The property of molecules to evade water-based surroundings, known as hydrophobicity, determines fundamental aspects of protein folding and how biomacromolecules associate with membranes and dissolve. The principal protein components known as amino acids demonstrate a range of hydrophobic characteristics, which depend on the chemical composition of their side chains and their ionization states. The hydrophobicity index measures these properties quantitatively and enables predictions about protein structure and functionality.
This table presents the comparative hydrophobicity scale of common amino acids measured at two physiological pH levels—pH 2 and pH 7—using normalized empirical data from Sereda et al. and Monera et al., respectively. Normalization of the values assigns glycine, which has a simple structure and neutral character, an index value of zero. The most hydrophobic amino acid receives a score of 100 at each pH level, but amino acids with greater hydrophilicity obtain negative values to indicate their poor interactions with nonpolar environments.
| Classification | Amino Acid | Index @ pH 2[1] | Index @ pH 7[2] |
| Very Hydrophobic | Leucine (Leu) | 100 | 97 |
| Isoleucine (Ile) | 100 | 99 | |
| Phenylalanine (Phe) | 92 | 100 | |
| Tryptophan (Trp) | 84 | 97 | |
| Valine (Val) | 79 | 76 | |
| Methionine (Met) | 74 | 74 | |
| Hydrophobic | Cysteine (Cys) | 52 | 49 |
| Tyrosine (Tyr) | 49 | 63 | |
| Alanine (Ala) | 47 | 41 | |
| Neutral | Threonine (Thr) | 13 | 13 |
| Glutamate (Glu) | 8 | -31 | |
| Histidine (His) | -42 | 8 | |
| Glycine (Gly) | 0 | 0 | |
| Serine (Ser) | -7 | -5 | |
| Glutamine (Gln) | -18 | -10 | |
| Aspartate (Asp) | -18 | -55 | |
| Hydrophilic | Arginine (Arg) | -26 | -14 |
| Lysine (Lys) | -37 | -23 | |
| Asparagine (Asn) | -41 | -28 | |
| Proline (Pro) | -46 | 5 (pH 6.5) |
Interpretation of Hydrophobicity Classes
- Very Hydrophobic Residues
At pH 2, leucine and isoleucine dominate with an index of 100, followed closely by phenylalanine (92), tryptophan (84), valine (79), and methionine (74). These amino acids possess large, nonpolar, aliphatic or aromatic side chains that strongly partition into hydrophobic phases, such as lipid membranes or protein cores. Their preference for hydrophobic environments remains consistent at pH 7, although slight rearrangements in ranking occur. Notably, phenylalanine retains the highest index (100), while isoleucine (99), tryptophan (97), leucine (97), valine (76), and methionine (74) follow closely.
The consistency shown here indicates that these residues serve as strong contributors to hydrophobic cores in globular proteins while also remaining essential for membrane-spanning α-helices.
- Hydrophobic Residues
The moderately hydrophobic category includes cysteine, tyrosine, and alanine. At pH 2, cysteine (52), tyrosine (49), and alanine (47) exhibit moderate nonpolar behavior. Upon transition to pH 7, tyrosine's index increases significantly (to 63), reflecting reduced polarity due to phenolic group deprotonation. Meanwhile, cysteine maintains its intermediate index (49), and alanine decreases slightly to 41, aligning with its small methyl side chain and minimal dipolar interaction.
These residues frequently reside at protein-solvent interfaces or lipid-water boundaries, where partial burial occurs without complete desolvation.
- Neutral Residues
Neutral residues are characterized by values ranging from -18 to +13, indicating minimal energetic preference for either polar or nonpolar environments. Threonine remains stable at an index of 13 across both pH values. Glutamic acid (Glu) and histidine (His) both score 8, suggesting moderate amphipathic character. Serine, a polar uncharged amino acid, shows weak hydrophilicity (-7 at pH 2 and -5 at pH 7).
Interestingly, glutamine (-18 at pH 2, -10 at pH 7) and aspartic acid (-18 at pH 2) straddle the neutral-to-hydrophilic boundary, influenced by the ionization states of their amide or carboxylic side chains.
- Hydrophilic Residues
Hydrophilic residues exhibit negative hydrophobicity indices, denoting a preference for aqueous surroundings. Arginine, lysine, asparagine, histidine, proline, glutamic acid, and aspartic acid fall into this category, with their indices decreasing markedly at neutral pH due to enhanced ionization.
At pH 2, proline (-46) and histidine (-42) are among the most hydrophilic, while arginine and lysine exhibit values of -26 and -37, respectively. Asparagine and glutamic acid also reflect substantial aqueous solubility. Upon reaching pH 7, glutamic acid (-31) and aspartic acid (-55) become significantly more hydrophilic, a direct result of complete deprotonation and generation of negatively charged carboxylate groups. Similarly, proline shifts from -46 to a highly hydrophilic index of -55, underscoring its unique secondary amine structure and conformational rigidity.
These residues are predominantly surface-exposed in globular proteins and participate in ionic interactions and hydrogen bonding with water and other polar biomolecules.
Effect of pH on Hydrophobicity Behavior
The observed variations across pH levels reveal the pH dependence of amino acid hydrophobicity. Ionizable side chains such as those of glutamic acid, aspartic acid, lysine, and histidine undergo protonation-deprotonation transitions between acidic and neutral pH. These transitions significantly alter side chain polarity, hence modulating their solubility behavior and protein conformational dynamics.
For instance, glutamic acid shifts from marginal neutrality (8 at pH 2) to a highly hydrophilic value (-31 at pH 7), reflecting ionization of its γ-carboxyl group. Similarly, histidine becomes more hydrophobic upon deprotonation of its imidazole group, transitioning from -42 to +8.
These dynamic shifts highlight the importance of incorporating environmental pH in computational modeling of protein structures, especially in predicting membrane protein insertion, pKa shifts, and ligand binding in enzymatic active sites.
Applications and Implications
The hydrophobicity index table is extensively utilized in:
- Protein folding simulations: Predicting residue burial and core formation.
- Membrane protein engineering: Selecting residues for transmembrane domains.
- Peptide design and drug delivery: Tailoring amphiphilicity for enhanced bioavailability.
- Solubility prediction: Optimizing recombinant protein expression systems.
The hydrophobicity index table serves as a foundational resource for understanding the physicochemical behavior of amino acids in varied pH environments. Through mapping the solvation preference of each residue, the tool delivers essential knowledge about protein structure stability and functionality. The significant changes in hydrophobic behavior across different pH levels demonstrate how chemical structure interacts with environmental factors in complex ways. This research data serves to expand theoretical knowledge and simultaneously supports practical biotechnological and pharmaceutical applications.
References
- Sereda, T. J., et al. Reversed-Phase Chromatography of Synthetic Amphipathic Alpha-Helical Peptides as A Model for Ligand/Receptor Interactions. Effect of Changing Hydrophobic Environment on the Relative Hydrophilicity/Hydrophobicity of Amino Acid Side-Chains. J Chromatogr A. (1994).
- Monera, O. D., et al. Relationship of Sidechain Hydrophobicity and Alpha-Helical Propensity on the Stability of the Single-Stranded Amphipathic Alpha-Helix. J Pept Sci. (1995).
