Dominant structural factors for complexation and denaturation of proteins using carboxylic acid receptors

Complexation accompanied by denaturation of protein with synthetic carboxylic acid receptors was investigated, to evaluate the key factors for recognition of proteins. The synthetic receptors used were tetraphenylporphyrin (TPP) derivatives and receptors bearing multiple (2–8) carboxylic acid groups. The complexation behavior was quantified from the absorption in the far UV CD spectrum attributed to the secondary structure of the protein. TPP derivatives bearing multiple carboxylic acid groups in the side chains exhibited higher affinity than other receptors that were smaller and had fewer carboxylic acid groups. As the degree of complexation was influenced by the pH and ionic strength in aqueous solution, electrostatic interaction was one of the most important factors for the recognition of proteins. Complexation was also estimated by observation of fluorescence quenching of the TPP derivatives. The stoichiometry of the complexes between lysozyme and the porphyrins was investigated by quantitative analysis of the denaturation using CD spectra. From the results of Job plots and slope analysis for the amount of denatured protein, formation of 1:1 complexes was confirmed. The equilibrium association constants (Kass) for lysozyme and the TPP receptors ranged from 0.6 × 106 to 1.1 × 106 M−1. The lytic activity of lysozyme was partially lost in the presence of anionic TPP derivatives, due to complexation and denaturation.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slideHighlights► Complexation accompanied by denaturation of protein with synthetic carboxylic acid receptors was investigated, to evaluate the key factors for recognition of proteins. ► TPP derivatives bearing multiple carboxylic acid groups in the side chains exhibited higher affinity than other receptors that were smaller and had fewer carboxylic acid groups. ► From the results of Job plots and slope analysis for the amount of denatured protein, formation of 1:1 complexes was confirmed.