Amino Acids, Peptides, and Proteins

Introduction to Proteins in Biological and Food Systems

• Proteins are central to biological systems, acting as the primary agents that sustain life. While DNA carries genetic codes, proteins (as enzymes) perform the biochemical reactions and processes required for cellular function.

• Thousands of enzymes exist, each catalyzing highly specific reactions. Beyond catalysis, proteins serve structural roles in collagen, keratin, and elastin, forming the basis of cells, bones, nails, hair, tendons, and skin.

• Chemically, proteins are complex polymers of 20 different amino acids linked in linear sequences via substituted amide bonds. A unique property of the amide linkage is its partial double-bond character, distinguishing it from the single bonds in polysaccharides (glycosidic) and nucleic acids (phosphodiester).

• The functional diversity of proteins arises from the nearly infinite number of three-dimensional conformations. For a small protein of 200 residues, there are $20^{200}$ possible sequences, each with distinct structures and biological functions.

• Etymology: The word "protein" is derived from the Greek word "proteois," meaning "of the first kind."

• Elemental Composition: Proteins typically consist of $50\%-55\%$ carbon, $6\%-7\%$ hydrogen, $20\%-23\%$ oxygen, $12\%-19\%$ nitrogen, and $0.2\%-3.0\%$ sulfur on a w/w basis.

• Protein synthesis occurs in ribosomes. Proteins that remain unmodified after synthesis are called "homoproteins." Those covalently modified or complexed with nonprotein components ("prosthetic groups") are called "conjugated proteins" or "heteroproteins."

• Examples of conjugated proteins include:

  • Nucleoproteins: complexed with nucleic acids (e.g., ribosomes).

  • Glycoproteins: containing carbohydrate groups (e.g., ovalbumin, $\kappa$-casein).

  • Phosphoproteins: containing phosphate groups (e.g., $\alpha$- and $\beta$-caseins, kinases).

  • Lipoproteins: complexed with lipids (e.g., egg yolk proteins, plasma proteins).

  • Metalloproteins: containing metal ions (e.g., hemoglobin, myoglobin, cytochromes).

• Structural Classification:

  • Globular Proteins: Spherical or ellipsoidal shapes resulting from the polypeptide chain folding on itself (includes most enzymes).

  • Fibrous Proteins: Rod-shaped molecules made of twisted linear chains (e.g., tropomyosin, collagen, keratin).

• Functional categories include enzymes, structural proteins, contractile proteins (myosin, actin), electron transporters (cytochromes), ion pumps, hormones (insulin), transfer proteins (hemoglobin), antibodies (immunoglobulins), storage proteins (egg albumen, seed proteins), and toxins.

• Food Proteins: Biologically, all proteins can be food, but practically they must be digestible, nontoxic, nutritionally adequate, functionally usable, available in abundance, and sustainable. Major sources include milk, meats, eggs, cereals, legumes, and oilseeds.

Physicochemical Properties of Amino Acids

• $\alpha$-Amino Acids are the basic units of proteins. They consist of an $\alpha$-carbon covalently attached to a hydrogen atom, an amino group ($NH_2$), a carboxyl group ($COOH$), and a unique side chain (R group).

• Proline is unique as it contains a secondary imine group (pyrrolidine ring) rather than a primary amine.

• Selenocysteine is recognized as the 21st natural amino acid. It is incorporated into select proteins (e.g., glutathione peroxidase) using the stop codon UGA through a mechanism called "translational recoding." There are at least 25 genes for selenocysteine proteins in the human genome.

• Derived Amino Acids: These result from posttranslational enzymatic modifications. Examples include:

  • Cystine: S-S cross-linked cysteine residues.

  • Desmosine and Isodesmosine: Found in elastin.

  • 4-Hydroxyproline and 5-Hydroxylysine: Found in collagen.

  • Phosphoserine and Phosphothreonine: Found in caseins.

  • $\gamma$-Carboxyglutamate: Found in blood-clotting factors.

• Stereochemistry: Except for Glycine, the $\alpha$-carbon of all amino acids is chiral. Natural proteins contain only L-amino acids. The L-nomenclature is based on the configuration of L-glyceraldehyde, not the direction of light rotation; most L-amino acids are actually dextrorotatory.

Acid-Base Properties and Polarity

• Amino acids are ampholytes (behave as acids and bases) and exist as dipolar ions (zwitterions) at neutral pH.

• Isoelectric Point (pI): The pH at which the dipolar ion is electrically neutral.

• Dissociation Constants ($pKa$):

  • $pKa_1$ refers to the $\alpha$-carboxyl group.

  • $pKa_2$ refers to the $\alpha$-amino group.

  • $pKa_3$ refers to side chain ionizable groups (Lys, Arg, His, Asp, Glu, Cys, Tyr).

• pI Calculation:

  • No charged side chain: $pI = \frac{pKa_1 + pKa_2}{2}$

  • Acidic amino acids: $pI = \frac{pKa_1 + pKa_3}{2}$

  • Basic amino acids: $pI = \frac{pKa_2 + pKa_3}{2}$

• Henderson-Hasselbalch Equation: $pH = pKa + \log\left(\frac{\text{[Conjugated base]}}{\text{[Conjugated acid]}}\right)$

• Fractional charge calculation:

  • Negative charge: $\text{Negative charge} = \frac{-1}{1 + 10^{(pKa - pH)}}$

  • Positive charge: $\text{Positive charge} = \frac{1}{1 + 10^{(pH - pKa)}}$

• In proteins, the $pKa$ values of ionizable groups shift significantly compared to free amino acids due to the altered electronic and dielectric environments within the 3D structure.

• Classification by Side Chain Interaction with Water:

  • Hydrophobic (Nonpolar): Aliphatic (Ala, Ile, Leu, Met, Pro, Val) and Aromatic (Phe, Trp, Tyr).

  • Hydrophilic (Polar): Charged (Arg, Asp, Glu, His, Lys) and Uncharged (Ser, Thr, Asn, Gln, Cys).

Hydrophobicity and Optical Properties

• Hydrophobicity is defined as the excess free energy of a solute in water compared to an organic solvent (typically octanol). It is measured as the free energy of transfer: $\Delta G_{tr, \text{oct} \rightarrow w} = -RT \ln\left(\frac{S_{AA, w}}{S_{AA, \text{oct}}}\right)$, where S represents solubility in mole fraction units.

• This parameter is additive: $\Delta G_{tr, \text{side chain}}^0 = \Delta G_{tr, AA}^0 - \Delta G_{tr, Gly}^0$.

• Aromatic amino acids (Trp, Tyr, Phe) absorb light in the near-UV region ($250-300\,\text{nm}$). trP and Tyr also fluoresce.

  • Phenylalanine: $\lambda_{\text{max}} = 260\,\text{nm}$, $\epsilon = 190\,\text{L}\,\text{mol}^{-1}\,\text{cm}^{-1}$.

  • Tryptophan: $\lambda_{\text{max}} = 278\,\text{nm}$, $\epsilon = 5500\,\text{L}\,\text{mol}^{-1}\,\text{cm}^{-1}$, Fluorescence $\lambda_{\text{max}} = 348\,\text{nm}$.

  • Tyrosine: $\lambda_{\text{max}} = 275\,\text{nm}$, $\epsilon = 1340\,\text{L}\,\text{mol}^{-1}\,\text{cm}^{-1}$, Fluorescence $\lambda_{\text{max}} = 304\,\text{nm}$.

Chemical Reactivity of Amino Acids

• Ninhydrin Reaction: Used to quantify free amino acids. Reacts with amino groups to form Ruhemann's purple ($570\,\text{nm}$). Proline and hydroxyproline produce a yellow color ($440\,\text{nm}$).

• O-phthaldialdehyde Reaction: Yields a fluorescent derivative (Ex $\lambda_{\text{max}} = 380\,\text{nm}$, Em $\lambda_{\text{max}} = 450\,\text{nm}$) in the presence of 2-mercaptoethanol.

• Fluorescamine: Reacts with primary amines to yield a fluorescent derivative (Ex $\lambda_{\text{max}} = 390\,\text{nm}$, Em $\lambda_{\text{max}} = 475\,\text{nm}$).

• Reductive Alkylation: Reaction with formaldehyde ($HCHO$) and sodium borohydride ($NaBH_4$) to modify amino groups.

• Succinylation: Reaction with succinic anhydride to introduce a negative charge at lysyl residues.

• FDNB (1-Fluoro-2,4-dinitrobenzene) and TNBS (2,4,6-Trinitrobenzene sulfonic acid): Used for determining reactive amino groups.

Structural Hierarchy in Proteins

• Primary Structure: The linear sequence of amino acids linked by peptide bonds. The sequence is the code for all subsequent folding levels.

• Peptide Bond Characteristics:

  • Partial double bond character due to resonance delocalization of electrons.

  • Restricted rotation ($\omega$-angle limited to approximately $6^\circ$).

  • The six-atom segment ($C_{\alpha}-CO-NH-C_{\alpha}$) is planar.

  • Trans configuration is $\sim 8.3\,\text{kcal/mol}$ more stable than cis, though Proline peptide bonds may undergo trans-to-cis isomerization ($\Delta G \approx 1.86\,\text{kcal/mol}$).

  • Only the $N-C_{\alpha} (\phi)$ and $C_{\alpha}-C (\psi)$ bonds have rotational freedom.

• Secondary Structure: Periodic spatial arrangements including:

  • $\alpha$-helix: Stabilized by intrachain hydrogen bonds (parallel to the axis). 3.6 residues per turn, pitch of $5.4\,\text{Å}$. Proline acts as an $\alpha$-helix breaker.

  • $\beta$-sheet: Extended strands. Antiparallel $\beta$-sheets are more stable because hydrogen bonds are linear ($0^\circ$ angle).

  • $\beta$-turns (or bends): $180^\circ$ reversals in the chain, common with Gly and Pro.

• Tertiary Structure: Compact 3D form. Driven by the "excluded volume effect" or "solvophobic force" to minimize the protein-water interfacial area.

  • Water-accessible surface area ($A_s$) in $\text{Å}^2$ related to molecular weight ($M$): $A_s = 6.3 \times M^{0.73}$.

  • Total accessible area of an unfolded polypeptide ($A_t$): $A_t = 1.48 \times M + 21$.

• Quaternary Structure: Spatial arrangement of multiple polypeptide chains (subunits). Driven by the burial of surface hydrophobic patches, especially when nonpolar residue content exceeds $30\%$.

Forces Involved in Stability of Protein Structure

• van der Waals Interactions: Dipole-induced dipole and induced dipole-induced dipole forces. Energy at distance $r$: $E_{\text{vdW}} = \frac{A}{r^{12}} + \frac{B}{r^6}$.

• Hydrogen Bonds: Strength ranges from $2-7.9\,\text{kcal/mol}$. Maximum strength when the bond angle is $0^\circ$. Distance is typically $2.9\,\text{Å}$.

• Electrostatic Interactions: $E_{\text{ele}} = \frac{1}{4\pi \epsilon_0} \frac{q_1 q_2}{\epsilon r}$. In water ($\epsilon = 80$), the interaction energy is very low ($\pm 0.84\,\text{kcal/mol}$), becoming significant only in the low-dielectric protein interior.

• Hydrophobic Interactions: The major driving force for folding. Endothermic and stronger at higher temperatures. Free energy change follows: $\Delta G_{H\phi} = a + bT + cT^2$. Energy reduces by $\sim 24\,\text{cal/mol}$ for every $1\,\text{Å}^2$ of nonpolar surface buried.

• Disulfide Bonds: The only covalent side chain cross-links, formed by oxidation of Cys residues. They stabilize folded structures but can be involved in sulfhydryl-disulfide interchange reactions.

• Conformational Entropy ($\Delta S_{\text{conf}}$): Acts as a destabilizing force ($T\Delta S_{\text{conf}} \approx -145.7\,\text{kcal/mol}$ for 100 residues). Net stability of proteins is marginal ($5-20\,\text{kcal/mol}$).

Protein Denaturation

• Definition: Major changes in secondary, tertiary, and quaternary structures without cleavage of the peptide backbone. Denaturation is a cooperative, "two-state transition."

• Thermodynamics: $\Delta G_{D, \text{app}} = -RT \ln(K_{D, \text{app}})$ where $K_{D, \text{app}} = \frac{[D]}{[N]}$.

• Denaturing Agents:

  • Temperature: Heat destabilizes exothermic hydrogen/electrostatic bonds but initially stabilizes hydrophobic bonds. Most proteins denature between $40-80^\circ\text{C}$. Cold denaturation occurs due to the weakening of hydrophobic interactions below $\sim 0^\circ\text{C}$.

  • Hydrostatic Pressure: Denaturation occurs between $1-12\,\text{kbar}$. Pressure eliminates void spaces ($V_{\text{cav}}$). Proteins are compressible but only partially unfold even at $10\,\text{kbar}$.

  • Shear: Mechanical shear (whipping, shaking) exposes proteins to air-liquid interfaces, causing denaturation.

  • pH: Extreme pH causes intramolecular repulsion. Proteins are most stable at their isoelectric point ($pI$).

  • Chemical Agents: Urea and Guanidinium Hydrochloride ($GuHCl$) solubilize nonpolar residues. Detergents like $SDS$ ($3-8\,\text{mM}$) cause irreversible denaturation.

  • Chaotropic Salts: Hofmeister series: F^- < SO_4^{2-} < Cl^- < Br^- < I^- < ClO_4^- < SCN^-. Salts like $NaSCN$ are destabilizers; $Na_2SO_4$ is a stabilizer.

Functional Properties of Food Proteins

• Functionality refers to physical and chemical properties influencing performance in food systems (processing, storage, consumption).

• Hydration Properties: Includes water binding capacity (g water per g protein). Charged residues bind $6\,\text{mol}$ water; nonpolar bind $1\,\text{mol}$. Bound water is unfreezable and exhibits hindered mobility.

• Solubility: Calculated using the DLVO theory, balancing electrostatic repulsion and attractive van der Waals/hydrophobic forces. Hamaker constant ($A$) is typically $10^{-21}\,\text{J}$.

• Emulsifying Properties:

  • Emulsifying Activity Index ($EAI$): Interfacial area created per unit mass of protein ($\text{m}^2/\text{g}$). $EAI = \frac{2T}{(1-\phi)C}$, where $T$ is turbidity, $\phi$ is oil volume fraction, and $C$ is protein concentration.

  • Emulsion Capacity ($EC$): Volume of oil emulsified per gram of protein before phase inversion.

• Foaming Properties: Evaluated by Overrun (percent foam volume) and Foam Stability (time for liquid drainage).

• Viscosity: Measure of resistance to flow ($\eta$). Protein solutions are often pseudoplastic (shear-thinning).

• Gelation: Transformation from sol to progel (denatured) to gel (network). Requires a Minimum Protein Concentration ($LCE$). Hardness follows: $G \propto (C - C_o)^n$.

• Texturization: Includes spun-fiber texturization (pH 12-13 dope through a spinneret) and thermoplastic extrusion ($150-180^\circ\text{C}$ pressure cooking).

• Dough Formation: Wheat gluten (Gliadins and Glutenins). Unique high Gln and Pro content. Kneading involves hydration, $\beta$-sheet to $\beta$-spiral transition, and sulfhydryl-disulfide interchange.

Nutritional Evaluation and Processing Changes

• Biological Evaluation:

  • Protein Efficiency Ratio ($PER$): Weight gain per unit of protein consumed.

  • Biological Value ($BV$): Percentage of nitrogen intake utilized.

  • Net Protein Utilization ($NPU = TD \times BV$).

• Chemical Score: Comparison with reference patterns ($FAO/WHO/UNU$). Limiting amino acids are usually Lys, Thr, Trp, or Met + Cys.

• Processing-Induced Toxicity:

  • Racemization: L-to-D via carbanion intermediate; reduces digestibility.

  • Mutagens: Pyrolysis above $200^\circ\text{C}$ forms IQ compounds (imidazoquinolines).

  • Lysinoalanine ($LAL$): Formed by reaction of dehydroalanine with lysine in alkali-treated proteins; potentially nephrotoxic in rats.

  • Nitrosamines: Reaction of nitrites with secondary amines under acidic/high-temp conditions; highly carcinogenic.

• Maillard Reaction: Carbonyl-amine reaction between reducing sugars and $\epsilon$-amino groups (Lys). Reduces bioavailability of lysine and produces Strecker aldehydes (flavors).

• Antioxidative Activity: Some Maillard products (reductones) act as antioxidants by chelating metals.

Chemical and Enzymatic Modifications

• Acylation: Succinylation replaces positive charges with negative charges, increasing solubility but potentially decreasing heat gelation and bioavailability.

• Phosphorylation: Used to increase calcium sensitivity.

• Sulfitolysis: Cleaves disulfide bonds to S-sulfonates using $\text{SO}_3^{2-}$ and copper.

• Transglutaminase: Catalyzes $\epsilon$-($\gamma$-glutamyl)lysyl cross-links to form gels or improve nutritional quality by attaching essential amino acids.