Protein chromatography hydrophobic

Reversed-phase chromatography is widely used as an analytical tool for protein chromatography, but it is not as commonly found on a process scale for protein purification because the solvents which make up the mobile phase, ie, acetonitrile, isopropanol, methanol, and ethanol, reversibly or irreversibly denature proteins. Hydrophobic interaction chromatography appears to be the least common process chromatography tool, possibly owing to the relatively high costs of the salts used to make up the mobile phases. 

Benedek, K., Dong, S., and Karger, B. L., Kinetics of unfolding of proteins on hydrophobic surfaces in reversed-phase liquid chromatography, /. Chromatogr., 317, 227, 1984. 

M de Frutos, A Cifuentes, JC Diez-Masa. Behavior of whey proteins in hydrophobic interaction chromatography. J High Resol Chromatogr 19 521-526, 1996. 

The surfaces of proteins are mostly hydrophilic. Although the majority of the hydrophobic residues tend to be buried in the interior of the protein, some hydrophobic regions are also found on the surface (Voet and Voet, 1995 Ladisch, 2001). The level of surface hydrophobicity differs from one protein to another, mainly as a consequence of the amino acid composition and sequence. Difference in surface hydrophobicity is the property exploited in hydrophobic interaction chromatography (HIC) and reverse phase chromatography (RPC). 

Meliander, W. R., Corradini, D., and Horvath, C. (1984). Salt-medated retention of proteins in hydrophobic-interaction chromatography. Application of solvophobic theory. J. Chromatogr. 317, 67-85. 

The VERSE method was extended to describe the consequences of protein denat-uration on breakthrough curves in frontal analysis and on elution band profiles in nonlinear isocratic and gradient elution chromatography [69]. These authors assumed that a unimolecular and irreversible reaction taking place in the adsorbed phase accoimts properly for the denaturation. The choice of this reaction was based on numerous literature observations of the irreversible adsorption of proteins on hydrophobic surfaces [70]. This model (VERSE-LC) was verified using... 

Reverse-Phase (RP) and High-Performance Liquid Chromatography (HPLC). Reverse-phase chromatography separates proteins based on the hydrophobicity of the molecules. It results in the separation of molecules based on the mass of proteins, because hydrophobicity of the... 

A reverse-phase chromatography matrix is a hydrophobic surface, such as silica. The protein mixture is loaded in a relatively hydrophilic solvent, so that proteins with hydrophobic patches on their surfaces will bind to the column. The column is then eluted with a solution of increasing hydrophobicity and the proteins are eluted in order of their hydrophobicity as they are displaced by the solvent gradient. This method also separates protein mixtures into many different groups. 

Figure 7 Correlation of plateau values with protein surface hydrophobicity (from hydrophobic interaction chromatography data) for the adsorption of egg-white lysozyme ( ), bovine pancrease ribonuclease (A), a-lact-albumin (x), sperm whale myoglobin ( ), and superoxide dismutase ( ) on negatively charged polystyrene in 50 mM KCI at 25°C and pH equal to pi of each protein. (From Ref. 17. Reprinted with permission.)...

Mevalonate kinase (EC 2.7.1.36) phosphorylates mevalonic acid, the NADPH-reduced form of HMG-CoA. The reaction is ATP and Mg dependent. The enzyme was purified from a C. roseus cell suspension culture, in which, after induction a specific activity of 1.5 nkat/mg protein is found [12]. The purification protocol comprised ion-exchange chromatography, hydrophobic interaction chromatography and gel filtration. By gel filtration an M,. of 105,000 was found for mevalonate kinase. 

Proteins can be separated by any of a number of different HPLC techniques size-exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, or reversed-phase chromatography. Affinity chromatography is not usu y considered to be an HPLC technique, and is not covered in this book. 

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