Hydrolysis of Proteins

Hydrolysis is the reverse of the condensation reaction that forms peptide links. Water breaks each peptide link, -CONH-, giving back -COOH and -NH₂ groups, so a protein is broken down into its constituent amino acids.With water alone the reaction is extremely slow, so proteins are hydrolysed by heating under reflux with hydrochloric acid (about 6 mol dm⁻³) for many hours. In the body, enzymes do the same job at 37 °C.H₂NCH₂CONHCH(CH₃)COOH + H₂O → H₂NCH₂COOH + H₂NCH(CH₃)COOHIn the acidic mixture the amine groups are protonated, so the amino acids are actually present as positive ions, such as ⁺H₃NCH₂COOH. If hot aqueous sodium hydroxide is used instead, the products are sodium salts, such as H₂NCH₂COO⁻Na⁺.Identifying the amino acids. The mixture can be separated by thin-layer chromatography (TLC). Amino acids are colourless, so the spots are located by spraying with ninhydrin (which makes them coloured) or by using UV light. Each amino acid is identified by its R_f value, compared with known values measured under the same conditions:R_f = distance moved by spot ÷ distance moved by solvent frontWorked example. A spot moves 3.6 cm while the solvent front moves 8.0 cm. R_f = 3.6 ÷ 8.0 = 0.45.