59
Previous experiments had shown that ~23% of the Thr was incompetent to binding AT [2]; thus, the observed binding could have resulted from Ab interacting with this incompetent Thr left in solution rather than the Thr-AT complex. Under the experiment conditions, it was impossible to tell from which mechanism the measured Mw resulted.
Interaction of Ab with Purified Thr-AT Complex
In order to distinguish between the two possible mechanisms, bound Thr-AT complex was separated from unbound Thr and AT via SEC (Figure 3). The fraction of Thr that is incompetent for binding to AT is evident as a secondary peak in the SEC chromatogram for the pre-mixed solution. After collecting the appropriate peak, an aliquot was re-applied to the column to confirm that no dissociation had occurred and there was negligible contamination by free Thr.
Figure 5. The best fit of LS and concentration data for crossover gradient with purified Thr-AT complex and Ab indicates 1:2 (Ab):(Thr-AT complex) stoichiometry with decreased affinity compared to Thr:Ab alone. Based on these data, the molar composition of each species can be calculated.
Conclusion
Figure 3. Overlay of size exclusion chromatograms for pure Thr (green), pure AT (blue), and a mixed solution of Thr and AT (magenta). Molecular weights measured by MALS correspond to the expected values. Fractions of covalently bound Thr-AT complex were collected for CG-MALS analysis.
A CG-MALS experiment, consisting of a single crossover gradient with Ab and purified Thr-AT complex, revealed the antibody bound the Thr-AT complex with the expected 1:2 stoichiometry (Figures 4 & 5). Since AT was covalently bound to the Thr active site, this potential binding site was inaccessible to the Ab, indicating that the antibody recognises an epitope on thrombin other than the active site. Previous binding assays also support this assertion. The analysis of Thr- Ab binding indicates no incompetent Thr, suggesting the binding epitope is always accessible. On the other hand, the binding of AT to Thr, which requires the active site to be accessible and/or properly folded, indicates that ~23% of Thr presented an active site that was incompetent for binding AT [2]. Taken together, these data confirm that the antibody binds thrombin away from the active site.
MALS is unique among biophysical characterisation techniques because it enables the simultaneous quantification of multiple species present in solution. SEC-MALS confirmed the irreversible binding of AT and Thr and enabled unequivocal separation of the purified complex for further characterisation. CG-MALS quantified the affinity and stoichiometry of the multivalent, equilibrium interaction between Ab and the Thr-AT complex, confirming the binding of the antibody to AT-inactivated thrombin but with decreased affinity. Thus, probing macromolecular interactions by light scattering provides a robust, complementary biophysical technique for understanding complex protein-interaction networks.
References
(1) “Measuring the Interaction Between Thrombin-α and an Anti-Thrombin Antibody.” Wyatt Technology Corp. Published 7 Jan 2013.
www.wyatt.com/files/literature/Calypso_antithrombinantibody.pdf
(2) “Determining the Kinetics of Covalent Thrombin-Antithrombin Association.” Wyatt Technology Corp. Published 7 Jan 2013.
www.wyatt.com/files/literature/Calypso_covalentantithrombin.pdf
(3) Some, D. and Kenrick, S. (2012) “Characterization of Protein-Protein Interactions via Static and Dynamic Light Scattering. In Protein Interactions, Cai, J. And Wang, R.E. (Eds.), InTech, DOI: 10.5772/37240. Available at:
www.intechopen.com/books/protein-interactions/characterization- of-protein-protein-interactions-via-static-and-dynamiclight-scattering (4) Gandhi, P.S., et al. Proc. Natl. Acad. Sci. 2008 105, 1832.
Time-Resolved Single Photon Counting Measurement Option Introduced
At Pittcon 2013 WITec launched StrobeLock, a time-correlated single photon counting measurement option. The imaging modes include Fluorescence Lifetime Imaging and Time-resolved Luminescence Microscopy, which can be integrated with the WITec alpha300 and alpha500 microscope series.
StrobeLock facilitates the acquisition of additional material contrasts hidden in the time function of a fluorescence or luminescence signal and allows them to be perfectly linked with Raman, SNOM or AFM imaging. It enables a variety of measurement possibilities for an improved and more comprehensive understanding of a sample’s properties and is specifically suited for materials science.
Figure 4. Measured Mw by MALS for composition gradient with varying concentrations of purified Thr-AT complex (10-70µg/mL) and Ab (1-10 µg/mL), indicating an interaction between the Ab and
Thr-AT complex. The Mw measured by light scattering increases as the antibody binds the Thr-AT complex. The maximum molecular weight occurs at an overall molar ratio of 1 Ab: 2 Thr-AT
complex, indicating a 1:2 stoichiometry.
Although binding of the antibody to the Thr-AT complex is evident, the measured affinity of the antibody for the Thr-AT complex is ~28x decreased as compared to the affinity for pure Thr,
with KD = 250 nM. This may indicate that AT imparts some steric hindrance for antibody- binding. Alternatively, the binding of Thr to AT may lock the enzyme in an inactive conformation [4] which has lower affinity for the antibody than the native or allosterically activated Thr.
The modular design of the WITec microscopes facilitates user-friendly combination of the StrobeLock module with the WITec confocal microscope series. StrobeLock is comprised of a pulsed excitation laser system combined with a Time-Correlated Single-Photon Counting (TCSPC) detector. The possibility to switch between time-resolved and conventional mode enables the microscope user to conveniently choose the preferred measurement technique.
“StrobeLock complements the modular WITec microscope systems with customised solutions for the most accurate Fluorescence Lifetime Imaging and Time-Resolved Luminescence Microscopy.” said Dr Joachim Koenen, WITec co- founder and Managing Director “This exceptional development significantly extends the capabilities of the WITec microscope series and opens a new field of application for a more comprehensive sample characterisation.”
MORE INFO. 172
INTERNATIONAL LABMATE - APRIL 2013
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