Evaluation of temperature effect on the interaction between β-lactoglobulin and anti-β-lactoglobulin antibody by atomic force microscopy.

Molecular recognition such as antigen-antibody interaction is characterized by the parameters of kinetics and the energy landscape. Examinations of molecules involved in the interaction at different temperatures using atomic force microscopy (AFM) can provide information on not only the effects of temperature on the unbinding force between a molecule of interest and a complementary molecule but also the parameters of kinetics and the energy landscape for dissociation of the molecular complex. We investigated the effect of temperature on the dissociation process of the complex of β-lactoglobulin and anti-bovine β-lactoglobulin IgG polyclonal antibody using AFM. Measurements of the unbinding forces between β-lactoglobulin and the antibody were performed at 25, 35, and 45 °C. The following results were obtained in our present study: (i) The unbinding forces decreased as temperature increased, suggesting that the binding force between β-lactoglobulin and the antibody includes the force originating from temperature-dependent interactions (e.g., hydrogen bonding). (ii) At each temperature, the unbinding force exhibited two linear regimes in the force spectra, indicating that the dissociation process of the β-lactoglobulin-antibody complex passes at least two energy barriers from the bound state to the dissociated state. (iii) The dissociation rates at zero force and the position of energy barriers increased as temperature increased. (iv) The heights of the two energy barriers in the reaction coordinates were 49.7 k(B)T and 14.5 k(B)T. (v) The values of roughness of the barriers were ca. 6.1 k(B)T and 3.2 k(B)T. Overall, the present study using AFM revealed more information about the β-lactoglobulin-antibody interaction than studies using conventional bulk measurement such as surface plasmon resonance.