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Chemistry · Organic chemistry · Year 3

Amino acids and proteins

Why does casein curdle when acid is added to milk, turning it into yoghurt?

Open the experiment

The situation

In the laboratory of the Tashkent dairy plant, the morning milk is being tested. If a little ethanoic (acetic) acid is dripped into milk, it suddenly curdles and separates into white clots — casein — and yellowish whey; if we boil egg white, the clear liquid turns white and sets. How are proteins built from amino acids, why do they precipitate at a particular pH, and what is damaged in them when they are heated?

Topic

Amino acids, peptides and proteins. Tashkent dairy plant: when acid is added to milk, casein curdles into yoghurt and cheese; egg white turns white when heated. In the laboratory, an amino acid…

Key ideas

  • Isoelectric point
  • Peptide bond
  • α-helix and β-sheet

Formulas

pI=12(pKa1+pKa2)

The isoelectric point is the pH at which the net charge is zero: the midpoint between the pKₐ values on either side of the zwitterion.

pH=pKa+lg⁡[A−][HA]

Henderson–Hasselbalch: at the half-equivalence point pH = pKₐ.

C3H7NO2+C2H5NO2→C5H10N2O3+H2O

Alanine + glycine → Ala-Gly dipeptide + water (formation of a peptide bond; in practice via protection and activation).

n=3.6 residues/turn,p=0.54 nm

α-helix (Pauling–Corey, 1951): 3.6 residues per turn, pitch 0.54 nm, 0.15 nm per residue; hydrogen bond C=O(i)···H–N(i + 4).

v=μE,μ∝zr

Electrophoresis: speed is proportional to the field strength and the charge of the particle; at pI z = 0 and the particle does not move.

Theory

Bioorganic chemistry, the cell and amino acids

Bioorganic chemistry studies the structure and function of the substances of living nature — proteins, nucleic acids, carbohydrates, lipids and bioregulators. In the elemental composition of the cell four elements — O, C, H and N — make up ≈ 98 % (the rest is P, S, K, Na, Ca, Mg, Fe and trace elements); in its molecular composition most is water, and in the dry matter proteins predominate. Prokaryotic cells (bacteria) have no nuclear envelope; in eukaryotes the DNA is in the nucleus and the organelles are separated by membranes. A membrane is a lipid bilayer with proteins embedded in it (the Singer–Nicolson “fluid mosaic” model, 1972): a barrier, transport and signal reception. In Uzbekistan the school of bioorganic chemistry is linked with the name of Academician O. S. Sadykov — the Institute of Bioorganic Chemistry in Tashkent bears his name. Heterofunctional compounds: hydroxy acids (lactic, malic, citric acid), oxo acids (pyruvic, acetoacetic acid) and amino acids; in the Reformatsky reaction an α-bromoester gives a β-hydroxy ester with zinc and an aldehyde (ketone). The 20 α-amino acids in proteins are divided by side chain into nonpolar (Gly, Ala, Val, Leu, Ile, Pro, Met), aromatic (Phe, Tyr, Trp), polar (Ser, Thr, Cys, Asn, Gln), negatively charged (Asp, Glu) and positively charged (Lys, Arg, His) groups; Trp, His and Pro are heterocyclic. For adults the essential amino acids (not synthesised in the body, they must come with food) are Val, Leu, Ile, Thr, Met, Phe, Trp, Lys (and His); the others are non-essential. In water an amino acid is a zwitterion H₃N⁺–CHR–COO⁻: a cation in acid and an anion in alkali; the pH at which the net charge is zero is the isoelectric point pI. In medicine, mixtures of amino acids are used for intravenous feeding, and some are used as drugs.

Chemical properties and stereochemistry

All the protein amino acids except glycine are chiral; only the L-series occurs in proteins (in the Fischer projection NH₂ is on the left; compared with glyceraldehyde). Most have the (S) configuration, but cysteine is (R) — because of the sulfur the CIP priority changes, while the spatial arrangement is the same. L-amino acids can be recognised by the “CORN” rule. Resolution of enantiomers: diastereomeric salts, enzymes (acylase hydrolyses only the L-form), chiral chromatography. Reactions: the carboxyl gives esters (alcohol + HCl), the amine gives N-acyl derivatives, Schiff bases (with aldehydes; pyridoxal phosphate enzymes work this way), a yellow DNP derivative with 2,4-dinitrofluorobenzene (Sanger), a phenylthiohydantoin with phenyl isothiocyanate (Edman), and a blue-violet colour with ninhydrin (Ruhemann's purple; proline gives yellow). Within a protein, amino acids are modified: hydroxyproline (collagen), phosphoserine (casein), cystine (S–S bridge), glycosylated asparagine.

The peptide bond and peptide synthesis

A peptide bond –CO–NH– forms when water is lost from the carboxyl of one amino acid and the amino group of another. Because of resonance the C–N bond is partly double: its length is ≈ 0.133 nm (an ordinary C–N is 0.147 nm, C=N 0.128 nm), there is no rotation about it, and the six atoms Cα–C(O)–N(H)–Cα lie in one plane, usually trans (ω = 180°). The chain rotates only about Cα: the angles φ and ψ. The polypeptide theory of protein structure (E. Fischer, F. Hofmeister, 1902): a protein is a chain of amino acids joined by peptide bonds. In chemical synthesis two amino acids cannot simply be mixed — each is both an amine and an acid, and the result is a mixture. So protecting groups are put on: on the amino group — Boc, Z (benzyloxycarbonyl), Fmoc; on the carboxyl — methyl or benzyl esters. The carboxyl is activated: acid chloride (SOCl₂), azide (Curtius), mixed anhydride, activated esters (p-nitrophenyl, N-hydroxysuccinimide), carbodiimide (DCC — dicyclohexylcarbodiimide: an O-acylisourea forms, and the by-product is insoluble dicyclohexylurea). Then the protection is removed (Boc with trifluoroacetic acid, an ester by alkaline hydrolysis). R. Merrifield (1963) invented assembling the chain in the solid phase on polymer beads (Nobel Prize, 1984).

Protein structure and denaturation

Simple proteins (proteins proper) consist only of amino acids, conjugated proteins (proteids) have an additional group: glycoproteins, lipoproteins, nucleoproteins, metalloproteins, phosphoproteins (milk casein). The primary structure is the sequence: F. Sanger was the first to determine the sequence of insulin in 1955 — 51 residues (A chain 21, B chain 30), 3 disulfide bridges (A6–A11, A7–B7, A20–B19). Secondary: the α-helix (Pauling and Corey, 1951) — a right-handed helix, 3.6 residues per turn, pitch 0.54 nm, with C=O(i)···H–N(i + 4) hydrogen bonds along the axis; the β-sheet — extended chains side by side, with hydrogen bonds between the chains (parallel or antiparallel), and β-turns. Tertiary is the spatial folding of the chain: a hydrophobic core, hydrogen bonds, ion pairs, S–S bridges. Quaternary — several chains (haemoglobin α₂β₂; insulin is stored as a hexamer with Zn²⁺ ions). Denaturation — by heating, acid/alkali, heavy metals, alcohol or urea the secondary to quaternary structure is destroyed, while the peptide bonds remain; egg white sets irreversibly at about 60–80 °C. Determining the N- and C-termini: Sanger (FDNB → DNP-amino acid), Edman (PITC → PTH-amino acid, the chain is shortened by one residue and the cycle repeats), dansyl chloride (fluorescent), Akabori (hydrazinolysis → the C-terminus); enzymatic — aminopeptidase (N-terminus), carboxypeptidase (C-terminus).

Enzymes and peptide bioregulators

Enzymes are catalysts of protein nature: unlike inorganic catalysts they are highly selective and work under mild conditions (37 °C, pH ≈ 7). The active site: the part where the substrate binds and the catalytic part (for example, the imidazole of histidine carries H⁺); an effector binds to the allosteric site and changes the activity. Many enzymes need a cofactor (Mg²⁺, Zn²⁺, Fe²⁺) or a coenzyme (NAD⁺, FAD, pyridoxal phosphate, coenzyme A). Isoenzymes are forms with different structure that speed up the same reaction (lactate dehydrogenase — five). Multienzyme systems carry out chains of metabolism (the pyruvate dehydrogenase complex). Enzyme kinetics is in the “Catalysis and enzymes” experiment. Peptide hormones: insulin and glucagon (pancreas), somatotropin and prolactin (pituitary), parathyroid hormone and calcitonin (Ca²⁺ metabolism), angiotensin (blood pressure), bradykinin, gastrin, secretin. Neuropeptides: enkephalins, endorphins, oxytocin, vasopressin. Toxins: fungal (α-amanitin), honeybee (melittin), snake (α-bungarotoxin), scorpion (chlorotoxin). Peptide antibiotics: gramicidin S, polymyxin, actinomycin D. All are in the “Gallery”.

On separation, lab work and the model

Separating proteins: homogenisation, extraction, salting out (ammonium sulfate: globulins precipitate at half saturation, albumins at full saturation), dialysis, chromatography (adsorption, partition, ion exchange, affinity, HPLC — “Extraction and chromatography”), electrophoresis (serum proteins at pH 8.6 separate into the albumin, α₁, α₂, β and γ fractions), mass spectrometry. Proteins are amphoteric polyelectrolytes (polyampholytes): positively charged below pI and negatively charged above; at pI the chain coils most tightly, and its solubility, viscosity and swelling are at a minimum — casein precipitates at pH ≈ 4.6 (L5). The hydrodynamics of polyelectrolytes, the isoelectric point of gelatin and the Donnan equilibrium are in the “Physics of macromolecules” experiment. Laboratory: L1 — colour reactions (ninhydrin, biuret, xanthoproteic, Millon, Fohl), L2 — solubility and precipitation (heating, heavy-metal salts, alcohol, acid, salt), L3 — albumin and globulins from egg white, L4 — egg and blood serum proteins, acid hydrolysis (6 M HCl, 110 °C) and amino acid composition, L5 — casein from milk. Safety: with concentrated HNO₃ and NaOH wear goggles and gloves; Millon's reagent contains mercury — it is poisonous; do not pour heavy-metal salts down the drain; ninhydrin leaves a dye on the skin. Model: the pKₐ values are table values at 25 °C, the groups are independent; the titration is calculated exactly from the charge balance; in electrophoresis the speed is proportional to charge (size is not taken into account); the milk buffer curve and the precipitation curves are approximate; the insulin Cα trace is a scheme calculated from constraints, not crystal coordinates; the gallery models are schematic “bead necklaces”.

Did you know?

  • Frederick Sanger received the Nobel Prize in 1958 for determining the amino acid sequence of insulin, and in 1980 for a method of reading DNA sequences — the first scientist to win two Nobel Prizes in chemistry (the second being Barry Sharpless, in 2001 and 2022).
  • Linus Pauling and Robert Corey proposed the α-helix and β-sheet models in 1951 — before the first accurate structures of protein crystals, basing them on the planarity of the peptide bond and on hydrogen bonds.

Inside the experiment

The experiment starts from a real-life situation: you predict the result, change the controls, run the experiment and draw a conclusion. Inside are missions, theory, a quiz and a guided tour in which the camera moves and explains the key points.

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