GPI activity seems to reflect clinical severity, such as the degree of anemia and episodes of hemolytic crisis. GPI Matsumoto, associated with severe anemia and hemolytic crisis, was extremely unstable, and GPI Iwate, which is associated with compensated hemolytic anemia, showed moderate heat instability. Affinity for substrate, fructose-6-phosphate, was slightly decreased in GPI Narita and GPI Fukuoka, which were associated with moderate anemia and hemolytic crisis. 

HPLC log P techniques, first described by Mirrlees et al. [374] and Unger et al., [375], are probably the most frequently used methods for determining log/1. The directly measured retention parameters are hydrophobicity indices, and need to be converted to a log P scale through the use of standards. The newest variants, breadths of scope, and limitations have been described in the literature [292-298]. A commercial automated HPLC system based on an extension of the approach described by Slater et al. [150] has just introduced by Sirius (www. sirius-analytical. com). 

---