An organic monolithic capillary column functionalized with human serum albumin and its application for the nano - chromatography study of its binding with universal cancer peptides and its impact on immunogenicity

Sixty picomoles of human serum albumin (HSA) was immobilized on a glycidylmethacrylate (GMA) capillary column previously developed by our group to study its binding with two universal cancer peptides (UCPs). Zonal HPLC experiments demonstrated that UCP2 and UCP4 have 1:1 interaction at the well-known site I of HSA with similar affinities in the micromolar range. At pH 7.4 UCP2 and UCP4 bound to HSA site I with negative Delta H values increasing with increasing temperature leading to positive heat capacity changes (UCP2: Delta C-p=1.06 kJ/mol/K; UCP4: Delta C-p=0.53 kJ/mol/K). The sign and magnitude of these thermodynamic data were explained by the burial of the polar groups of the peptide inside the hydrophobic binding pocket of HSA leading to coil helix transition upon the binding. The strong linear coefficient (r(2)= 0.995) for the plot comparing the immunogenicity index (II) between a series of four UCPs against the enthalpy gain confirmed a high degree of similarity for the mechanism of immunogenicity of these short peptides. The positive slope (+2.59) indicated that at physiological pH (7.4), the helix gain upon UCPs/HSA binding promoted the immunogenicity of the peptide vaccine.