Ampholytes fractionation

Figure 3. Preparative isoelectric focusing. The PNL eluted from gel filtration was subjected to isoelectric focusing using a column of 110 ml capacity (LKB) with ampholytes pH 7-11. After 48 h (9.6 W constant power), fractions of 3 ml were removed and assayed for PNL activity (- - ) and pH (- -).

Figure 5. Analytical isoelectric focusing. Ultrathin layers (0.4 nun) of polyacrylamide with ampholytes pH 2-11 were used. Samples of 10 pg of protein in 10 pi of 1 % glycine were applied. A.- Silver staining. B.- Stain for activity on overlays containing pectin in tris/HCl buffer at pH 8.0 with CaClj M.- Broad pi Calibration Kit protein (Pharmacia), samples of 5 pg of protein were applied. 1.-Ammonium sulphate precipitated proteins from cultures on pectin. 2.- Fractions with PNL activity eluted from the Superdex 75HR1030 column. 3.- Purified PNL.

Because the pi of a protein is based on its amino acid sequence, this technique has good resolving power. The resolution can be adjusted further by changing the range of the pH gradient. The use of immobilized pH gradient (IPG) strips has enabled reproducible micropreparative fractionation of protein samples, which is not consistently possible when ampholytes are used in the first dimension (Gorg et al., 2000). 

The development of modem methods, suitable for the analysis of ampholytes in biological fluids, provided means for isolating from urine some chemically better defined fractions containing peptide compounds. The methods used did not, however, exclude the existence of some other forms of combined amino acids in the fractions studied. 

The first fractionation of urinary ampholytes in this way was carried out by Boulanger et al. (BIO) in 1952 with the use of ion-exchange resins. They had designed this procedure previously for the fractionation of ampholytes in blood serum (B8). According to this method, deproteinized urine was subjected to a double initial procedure aiming at the separation of low-molecular weight substances from macro-molecular ones. One of the methods consisted of the fractionation of urinary constituents by means of dialysis, the second was based on the selective precipitation of urinary ampholytes with cadmium hydroxide, which, as had previously been demonstrated, permits separation of the bulk of amino acids from polypeptides precipitated under these circumstances. Three fractions, i.e., the undialyzable part of urine, the dialyzed fraction, and the so-called cadmium precipitate were analyzed subsequently. 

In 1952 Carsten (Cl) developed a method, which allowed him to isolate and characterize several lower peptides contained in normal and pathological urine. According to this procedure, urine was desalted on the Amberlite IR-100 column and the adsorbed substances washed out with 2 M ammonia solution. The eluate was then passed through the column of Amberlite IRA-400. This column retained the ampholytes and rejected the weak bases. The former were recovered by elution with 1 M hydrochloric acid and the eluate was subsequently fractionated on Dowex 50 resin with 2M and later 4M hydrochloric acid as the eluents. By applying two-dimensional paper chromatography to further analysis of... 

IFF is a high-resolution technique that can routinely resolve proteins differing in pi by less than 0.05 pH unit. Under nondenaturing conditions, antibodies, antigens, and enzymes can retain their activities during IEF. The proper choice of ampholyte or IPG range is very important to the success of a fractionation. 

Figure 1. Fractionation of proteins in the culture filtrate of Trichoderma reesei according to their pi values Xyl, xylanase Ara, arabinosidase AE, acetyl esterase / X, /3-xylosidase aG, a-glucuronidase / G, / -glucosidase CBH, cellobiohydrolase EG, endoglucanase. Chromatofocusing was performed in a PBE-94 anion exchange resin (Pharmacia) with a pH-gradient created by ampholyte buffers (Pharmacia). Solid line, A dotted line, pH. (Reproduced with permission from ref. 24. Copyright 1988.)...

Svensson H (1961), Isoelectric fractionation, analysis, and characterization of ampholytes in natural pH gradients, the differential equation of solute concentrations at a steady state and its solution for simple cases, Acta Chem. Scand. 15 325-341. 

The proper choice of ampholyte range is very important to the success of an IEF fractionation. Ideally, the pH range covered by the focused carrier ampholytes should be centered on the pis of the proteins of interest. This insures that the proteins of interest focus in the linear part of the gradient with many extraneous proteins excluded from the separation zone. Carrier ampholyte concentrations of about 2% (w/v) are best. Concentrations of ampholytes below 1% (w/v) often result in unstable pH gradients. At concentrations above 3% (w/v) ampholytes are difficult to remove from gels and can interfere with protein staining. 

Samples for the Rotofor need not be completely desalted before fractionation. Ions in the sample solution are electrophoresed into the two end compartments in the early stages of the run. Two percent (w/v) carrier ampholyte in the initial sample solution supplies enough ampholyte for refractionation of pooled material. After the tubes containing the protein of interest have been identified, they can be pooled for a second run. The pooled material need only be diluted enough (usually with water) to fill the separation chamber. The ampholytes in the pooled fractions cover pH ranges centered on the pis of the proteins of interest and are generally less than 1 pH unit. Thousand-fold purification has been achieved on refractionation. 

A representative IEF purification with the Rotofor that demonstrates the effectiveness of refractionation is shown in Fig. 8. The starting material for this fractionation was 150 mg of crude snake venom containing 2% (w/v) pH 3-10 carrier ampholytes. Aliquots of each of the 20 fractions from the run were analyzed in polyacrylamide IEF gels in pH 3-10 gradients. The focusing patterns of the proteins in the odd-numbered fractions are shown in Fig. 8a. The protein of interest (outlined with an oval) was found in fractions 10, 11, and 12. These three fractions were pooled, diluted with water to the volume of the separation chamber and refractionated. No additional ampholytes were added to the pooled material. IEF analysis of the refractionated material (Fig. 8 b) revealed that fraction 13 contained the protein of interest in nearly pure form. 

H. Haglund, Isoelectric Focusing in pH Gradients— A Technique for Fractionation and Characterization of Ampholytes, in Methods of Biochemical Analysis, Vol. 19 (D. Glick, Ed.), Wiley, New York, 1971, p. 1. 

Zhou and Johnston [55] reported protein characterization by capillary isoelectric focussing (CIEF) on-hne coupled to RPLC-MS. Direct coupling of CIEF to ESl-MS is limited by interferences by the ampholytes. Inserting RPLC in-between can help removing these interferences. CIEF is performed in combination with a microdialysis membrane-based cathodic cell to remove the ampholyte and to collect protein fractions by stop-and-go CIEF prior to transfer to a 5><0.3-pm-ID C,8 trapping colunm and RPLC separation on a 50><0.3-pm-ID C4 column. The separation is performed using an acetonitrile-water gradient (0.1% acetic acid). ESI-MS is performed on a quadrupole-TOF hybrid (Q-TOF) instrument. 

Each stored sample is then loaded on a reversed-phase protein trap column attached to a second six-port valve located on the capillary LC system. Proteins and peptides in the stored sample are captured on the trap column and washed with a solution of water-acetonitrile-fomic acid (94.9 5.0 0.1) at a flow-rate of 20pLmin for 3 min. Most of the ampholyte, urea and DTT are removed during this washing step. The protein fraction is then eluted from the trap column with mobile phase and further separated on a C4 reversed-phase column (5 cm X 300 pm i.d.). Mobile phase A [water-acetonitrile-fomic acid (94.9 5.0 0.1)] and mobile phase B [acetonitrile-water-acetic acid (94.9 5.0 0.1)] are delivered at a flow-rate of lOpLmin using a two-step gradient of 40min (phase B from 20 to 60%) and 2 min (phase B from 60 to 90%). The sample eluted from the RPLC column is sent directly into the ESI-QTOF mass spectrometer. 

Haglund, Herman, Isoelectric Focusing in pH Gradients—A Technique for Fractionation and Characterization of Ampholytes 19 1... 

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