Protein hydrophobicity

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. 

Amphoteric hydrophobic Blue dextran, collagen, gelatin, hydrophobic proteins Hydrophobic peptides Buffer or salt solution with organic solvent (e,g, 20% CH3CN in 0.1 M NaNOi) 35-45% CH3CN in 0.1% TFA... 

Common bean procyanidins are capable of both hydrophilic and hydrophobic interaction with protein. Hydrophilic interactions are favored with a hydrophilic glycoprotein like common bean globulin Gl, while hydrophobic interactions are favored after protein denaturation, when protein hydrophobic groups are exposed to the solvent. 

Interactions between proteins and salts in the binding buffer are also a major determinant of selectivity. Salts that are strong retention promoters in HIC are excluded from protein surfaces by repulsion from their hydrophobic amide backbones and hydrophobic amino acid residues.8,9 This causes the mobile phase to exert an exclusionary pressure that favors the association of proteins with the column, regardless of stationary-phase hydrophobicity.1(W2 Because this mechanism involves the entire protein surface, the degree of exclusion is proportional to average protein hydrophobicity, regardless of the distribution of hydrophobic sites. 

Physical properties of the protein structure should be considered in designing strategies to achieve stable formulations because they can often yield clues about which solution environment would be appropriate for stabilization. For example, the insulin molecule is known to self-associate via a nonspecific hydrophobic mechanism66 Stabilizers tested include phenol derivatives, nonionic and ionic surfactants, polypropylene glycol, glycerol, and carbohydrates. The choice of using stabilizers that are amphiphilic in nature to minimize interactions where protein hydrophobic surfaces instigate the instability is founded upon the hydro-phobic effect.19 It has already been mentioned that hydrophobic surfaces prefer... 

Munson, M., S. Balasubramanian, K.G. Fleming, A.D. Nagi, R. O Brien, J.M. Sturtevant, and L. Regan. 1996. What makes a protein a protein Hydrophobic core designs that specify stability and structural properties. Protein Sci 5 1584—1593. 

A study of protein resistance of terminally attached PEG chains was performed. Steric repulsion between the PEG chain and the approaching protein, hydrophobic, and van der Waals forces were calculated. High surface density and long chain PEGs were found to favor protein resistance. 

In small proteins, hydrophobic residues are less likely to be sheltered in a hydrophobic interior—simple geometry dictates that the smaller the protein, the lower the ratio of volume to surface area. Small proteins also have fewer potential weak interactions available to stabilize them. This explains why many smaller proteins such as those in Figure 4—18 are stabilized by a number of covalent bonds. Lysozyme and ribonuclease, for example, have disulfide linkages, and the heme group in cytochrome c is covalently linked to the protein on two sides, providing significant stabilization of the entire protein structure. 

B5.2 Measurement of Protein Hydrophobicity B5.3 Water Retention Properties of Solid Foods... 

There are two definitions of protein hydrophobicity average hydrophobicity and surface hydrophobicity. The average hydrophobicity was defined by Bigelow (1967) as the total hydrophobicity of all amino acid residues comprising a protein divided by the number of amino acids in the protein. There is no standard definition of surface (or effective) hydrophobicity except the concept that there must be hydrophobic regions on the molecular surface that play an effective role in protein function. Readers who are interested in a more detailed discussion are referred to Nakai and Li-Chan (1988). 

Limitations in using anionic probes such as ANS and CPA to determine protein hydrophobicity include the possibility that electrostatic as well as hydrophobic interactions may contribute to the interaction between the protein and the probe (Greene, 1984). The use of charged but neutral probes (having both electron donor and acceptor groups e.g., prodan) or uncharged probes (e.g., DPH Davenport,... 

Boatright and Hettiarachchy (1995) reported that solubility improvement in soy protein resulted from preventing its oxidation by adding antioxidants and corresponded with the increase in total protein surface hydrophobicity as determined by the SDS binding method (see Basic Protocol 2). This hydrophobicity was not the total protein hydrophobicity but is similar to the exposable hydrophobicity, Se, which is Sq measured in the presence of SDS (Townsend and Nakai, 1983). This change in the exposure of hydrophobic sites in protein molecules is dependent on the concentration of SDS used in the analysis. One of the advantages of the SDS binding method is that there is no need for an expensive spectrofluorometer. 

Comparison of protein hydrophobicity measured using different methods is tabulated in Nakai et al. (1996). 

Protein hydrophobicity has been most frequently expressed as relative values measured by the methods used, since no standardized unit has ever been established. These relative values are incorporated directly into correlation studies with protein functions. Therefore, the correlation coefficient of a measured functionality against the counterpart predicted from the measured hydrophobicity is the most reliable parameter to use for comparing different methods for hydrophobicity measurement. 

Kato, A., Matsuda, T., Matsudomi, N., and Ko-bayashi, K. 1984. Determination of protein hydrophobicity using a sodium dodecyl sulfate binding method. J. Agric. Food Chem. 32 284-288. 

Detergent binding capacity, determination of protein hydrophobicity, 304-305 Dew-point determination method, water activity... 

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