Proteins size exclusion HPLC

Size-exclusion HPLC (SE-HPLC) separates proteins on the basis of size and shape. As most soluble proteins are globular (i.e. roughly spherical in shape), separation is essentially achieved on the basis of molecular mass in most instances. Commonly used SE-HPLC stationary phases include silica-based supports and cross-linked agarose of defined pore size. Size-exclusion systems are most often used to analyse product for the presence of dimers or higher molecular mass aggregates of itself, as well as proteolysed product variants. 

Fractionation of proteins according to size utilizing cross-linked dextran or polyacrylamide gel columns was first demonstrated by Porath and Flodin 63 in 1959. This technique has become the most widely accepted method for separation and molecular weight determination of hydrophilic and some hydrophobic macromolecules using aqueous buffers with or without organic modifier. While this technique might not be unique in its ability to resolve and separate proteins, it is one additional simple and effective tool in the chemist s armamentarium. The theories behind size-exclusion HPLC and size-exclusion chromatography at low pressure are identical and are described in several publications. 31 34 36 39 44 64 65 ... 

Note that proteins are now synthesized routinely by stepwise assembly or fragment condensation (see Vol. E22a, Section 4.1 ) 67 68 and chances of product contamination with shorter fragments that may have similar retention times under RP-HPLC should be considered 69 Additionally, size-exclusion HPLC was found to be particularly useful in distinguishing linear from cyclic peptides. 

Table 6 Survey of Commercially Available Prepacked Columns for the Size-Exclusion HPLC of Proteins ... 

GW Welling, S Welling-Wester. Size-exclusion HPLC of proteins. In RWA Oliver, ed. HPLC of Macromolecules, a Practical Approach. Oxford IRL Press, 1989, pp 77-89. 

CE Davis, JB Anderson. Size exclusion/HPLC of heated water soluble bovine and porcine muscle proteins. J Food Sci 49 598-602, 1984. 

Size-exclusion HPLC is particularity useful in either direct pharmacodynamic studies of the radiolabeled product or indirect studies that employ a labeled monoclonal antibody. In order to observe shifts in apparent MW due to noncovalent binding interactions, the mobile phase for these analyses should be a physiological buffer and the ligand size cannot be less than half that of the labeled protein. In cases where complexation may interfer with in vivo targeting, size-exclusion HPLC can be used prior to clinical administration of the potential or existing biotechnology product to establish the most effective regimen or dose. 

The silica-based TSK-2000 size exclusion HPLC column has excellent separation properties for proteins in MW range 2000-20,000, when 0.1% aqueous TFA is used as eluent (3,10). We have not seen this property when solvents at neutral pH were used or with other size-exclusion HPLC columns (resin-based as well as zirkoniumoxide-based). The use of 0.1% aqueous TFA solution also dramatically increases the recovery of chemokines. Nevertheless, the TSK-2000 column should be used only when other methods of purification cannot be applied. 

FIGURE 21.15 Size exclusion HPLC chromatograms of (a) chemically and (b) electrochemically acidified soybean protein solutions, with 0.06 M KCl added and maintained at 25°C, at different pH values. (Reprinted with permission from Bazinet, L., Lamarche, F., and Ippersiel, D., i. Agric. Food Chem., 46, 2013, 1998. Copyright 1998 American Chemical Society.)... 

Figure 4 Equilibrium association of CAB as a function of final protein concentration ([CAB]f) as measured by size exclusion HPLC. Equilibrium refolding was performed by rapid dilution of unfolded CAB in 5 M GuHCl to 2.0 M GuHCl and a range of final protein concentrations. After equilibration for three to eight hours, monomer ( ), dimer (A), and trimer ( ) concentrations were determined for each final protein concentration as described in Experimental Procedures.

The fragments are therefore large molecules that fall within the glu-tenin size range, and the separation by size-exclusion HPLC (SE-HPLC) is unaffected if time and intensity of sonication are optimum. Thus, sonication combined with SE-HPLC provides a sound method for determining the relative proportions of the main protein classes. If the solubilized protein is to be used for measuring molecular weight distribution or functional properties, however, the changes induced by sonication must be taken into account. 

Pasaribu, S. J., J. D. Tomlinson, and G. J. McMaster. 1992. Fractionation of wheat flour proteins by size exclusion—HPLC on an agarose-based matrix. Journal of Cereal Science 15 121-136. 

Singh, N. K., G. R. Donovan, 1. L. Batey, and R MacRitchie. 1990. Use of sonication and size-exclusion HPLC in the study of wheat flour proteins. I. Dissolution of total proteins in unreduced form. Cereal Chemistry 67 150-161. 

High-performance liquid chromatography (HPLC) was first applied to cereal proteins by Bietz (1986). Size-exclusion HPLC (SE-HPLC) has been used to quantify cereal proteins it separates proteins according to molecular size. Wheat proteins have been the most studied of the cereals. The preferred solvent has been dilute sodium dodecyl sulfate (SDS)/buffer. A wheat flour suspension (-1.0 mg/mL) is sonicated under conditions (time, intensity) that solubilize almost all, if not all, of the protein without altering quantitation of the main protein classes (see discussion in Chapter 9). 

The complexes were further purified by size-exclusion HPLC. In this way colorless material was removed, and from the elution volume a molecular weight of approximately 335 kDa could be estimated for both complexes. The height of the protein absorbance in the UV as compared to that of other pigment-protein complexes suggested a BChl g content of 10 - 15 % by weight, corresponding to about 40 BChls per complex. This would indicate that the complexes contain the antenna complement... 

Gel permeation (size exclusion) hplc was performed on a Waters Protein PAK 300SW column, in a buffer containing lOOmM sodium phosphate (pH 7.0). 5pg of protein was injected. The flow was driven at 0.5 mg/ml by a Beckman HOB pump and controller, and the eluate monitored at 220nm using a Beckman 165 flow-through spectrophotometer. 

Derivatization of cysteinyl residues was performed with 2.5 mM iodoacetamide (lAM) after 8 min. light activation. Thylakoids were removed by centrifugation then the protein components were separated by size exclusion HPLC and their radioactivity counted. 

Fig. 2. Fractionation of rat liver proteins modified by ADP-ribosylation at arginine. The add-insoluble fraction from rat liver was dissolved in a buffer containing 6 Mguanidinium chloride, adjusted to pH 7.0, and incubated at 37°C for 4 h. An aliquot was then subjected to column centrifugation to eliminate noncovalently bound nucleotides. Aliquots of the protein fraction were fractionated by size exclusion HPLC using two columns in series (Bio-Sil TSK 250 plus TSK 400, 300 mm X 7.5 mm i.d., each) preceded by a guard column (Bio-Sil TSK 125,75 mm X 7.5 mm i.d.). Fractions of 1 ml each were collected and either analyzed for protein or incubated with neutral hydroxylamine (1M, 37°C, 12 h) and analyzed for ADP-ribose [3)...

In addition to other size-based analytical techniques such as size-exclusion HPLC or slab gel electrophoresis, CGE can be adequately used for the determination of a protein s apparent molecular weight. Denaturing... 

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