Hydrophilic Protein Separation

Hydrophilic size separation columns for use with aqueous samples are very popular choices for purifying proteins and carbohydrates. Protein separation columns are available on both silica and polymeric supports. It is surprising that the best of these protein purification columns in terms of resolution and in recovery of native protein are silica-based columns. One would expect that protein release from silica would be a real problem. It certainly is in many other silica columns. These columns, however, especially the TSK family of columns, give excellent recovery of enzymatic activity. I have talked to other column manufacturers who have investigated the problem. They say that when you remove the bonded phases from these columns they appear to be identical to bonded phases from a number of other, less successful, columns designed for protein purification. All of these bonded phases are primarily diol ether polymers, very hydrophilic, but of intermediate polarity. Some modification of 

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Aqueous-detergent solutions of appropriate concentration and temperature can phase separate to form two phases, one rich in detergents, possibly in the form of micelles, and the other depleted of the detergent (Piyde and Phillips, op. cit.). Proteins distribute between the two phases, hydrophobic (e.g., membrane) proteins reporting to the detergent-rich phase and hydrophilic proteins to the detergent-free phase. Indications are that the size-exclusion properties of these systems can also be exploited for viral separations. These systems would be handled in the same way as the aqueous two-phase systems. 

If the virus is treated with proteolytic enzymes the fuzzy layer formed by the viral spikes is removed (Osterrieth, 1965 Compans, 1971 Gahm-berg et al, 1972 Sefton and Gaffney, 1974 Utermann and Simons, 1974). Remnants of both El and E2 are left in the bilayer. These have a hydrophobic amino acid composition, and are soluble in lipid solvents such as chloroform-methanol. The amphiphilic nature of the spike protein is also evident from its capacity to bind Triton X-100 (0.6 g/g protein) which binds to the hydrophobic part to form a water-soluble protein-detergent complex (Simons et al., 1973a). The ability of amphiphilic proteins to bind Triton can be used to separate them from hydrophilic proteins using an extraction procedure recendy described... 

Cloud point extraction from biological and clinical samples. The most frequent use of CPE is for the separation and purification of biological analytes, principally proteins. In this way, the cloud point technique has been used as an effective tool to isolate and purify proteins when combined with chromatographic separations. Most of the applications deal with the separation of hydrophobic from hydrophilic proteins, with the hydrophobic proteins having more affinity for the surfactant-rich phase, and the hydrophilic proteins remaining in the dilute aqueous phase. The separation of biomaterials and clinical analytes by CPE has been described [105,106,113]. 

The most effective supports used in the separation of proteins all have certain common characteristics. They should be hydrophilic as separations are almost always carried out in aqueous buffers. Supports must be inert in that nonspecific binding is minimized. It is also desirable that the support does not contribute to the separation in ways different from the active groups attached to it. This helps to insure predictability and reproducibility of the separations among different manufactured lots of chromatographic media. 

Hydrophilic size separation columns for use with aqueous samples have recently become very popular in purifying proteins and carbohydrates. These will be covered in size separation in Chapter 8. 

A plot of the temperatures required for clouding versus surfactant concentration typically exhibits a minimum in the case of nonionic surfactants (or a maximum in the case of zwitterionics) in its coexistence curve, with the temperature and surfactant concentration at which the minimum (or maximum) occurs being referred to as the critical temperature and concentration, respectively. This type of behavior is also exhibited by other nonionic surfactants, that is, nonionic polymers, // - a I k y I s u I Any lalcoh o I s, hydroxymethyl or ethyl celluloses, dimethylalkylphosphine oxides, or, most commonly, alkyl (or aryl) polyoxyethylene ethers. Likewise, certain zwitterionic surfactant solutions can also exhibit critical behavior in which an upper rather than a lower consolute boundary is present. Previously, metal ions (in the form of metal chelate complexes) were extracted and enriched from aqueous media using such a cloud point extraction approach with nonionic surfactants. Extraction efficiencies in excess of 98% for such metal ion extraction techniques were achieved with enrichment factors in the range of 45-200. In addition to metal ion enrichments, this type of micellar cloud point extraction approach has been reported to be useful for the separation of hydrophobic from hydrophilic proteins, both originally present in an aqueous solution, and also for the preconcentration of the former type of proteins. 

Hence DOLPA reverse micelles recognize the Arg-rich proteins through specific phosphate-guanidinium groups. Such noncovalent bonds of surfactant molecules lead to alterations in the hydrophilic protein surface into sufficiently hydrophobic surfaces to be solubilized in a nonpolar solvent. Molecular recognition on the protein surface facilitates protein transfer, a significant characteristic for the specific separation of biomolecules. 

Epoxy coating is a hydrophilic coating due to the high content of ether and hydroxyl groups. It is expected that this hydrophilic surface will reduce protein adsorption and, thus, is suited for protein separations. 

The second difference between SEC and LC occors when proteins or other biological materials are fractionated. To fractionnte large molecules the packing must have very large pures. For protein separations the packing must be hydrophilic and have minimal interaction with the solutes. Up to now this combination... 

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