The energetics of peptide bond formation at elevated temperatures.

  • Robert Tattersall

Student thesis: Doctoral ThesisPhD


Published data on the energetics of peptide bond formation are scarce and virtually none have been reported at elevated temperatures. The energetics of the peptide bond forming reaction between the partially protected amino acids carbobenzoxyglycine and L-phenylalanine amide at a range of temperatures were therefore investigated in detail. The choice of reaction components ensured the unambiguous formation of only one type of peptide bond. The free energies at temperatures up to 60°C were determined experimentally by equilibrium measurements and up to 100°C by extrapolation. The peptide bond forming reaction in buffered solution at pH 7.2 was catalysed enzymatically. The isolation and testing of a thermophilic enzyme from Bacillus stearo-thermophilus is described. The enzyme finally selected as catalyst was thermolysin. The equilibrium constant measurements were made using radiotracers and the synthesis of 14C labelled protected amino acids and dipeptides is described. An attempt was made to approach equilibrium from both directions. Ion-exchange chromatography and paper and thin layer chromatography and electrophoresis were tried for dipeptide isolation and purity assessment. Precipitation and crystallization methods were finally employed. Ionization constants were determined by potentiometry at temperatures up to 80°C. The Deltac° results at 25°C agreed favourably with the only previously published value derived from an equilibrium measurement. Although the free energy change with increasing temperature was found not to be large, the standard free energy for peptide bond o formation did appear to become negative at temperatures exceeding 60°C. The enzyme was found to affect the equilibrium at high dipeptide/enzyme molar ratios. The enthalpy of peptide bond formation was calculated from the van't Hoff equation and the entropy subsequently derived.
Date of Award1976
Original languageEnglish
Awarding Institution
  • University of Bath

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