Figure 1:- Tertiary structure of the protein cytochrome C. Yellow indicates the hydrophobic patches on the protein surface and the red indicates the hydrophilic residues.
Image from (GE Healthcare 2010)
It is generally known that proteins are separated by their differences according to charge, size or molecular weight. This difference is exploited by a common procedure called salt fractionation, which adopts ammonium sulphate precipitation. Wilson and Walker (2005); Jason and Osterlund (1997). The concentration of the ammonium sulphate is therefore increased in the protein solution causing the proteins to precipitate and this is due to the increased ionic strength. Wilson and Walker (2005) has also highlighted that ammonium sulphate precipitation is another form of hydrophobic interaction chromatography because they adopt the same principles. GE healthcare (2010), explains that the interactions between the proteins and the hydrophobic interaction chromatography medium are highly influenced by the presence of salt in the protein sample which means the higher salt concentration increases the interactions and lower salt concentrations decreases the interactions. Wilson and Walker (2005) have demonstrated the above literature with an example stating that the phenyl-Sapharose being the absorbent is transferred to the column which consists of high salt concentration. The hydrophobic interaction takes place between the phenyl groups and the hydrophobic patches that are exposed on the surface of protein structure. The elution of proteins occurs when the ionic strength is reduced and the interaction is reversed. The presence of salt aids the exposure of the hydrophobic regions and enhances the interactions between the hydrophobic ligands and the hydrophobic surface of the proteins. GE healthcare (2010); Poole (2003).
In the process of hydrophobic interaction chromatography, the hydrophobic surface of the resins attracts the proteins with hydrophobic properties which are attached to the porous matrix. The interactions between the HIC resins and the protein molecules are enhanced by the relatively high concentration of salt. High ionic strength buffer is essential, so that the target protein binds to the resins on the matrix and other protein molecules washes away. Bonner (2007); Biotecharticles b (2010). Bonner (2007) illustrates the binding conditions required for hydrophobic interaction chromatography (HIC) where temperature plays a significant role as most of the binding process takes place at room temperature. The absorption of the hydrophobic interaction resin is determined by the choice of the ligand which is of two types, alkyl and aryl. Remington (2005) states that straight chain alkyl ligands show more hydrophobic character than aryl ligands, as there is an invariable substitution degree which determines the protein binding capacity of the HIC resins. Consequently, it is generally known that there is a relationship between the alkyl chain length and the binding capacity of the hydrophobic interaction chromatography resins which is, the binding capacity increases with the increase of alkyl chain length. The base of the matrix to which the hydrophobic ligands are attached are generally hydrophilic carbohydrates. Synthetic copolymer and cross-linked agarose is the base matrixes that are commonly used however the selectivity of a copolymer support cannot be replaced with an agarose based support. Remington (2005); Builder (1993). The salt condition in the protein sample is one of the main binding factors since it promotes the exposure of the hydrophobic patches on the surface of the protein’s tertiary structure and fractionation with a common salt ammonium sulphate results with the presence of ammonium and sulphate ions in the sample. Wilson and Walker (2005). To maximise the process in a commercial level the pH factor can be integrated accordingly.
The chromatography procedure starts by equilibrating the high concentrated salt buffer into the column causing the binding of the hydrophobic proteins to the hydrophobic ligands attached to the matrix and the elution of the proteins with lowest hydrophobicity start to take place. The salt commonly used is ammonium sulphate due to its strong retention power and other examples of salts used include sodium chloride, ammonium citrate, and ammonium phosphate. Poole (2003). However it is essential to execute ammonium sulphate fractionation before the chromatographic procedure as it leaves with high concentrations of salt. Bonner (2007). With changes in the salt concentration gradient, the eluted proteins are purified in a concentrated form. Biotecharticles b (2010). The elution process of proteins continues as the salt concentration decreases, leaving the protein with the highest hydrophobicity to elute at the end. Finally, the remaining protein and ion molecules are washed away with a salt free buffer before the column is equilibrated. GE healthcare (2010). The elution of precipitated biomolecules such as lipoproteins and lipids are extremely difficult since they have strong interactions. Bonner (2007) explains that the hydrophobic interaction resins are washed with 0.5 to 1 M of sodium hydroxide, 70 percent of ethanol and 30 percent of isopropanol before the column is re-equilibrated with high concentration salt buffer.
Reverse phase chromatography (RPC) is another technique that uses the same principle of hydrophobic interaction chromatography as they both involve with the interactions between the hydrophobic patches (non polar) on the surface of the proteins and hydrophobic ligands on the matrix. Builder (1993). The proteins are strongly bonded to the Reverse phased chromatography resins than the Hydrophobic Interaction Chromatography resins as they consist of more ligands. This is because the hydrophobic interaction resins have smaller degree of substitution compared to the revered phase chromatography resins. Remington (2005). Reversed phased chromatography uses a three dimensional system, where the elution of the solutes takes place in a non polar solution and thus affects the biological activity. On the other hand, hydrophobic interaction chromatography resins undergoes a two dimensional system where the absorption and partition of protein molecules takes place on apolar solid phase and this allows lenient binding of the proteins and attains good conditions for the elution resultantly increasing the biological activity. Jennissen (2002). In summary, it proves that hydrophobic interaction chromatography is an effective way to separate and elute protein molecules on their hydrophobic character compared to reverse phase chromatography by working on a polar stationary phase and using lower density conditions. Bonner (2007).
References
Biotech articles, Uppangala, N.,(2010) Assessed on 10/10/10
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