Ovalbumin separation

New designs for axial flow process chromatography columns have been examined using ovalbumin separation on Whatman Express-Ion Exchanger Q with a 16 L Side-Pack and a 24 L IsoPak column.38 The Side-Pak column is packed in the transverse direction, so radial inhomogeneity is minimized. 

FIGURE 22.23 Electropherograms of (a) and (b) two batches of turkey ovalbumin and (c) chicken ovalbumin. Separation conditions fused-sUica capillary 72 cm (50 cm effective length), 100 mM H3B03/Na0H and... 

FIGURE 2.15 Influence of the pore size of Sephacryl HR on the separation of proteins of various molecular mass. The protein mixture is composed of ferritin, aldolase, ovalbumin, and chymotrypsinogen A. [Reproduced from Hagel et al. (1989), with permission.]... 

FIGURE 7.4 Separation of a standard protein mixture on a Fractogel EMD BioSEC-column (600-16 mm) after incubation with 30% acetonitrile. The sample contained BSA ( ), ovalbumin ( ), and cytochrome c (A) (sample volume 500 ftl flow rate 1.0 ml/min). No significant shifts of the retention times and no loss of the resolution were observed even after 900 hr of exposure. 

FIGURE 7.6 Effect of column length on the separation efficiency. Two different Fractogel EMD BioSEC columns (A 600 X 16 mm, B 1000 X 16 mm) were tested using BSA, ovalbumin, and cytochrome c (S/S/3 mg/ml) as sample (20 m/VI sodium dihydrogen phosphate, 300 m/VI NaCI, pH 7.2 0.5 ml/min). Better resolution can be achieved using longer columns. 

The chromatogram of the protein mixture should show the partial separation of serum albumin and ovalbumin with a trough of at least 30% of height between their peak signals and baseline separation between ovalbumin and cytochrome c. If present in the sample, the dimeric form of serum albumin should also appear as an individual peak signal before elution of the monomeric form. 

FIGURE 7.10 Dependence of the resolution on the sample volume. A preparative Superformance column 1000-200 (bed volume 20 liters) packed with Fractogel END BioSEC (S) (bed height 63 cm) was loaded with 60 ml (top) and 300 ml of a mixture of bovine serum albumin (5 mg/ml), ovalbumin (5 mg/ml), and cytochrome c (3 mg/ml) (bottom) (20 m/VI sodium phosphate buffer, 0.3 M NaCI, pH 7.2 flow rate 100 ml/min corresponding to 19 cm/hr). When the sample volume is 300 ml the separation efficiency for BSA and ovalbumin is similar. Thus the column can be loaded with larger sample volumes, resulting in reasonable separations. 

FIGURE 7.17 Separation of a complex mixture on Fractogel EMD BioSEC (S) with a column dimension of 1000 X 50 mm (Superformance glass column). The sample contained ferritin (I), immunoglobulin G (2), transferrin (3), ovalbumin (4), myoglobin (5), aprotinin (6), and vitamin B, (7). Five milliliters of the mixture was injected onto the column at a flow rate of 3 ml/min (eluent 20 mAI sodium phosphate buffer, 0.1 M NaCI, pH 7.2). 

Loading capacities in size exclusion chromatography are very low because all separation occurs within the liquid volume of the column. The small diffusion coefficients of macromolecules also contribute to bandspreading when loads are increased. The mass loading capacities for ovalbumin (MW 45,000) on various sizes of columns can be seen in Table 10.5. The maximum volume that can be injected in size exclusion chromatography before bandspreading occurs is about 2% of the liquid column volume. The maximum injection volumes for columns of different dimensions can also be seen in Table 10.5. 

Figure 9.10 Three-dimensional representation of the data volume of a tryptic digest of ovalbumin. Series of planar slices through the data volume produce stacks of disks in order to show peaks. Reprinted from Analytical Chemistry, 67, A. W. Moore Jr and J. W. Jorgenson, Comprehensive three-dimensional separation of peptides using size exclusion chromatogra-phy/reversed phase liquid chromatography/optically gated capillary zone electrophoresis, pp. 3456-3463, copyright 1995, with permission from the American Chemical Society.

Proteins. A chiral stationary phase with immobilized a -acid glycoprotein on silica beads was introduced by Hermansson in 1983 [18, 19]. Several other proteins such as chicken egg albumin (ovalbumin), human serum albumin, and cellohy-drolase were also used later for the preparation of commercial CSPs. Their selectivity is believed to occur as a result of excess of dispersive forces acting on the more retained enantiomer [17]. These separation media often exhibit only modest loading capacity. 

Continuous free flow electrophoresis has been used for the separation of biopolymers (e.g. ovalbumin and lysozyme) [20] as well as smaller inorganic species (e.g. [Co sepulchrate)] and [Co (CN)g] ) [21]. Sample processing rates of 15 mg h were reported for a mixture of Amaranth (MW 804) and Patent Blue VF (MW 1159) [22]. 

Separations in hydrophobic interaction chromatography have been modeled as a function of the ionic strength of the buffer and of the hydrophobicity of the column, and tested using the elution of lysozyme and ovalbumin from octyl-, butyl- and phenyl-Sepharose phases.2 The theoretical framework used preferential interaction analysis, a theory competitive to solvophobic theory. Solvophobic theory views protein-surface interaction as a two-step process. In this model, the protein appears in a cavity in the water formed above the adsorption site and then adsorbs to the phase, with the free energy change... 

FIGURE 1.3 Three-dimensional representation of a tryptic digest of ovalbumin. The three-dimensional separation consists of size-exclusion chromatography (first dimension), reversed-phase LC (second dimension), and capillary electrophoresis (third dimension). From Moore and Jorgenson, (1995) with permission of the American Chemical Society. 

FIGURE 8.7 2D chromatogram of a SEC x RPLC separation of a tryptic digest of ovalbumin. Reproduced with permission from Opiteck et al. (1997), copyright 1997, American Chemical Society. 

Fig. 3 SDS-PAGE Photograph Separation (Lane Mr and A) and activity staining (Lane B and C) of the crude filtrate of Funalia trogii. Lane Mr standard molecular weight markers ([3-galactosi-dase, 118.0 kDa bovine serum albumin, 79.0 kDa ovalbumin, 47.0 kDa carbonic anhydrase, 33.0 kDa P-lactoglobulin, 25.0 kDa and lysozyme, 19.5 kDa). Relative mobilities of the standard markers vs. common logarithms of their molecular masses were plotted.With the linear regression output, the molecular masses of the proteins in the crude filtrate were estimated (taken from [18])...

The breakthrough curves measured for the monolithic columns with different proteins are very sharp and confirm again the fast mass transport kinetics of the monoliths [133, 134]. The frontal analysis used for the determination of the breakthrough profile can also be used for calculation of the dynamic capacity of the column. For example, the capacity for the 60x16mm i.d. monolith at 1% breakthrough is 324 mg of ovalbumin and represents the specific capacity of 40.0 mg/g of separation medium or 21.6 mg/ml of column volume. 

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.

Two-dimensional SECxRP can be also used for protein and peptide separation, and this approach has been used for separation of peptides in tryptic digests of ovalbumin and serum albumin [14],... 

As a variant, activate the peptide separately first and couple then to the carrier Dissolve the peptide to 1 mg/ml in ddH20, add 10 mg EDAC hydrochloride per milligram of peptide, and adjust pH to 5.0. Incubate at RT for 5 min and correct the pH with diluted NaOH during this period. Then add the same volume of carrier protein solution. The amount of carrier protein should be in a ratio of 40 moles of COOH groups per mol peptide (ovalbumin 42.7 kD, 31 Asp, 48 Glu/Mole BSA 67.7 kD, 54 Asp, 97 Glu/Mole). Shake at RT for 4 h and stop the reaction by addition of 1/10 volume of 1 M sodium acetate buffer, pH 4.2. Free the sample from surplus reagents by gel filtration or dialysis and concentrate to about 1 ml by ultrafiltration. 

R. Montgomery and his colleagues108 fractionated, on Dowex 50, the aspartamidoglycan from ovalbumin. The five components obtained were each subjected to exhaustive hydrolysis with a-D-man-nosidase and 2-acetamido-2-deoxy-/3-D-glucosidase, used separately. The results are shown in Table X. One way in which to explain these results would be to change the position of the extra hexosamine residue in 1, to give the alternative structure 2. 

These fractions, altogether, account for only 30% of the mannose in the ovalbumin digest before separation by ion-exchange chromatography. 

Abbreviations IR inverted repeat DR direct repeat ER everted repeat NR no repeat . Numbers of the HREs give the number of pairs separating the half-sites. R purine, Y pyrimidine. NGFI Nerve growth factor induced receptor COLfP-TF Chicken ovalbumin upstream promoter transcription factor ROR Retinoic acid related orphan receptor, a, P und y are receptor subtypes coded by distinct genes. After Mangelsdorf et al., 1995. 

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