Bovine serum albumin phase

F W. Wainer and R. M. Stifhn, Direct resolution of the stereoisomers of leucovorin and 5-methylteti ahydrofolate using a bovine serum albumin liigh-performance liquid cliromatographic chiral stationary phase coupled to an acliiral phenyl column , 7. Chromatogr. 424 158-162 (1988). 

Recently, two examples of the separation of enantiomers using CCC have been published (Fig. 1-2). The complete enantiomeric separation of commercial d,l-kynurenine (2) with bovine serum albumin (BSA) as a chiral selector in an aqueous-aqueous polymer phase system was achieved within 3.5 h [128]. Moreover, the chiral resolution of 100 mg of an estrogen receptor partial agonist (7-DMO, 3) was performed using a sulfated (3-cyclodextrin [129, 130], while previous attempts with unsubstituted cyclodextrin were not successful [124]. The same authors described the partial resolution of a glucose-6-phosphatase inhibitor (4) with a Whelk-0 derivative as chiral selector (5) [129]. 

Microspheres were prepared from carboxymethyl chitosan and alginate by emulsion phase separation. The encapsulated bovine serum albumin was... 

FIG. 3 Time course of the solid-phase extraction of bovine serum albumin by an AOT-based w/o-Me solution. Conditions 500 mg of solid-phase protein 20 mL of iso-octane containing 25 mM AOT and 1000 mM water (w = 40). (Reprinted with permission from Ref. 58.)... 

FIG. 4 Relationship between the final AOT and protein amounts in the solid phase for several different experiments, using a w/o-ME extractant of 50 or 100 mM AOT (unifilled and filled symbols, respectively), Wg = 20. Proteins (upward-pointing triangles) bovine serum albumin (downward-pointing triangles) a -ch5miotrypsin (diamonds) lysozyme. (Reprinted with permission from Ref. 58.)... 

Membranes offer a format for interaction of an analyte with a stationary phase alternative to the familiar column. For certain kinds of separations, particularly preparative separations involving strong adsorption, the membrane format is extremely useful. A 5 x 4 mm hollow-fiber membrane layered with the protein bovine serum albumin was used for the chiral separation of the amino acid tryptophan, with a separation factor of up to 6.6.62 Diethey-laminoethyl-derivatized membrane disks were used for high-speed ion exchange separations of oligonucleotides.63 Sulfonated membranes were used for peptide separations, and reversed-phase separations of peptides, steroids, and aromatic hydrocarbons were accomplished on C18-derivatized membranes. 

In the method shown in Figure 9A, a biotin-labeled cDNA probe is first immobilized to a polyvinylchloride microtiter plate well that is coated with bio-tinylated-bovine serum albumin [33], The target DNA is hybridized in the liquid-phase with a digoxigenin-labeled probe, so that the biotin-labeled probe can capture a marker enzyme. An antibody-conjugated enzyme is then added, followed by a chemiluminescent substrate. 

Chiral stationary phases for the separation of enantiomers (optically active isomers) are becoming increasingly important. Among the first types to be synthesized were chiral amino acids ionically or covalently bound to amino-propyl silica and named Pirkle phases after their originator. The ionic form is susceptable to hydrolysis and can be used only in normal phase HPLC whereas the more stable covalent type can be used in reverse phase separations but is less stereoselective. Polymeric phases based on chiral peptides such as bovine serum albumin or a -acid glycoproteins bonded to... 

Although similar efforts have been devoted to related polymer systems (Overberger and Cho, 1968 Overberger and Dixon, 1977 Okamoto, 1978), large enantioselectivity has not been observed. Goldberg et al. (1978) conducted borohydride reduction of phenyl ketones in micelles of the chiral surfactant [44]. The result was disappointing, since the maximal enantioselectivity was only 1.66% for phenyl propyl ketone. A much better optical yield was reported when this reaction was carried out under phase-transfer conditions (Masse and Parayre, 1976). The cholic acid micelle and bovine serum albumin exhibited the relatively high enantioselectivity in the reduction of trifluoroacetophenone (Baba ef al., 1978). 

Fig. 17. Rapid reversed-phase separation of proteins at a flow-rate of 10 ml/min (Reprinted with permission from [127]. Copyright 1999 Elsevier). Conditions Column, 50x4.6 mm i.d. poly(styrene-co-divinylbenzene) monolith,mobile phase gradient 42% to 90% acetonitrile in water with 0.15% trifluoroacetic acid in 0.35 min, UV detection at 280 nm. Peaks ribonucle-ase (1), cytochrome c (2), bovine serum albumin (3), carbonic anhydrase (4), chicken egg albumin (5)...

Figure 19.9. Chromatogram for stepwise elution of bovine serum albumin on a Vistec diethyl aminoethyl cellulose ion-exchanger, using stepwise increases in sodium chloride concentration in the mobile phase to achieve selective desorption. Proteins 1, serum fraction not adsorbed by column (includes y-gobuhn) 2,3,...

Abbreviations BSA, bovine serum albumin PBS, phosphate buffered saline PMFS, phase-modulation fluorescence spectroscopy TCSPC, lime-correlated single photon counting. 

Figure 3.23 Selectivity of phenyl and alkyl bonded stationary phase materials for protein separation. Column A, TSK gel phenyl-5PW RP, 75 mm x 4.6 mm i.d. B, TSK gel TMS 250, 75 mm x 4.6 mm i.d. eluent, 60 min linear gradient elution from 5% of 0.05% trifluoroacetic acid in 5%> aqueous acetonitrile to 80% of 0.05% trifluoroacetic acid in 80% aqueous acetonitrile flow rate, lml min-1 detection, UV 220 nm. Peaks 1, ribonuclease 2, insulin-, 3, cytochrome c 4, lysozyme-, 5, transferrin-, 6, bovine serum albumin-, 1, myoglobin-, and 8, ovalbumin.

Antibody binding to HA or to virus was determined by direct solid phase RIA. Lyso yme and bovine serum albumin were used to correct for non-specific binding (3% or less). Values are means of triplicate with varied 1.4% or less. 

In order to obtain a thermodynamically stable micro emulsion, the analysis of the phase behaviour is indispensable. With bovine serum albumin instead of an enzyme (because of the cost of the bio-catalyst) phase behaviour studies are shown in Fig. 2. A strong shift of the phase boundary is observed, yielding a system that solubilises much less water in the presence of the protein. In case of hydrophobic enzymes, the addition of dry lyophilised protein to an already prepared reverse micellar solution can also work well [53]. 

Figure 8.12 Time-dependent surface shear viscosity rjS of bovine serum albumin (BSA) + dextran sulfate (DS) at the /7-tetradecane-water interface (pH = 7, ionic strength = 50 mM, 25 °C) ( ) 10 3 wt% DS ( ) 10 3 wt% BSA (A) 10"3 wt% DS added (11) to aqueous sub-phase below the BSA film after 24 h. Reproduced from Dickinson (1995) with permission.

Rodriguez Nino, M.R., Rodriguez Patino, J.M. (1998). Surface tension of bovine serum albumin and Tween 20 at the air-aqueous phase interface. Journal of the American Oil Chemists Society, 75, 1241-1248. 

Sengupta, T., Damodaran, S. (2000). Incompatibility and phase separation in a bovine serum albumin/p-casein/water ternary film at the air-water interface. Journal of Colloid and Interface Science, 229, 21-28. 

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