SPR data was obtained by measuring of the His-tagged C5a peptides to ScpAS512A was measured at 25 °C with a BIAcore X100 system (GE Healthcare, UK) in Hepes buffer (10 mM Hepes-KOH pH 7.5, 150 mM NaCl, 0.005% (v/v) Tween 20, 50 μM EDTA). The experiments were conducted in triplicate. Values of binding constants reported on Table 1 represent the mean and standard deviation from three experiments. Interactions with the human C5a peptides (rhC5a, rhC5adR, rhC5acore and point mutations) were assessed with an NTA sensor chip (GE Healthcare, UK) with approximately 30–40 response units (RU) of immobilized peptide ligand and a flow rate of 30 μL/min. The binding to human C5a peptides was measured over a range of ScpAS512A concentrations (32-fold) divided equally over 6 experiments (i.e. 2-fold change in ScpAS512A concentration between measurements). Association and dissociation phases were each monitored for 200 s. The highest ScpAS512A concentrations were 90, 500, and 1000 nM for rhC5a, rhC5adR, and rhC5acore binding respectively. The highest ScpAS512A concentration was 90 nM for rhC5aK4A,K5A, rhC5aK12A,K14A, and rhC5aK49A, 360 nM for rhC5aR46A, and 720 nM for rhC5aR37A and rhC5aR40A. Double referencing was used to remove the effects associated with buffer changes. In addition, the signal associated with non-specific ScpAS512A interactions with the chip surface was subtracted from each sensorgram. The sensorgrams were fit in a global analysis with the BIAevaluation 4.1 curve fitting software (GE Healthcare, UK). The best fit (lowest chi2) was obtained using 1:1 Langmuir model with a drifting baseline.

Kinetic and thermodynamic parameters for ScpAS512A binding to C5a peptides.

a Reported as mean and standard deviation from 3 experiments.

b ΔΔG° = −RTln[KD /KD (rhC5a, 150 mM NaCl)], R = 1.986 (cal mol−1 K−1), T = 298 K.

c Binding energy for rhC5a at 298 K , ΔG°bind = -RTln[1/KD] = -10.2 kcal/mol.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.