Chromatography of complex protein mixtures.

This review has shown that a variety of chromatographic techniques are available for fractionating proteins. Fortunately, high-quality columns of every type described in this review are commercially available. Most water-soluble proteins may be eluted from size-exclusion, hydrophobic-interaction, ion-exchange, metal chelate, and bioaffinity columns with ease. When this is the case, high recovery and retention of biological activity are the norm. The exception is reversed-phase chromatography where the organic solvents and acids used in polypeptide elution denature many proteins. When problems do occur, they are generally the result of unique structural features of the protein. Very hydrophobic proteins have presented the biggest problem in that they are difficult to solubilize, particularly with retention of biological activity. It has been found that zwitterionic and non-ionic detergents are the most suitable solubilizing agents, but urea has also been used in cases where hydrophobic interacts are not as strong. Unfortunately, there is still an element of trial-and-error in selecting the most suitable solubilizing agent. Heterogeneous glycosylation of proteins also presents a problem. Both neutral and charged monosaccharides can be incorporated into proteins through multiple steps at several sites. Thus, there is the potential in a sample for a large number of glycoprotein species which have the same polypeptide backbone and differing amounts of oligosaccharide. A problem arises when size-exclusion, ion-exchange, hydrophobic-interaction, reversed-phase and bioaffinity systems begin to discriminate between these very similar glycoprotein species. Chromatographic peaks can become very broad, due to incomplete fractionation, and the polypeptide chain of interest can be associated with multiple peaks. The separation of glycoproteins requires much more study before logical procedures can be suggested for column selection and operation. Aggregated species are another class of proteins which present occasional problems. Multimeric proteins are adsorbed to sorbents by a series of forces, among which are hydrogen bonding, hydrophobic interactions, and electrostatic forces. These forces are also responsible for the maintenance of quaternary structure in proteins. When the same forces dominate both retention of protein structure and adsorption at the sorbent surface, the quaternary structure of the protein can be disrupted during elution. Very basic proteins also present a problem in some cases. Columns with residual negative charges, such as a silica-based reversed-phase column, adsorb anionic species so strongly that they are difficult to elute.(ABSTRACT TRUNCATED AT 400 WORDS)