Structure and Properties of Amino Acids
The structure of an amino acid consists of a central carbon atom with a hydrogen attached, an acidic carboxyl group (-COOH), an amino group (-NH2), and an organic side chain (also known as an R group). There are 20 amino acids that make up proteins, and they all have the same basic structure, differing only in the R group or side chain. The basic structure of an amino acid is shown below:
Figure From Western Oregon University
Different R groups have different properties depending on the nature of the atoms contained in the functional group. Some R groups contain carbon and hydrogen and are non-polar or hydrophobic. Others contain polar, uncharged functional groups such as alcohols, amides, and sulfhydryl groups. A few amino acids are basic (containing amine functional groups) or acidic (containing carboxyl functional groups). These amino acids can form complete charges and generate ionic interactions. These properties affect how they interact with surrounding amino acids in peptides and proteins and thus affect the three-dimensional structure and properties of proteins.
Chemical structure for the 20 amino acids that are found in all naturally occurring proteins (Cowsill et al., 2012)
Nonpolar (Hydrophobic) Amino Acids
Nonpolar amino acids can be broadly classified into two more specific categories: aliphatic amino acids and aromatic amino acids. Aliphatic amino acids (glycine, alanine, valine, leucine, isoleucine, and proline) typically feature branched hydrocarbon chains, ranging from the simplest in glycine to the more complex structures in leucine and valine. Proline is also categorized as an aliphatic amino acid, but due to the cyclization of the hydrocarbon chain with the terminal amine, it forms a unique 5-membered ring structure with distinctive properties. The structural rigidity of proline's ring allows it to significantly alter the three-dimensional structure of proteins and is often found in regions of protein folding or turns.
Aromatic amino acids (phenylalanine, tyrosine, and tryptophan) contain aromatic functional groups, which, as their name suggests, make them mostly nonpolar and hydrophobic due to their high carbon/hydrogen content. However, it's important to note that hydrophobicity and hydrophilicity represent a sliding scale, and different amino acids can exhibit varying physical and chemical properties based on their structures. For example, compared to phenylalanine, tyrosine's presence of a hydroxyl group increases its reactivity and solubility.
Methionine, a sulfur-containing amino acid, is typically classified as a nonpolar hydrophobic amino acid because the terminal methyl group forms a thioether functional group, which usually cannot establish a permanent dipole moment within the molecule, maintaining low solubility.
Polar (Hydrophilic) Amino Acids
Polar hydrophilic amino acids can be broadly categorized into three main groups: polar uncharged, acidic, and basic functional groups. In the polar uncharged group, side chains contain heteroatoms (oxygen, sulfur, or nitrogen) capable of forming a permanent dipole moment within the R-group. These include amino acids with hydroxyl and sulfhydryl functional groups, such as serine, threonine, cysteine, as well as amino acids with amide groups, such as asparagine, glutamine, and aspartic acid. Aspartic acid (aspartate) and glutamic acid (glutamate) are two acidic amino acids containing side chains with carboxylic acid functional groups capable of complete ionization in solution. Basic amino acids, lysine, arginine, and histidine, contain amino groups capable of protonation to carry a full charge.
Many amino acids with hydrophilic R-groups can participate in the active sites of enzymes. The active site is the portion of the enzyme that directly binds to the substrate and facilitates the reaction. Enzymes derived from proteins have catalytic groups composed of amino acid R-groups that promote the formation and breakdown of bonds. Amino acids crucial for binding specificity in the active site often do not reside adjacent to each other in the primary structure but fold to create the active site during the formation of the tertiary structure.
Amino Acids as Zwitterions
In chemistry, zwitterions are molecules with two or more functional groups, where at least one carries a positive charge, and another carries a negative charge, resulting in a net charge of zero for the entire molecule at a specific pH. Due to the presence of at least one positive and one negative charge, zwitterions are sometimes referred to as internal salts. The charges on different functional groups balance each other, allowing the entire molecule to be electrically neutral at a particular pH. The pH at which this occurs is known as the isoelectric point.
Unlike simple amphiprotic compounds that can only form cations or anions, zwitterions simultaneously exhibit both ionic states. Amino acids serve as examples of zwitterions, existing in a balance between two acidic forms where protons (H+) shuttle between the amino group and the carboxyl group, as illustrated below:
In this equilibrium state, the weaker acid consistently predominates. Because the amine group is weaker than the carboxyl group, the equilibrium tends to shift to the left (towards the "zwitterion" side). Since amino acids exist as zwitterions and certain amino acid R-groups also have the potential for ionization, their charge states within the body may vary based on the local microenvironment, including pH, temperature, and solvent conditions.
Structure of common basic and acidic amino acids, with the pKa values of side chain functionalities shown (Dharmayanti et al., 2021)
Amino Acid Analysis Methods
Amino acid analysis is a pivotal technique used to identify and quantify the composition of amino acids within a sample. Several sophisticated methods have been developed, each offering unique advantages in terms of sensitivity, accuracy, and efficiency. Among the notable techniques are Reverse Phase High-Performance Liquid Chromatography (RP-HPLC), Liquid Chromatography-Mass Spectrometry (HPLC-MS), Gas Chromatography (GC), Gas Chromatography-Mass Spectrometry (GC-MS), and Capillary Electrophoresis (CE), each contributing to the comprehensive analysis of amino acids.
Reverse Phase High-Performance Liquid Chromatography (RP-HPLC):
RP-HPLC is a widely employed method for amino acid analysis. It utilizes a stationary phase with hydrophobic properties, separating amino acids based on their hydrophobicity. This technique is known for its high resolution and efficiency in separating complex mixtures of amino acids.
Liquid Chromatography-Mass Spectrometry (HPLC-MS):
HPLC-MS combines the separation power of HPLC with the mass analysis capabilities of mass spectrometry. This tandem technique allows for not only the identification but also the precise quantification of amino acids. It is particularly valuable for complex samples where high specificity is required.
Gas Chromatography (GC):
GC is a technique that separates amino acids based on their volatility. In this method, amino acids are vaporized and carried through a column by an inert gas, with separation occurring based on their retention times. Although GC is limited to volatile and thermally stable amino acids, it is highly efficient for specific applications.
Gas Chromatography-Mass Spectrometry (GC-MS):
GC-MS integrates the separation capabilities of GC with the mass analysis precision of MS. This method is well-suited for the identification and quantification of amino acids in volatile and semi-volatile samples, providing enhanced sensitivity and specificity.
Capillary Electrophoresis (CE):
CE is a powerful technique for separating amino acids based on their charge and size differences. It operates on the principle of electrophoretic mobility in a capillary, enabling the separation of amino acids with high resolution. CE is especially useful for analyzing small sample volumes.
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Applications of Amino Acid Analysis
Protein Structure and Function Studies:
Amino acid analysis is fundamental in characterizing proteins, offering insights into their structure and function. This information is pivotal for advancing fields like structural biology and biochemistry.
Nutritional Sciences:
Amino acid analysis is employed in nutritional analysis to evaluate the amino acid profiles of food products. This aids in assessing the nutritional quality of diets and formulating balanced nutritional plans.
Biotechnology and Pharmaceutical Research:
Amino acid analysis is crucial in biotechnological and pharmaceutical research to assess the purity of biotherapeutics. Understanding amino acid composition is vital for ensuring the efficacy and safety of these products.
Food and Beverage Industry:
Beyond nutritional analysis, amino acid analysis is used in the food and beverage industry to assess product quality and authenticity. It also contributes to the development of flavors and enhancers through amino acid content analysis.
Animal Nutrition and Feed Analysis:
Amino acid analysis is applied in animal nutrition to optimize feed formulations. By understanding the amino acid composition of feed ingredients, nutritionists can develop balanced diets for livestock, poultry, and aquaculture.
Environmental Monitoring:
Amino acid analysis is utilized in environmental science to study microbial activity and assess aquatic ecosystem quality. It provides insights into nutrient cycling, microbial community dynamics, and the impact of pollutants on ecosystems.
Metabolic Pathway Studies:
Researchers use amino acid analysis to elucidate metabolic pathways within living organisms. This technique helps trace the flow of amino acids through biochemical pathways, contributing to a deeper understanding of cellular metabolism.
Pharmaceutical Quality Control:
In the pharmaceutical industry, amino acid analysis is essential for quality control. It verifies the amino acid composition of peptide-based drugs and pharmaceutical proteins, ensuring consistency and compliance with regulatory standards.
References
- Aouniti, A., et al. "Amino acid compounds as eco-friendly corrosion inhibitor in acidic media-Review." Arabian Journal of Chemical and Environmental Research 4.1 (2017): 18-30.
- Cowsill, Benjamin James. The physics of pregnancy tests: a biophysical study of interfacial protein adsorption. The University of Manchester (United Kingdom), 2012.
- Dharmayanti, Cintya, et al. "Strategies for the development of pH-responsive synthetic polypeptides and polymer-peptide hybrids: recent advancements." Polymers 13.4 (2021): 624.
