Dynamic response of glucagon/anti-glucagon pairs to pulling velocity and pH studied by atomic force microscopy.
We used atomic force microscopy (AFM) to measure the unbinding force between antigen coupled to an AFM tip and antibody coated on the substrate surface. Dynamic responses of glucagon/anti-glucagon pairs with multiple pull-off steps to pH and pulling velocity were studied by AFM. Force-distance curves of a specific glucagon-anti-glucagon interaction system with mono-, di-, and multi-unbinding events were recorded, which may be attributed to a single, sequential or multiple breaking of interacting bond(s) between glucagon and anti-glucagon. We studied the dynamic response of glucagon-anti-glucagon pairs to various pulling velocities (16.7-166.7 nm/s). It was found that the mean value of the unbinding force was shifted toward higher values with increasing pulling velocity at each pH. This indicates that the friction force between glucagon and anti-glucagon may contribute to the unbinding force. Moreover, the dynamic response of glucagon-anti-glucagon pairs to pH (4-10) with different pulling velocities was studied. Within the acid range, the bond strength between the glucagon/anti-glucagon complex showed a rapid increase from pH 4 to 7 and reached a maximum (256.4+/-48.9 pN at 166.7 nm/s) at neutrality, followed by a sharp decrease with increasing pH (pH 7-10). This could be attributed to the conformational change that occurred in glucagon when the pH value in solution was varied from the reference level at neutrality. This study demonstrated that the pH dependence of multiple antigen-antibody bond-rupture forces could be measured by a force-based AFM biosensor. Unraveling the relationship between inter-molecular force and intra-molecular conformational change in acid, neutral, and alkaline environments may provide new directions for future application of force measurements by AFM in proteomics or in the development of a clinical cantilever-based mechanical biosensor.
Lin
Shiming
S
Center for Optoelectronic Biomedicine, National Taiwan University, College of Medicine, 1-1 Jen-Ai Road, Taipei, Taiwan. sml@ntumc.org
Wang
Yu-Ming
YM
Huang
Long-Sun
LS
Lin
Chii-Wann
CW
Hsu
Su-Ming
SM
Lee
Chih-Kung
CK
D000906:Antibodies / Q000737:chemistry*; D000936:Antigen-Antibody Complex / Q000737:chemistry*; D005934:Glucagon / Q000737:chemistry* / Q000276:immunology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008846:Micromanipulation / Q000379:methods*; D018625:Microscopy, Atomic Force / Q000379:methods*; D009038:Motion; D011485:Protein Binding; D013314:Stress, Mechanical
D000906:Antibodies; D000936:Antigen-Antibody Complex; D005934:Glucagon