Amino acids are organic compounds that serve as the building blocks of proteins. Each amino acid contains an amino group, a carboxyl group, a hydrogen atom, and a unique side chain (R-group) attached to a central alpha-carbon. Twenty standard amino acids are encoded by the genetic code.
Classification by Side Chain Properties
Nonpolar, Hydrophobic Amino Acids
These amino acids have side chains that are hydrophobic and tend to cluster in the interior of proteins. They include glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, and tryptophan. Proline is unique because its side chain forms a ring with the amino group.
Polar, Uncharged Amino Acids
These amino acids have side chains that can form hydrogen bonds with water. They include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Cysteine can form disulfide bonds that stabilize protein structure.
Positively Charged (Basic) Amino Acids
These amino acids have side chains that are positively charged at physiological pH. They include lysine, arginine, and histidine. Histidine has a pKa near physiological pH, allowing it to act as a proton donor or acceptor in enzyme active sites.
Negatively Charged (Acidic) Amino Acids
These amino acids have side chains that are negatively charged at physiological pH. They include aspartic acid and glutamic acid. Their carboxyl groups are often involved in salt bridges and metal binding.
Special Properties
Isoelectric Point
Each amino acid has a characteristic isoelectric point (pI) — the pH at which it carries no net charge. At pH values below the pI, the amino acid is positively charged; above the pI, it is negatively charged.
Optical Activity
All standard amino acids except glycine are chiral, existing as L- and D-enantiomers. Only L-amino acids are incorporated into proteins by the ribosome.
Practical Relevance of Amino Acid Properties in Protein Work
The isoelectric point (pI) of each amino acid determines the net charge of a protein at a given pH and governs charge-based separation methods such as ion-exchange chromatography and capillary zone electrophoresis (CZE), where proteins and peptides migrate at different velocities according to their charge-to-size ratio. Choose a cation-exchange column (negatively charged resin) when the target protein’s pI is above the buffer pH (protein net positive), or an anion-exchange column when the pI is below the buffer pH (protein net negative). For example, if a protein has pI 5.5, use anion-exchange at pH 8.0 to bind the negatively charged protein. Hydrophobicity of amino acid side chains drives reverse-phase HPLC separation — C18 columns retain nonpolar residues more strongly, so elution order correlates with the hydrophobicity index (Kyte-Doolittle scale). Leucine and isoleucine have the highest hydrophobicity, while arginine and lysine are most hydrophilic. Post-translational modifications (PTMs) alter the properties of specific amino acids. Phosphorylation of serine, threonine, or tyrosine adds negative charge and is detected by mass shift (+80 Da) or anti-phospho antibodies in Western blotting. Glycosylation of asparagine (N-linked) or serine/threonine (O-linked) increases molecular weight and hydrophilicity, which can be exploited for purification by lectin affinity chromatography. Ubiquitination targets lysine residues and marks proteins for proteasomal degradation — detected by a mass shift of 8.5 kDa per ubiquitin molecule. Peptide bonds form between the carboxyl group of one amino acid and the amino group of another, releasing water — this condensation reaction is catalyzed by the ribosome during translation and requires GTP energy.
Real-World Application
Recombinant human insulin is produced in E. coli as a fusion protein. After purification by ion-exchange chromatography exploiting the pI of insulin (5.3), the protein is refolded and the disulfide bonds between cysteine residues (CysA6-CysA11, CysA7-CysB7, CysA20-CysB19) are allowed to form by controlled oxidation. The correctly folded insulin is separated from misfolded forms by reverse-phase HPLC based on differences in surface hydrophobicity.
