Studying effects of capillary flow on membrane proteins by time-resolved fluorescence spectroscopy

Massimo Sassaroli; Katsumi Uchiyama; Heikki Vaananen; Robert E. Dale; Peter L. A. Giesen; J. B. Alexander Ross

doi:10.1117/12.307053

1 May 1998 Studying effects of capillary flow on membrane proteins by time-resolved fluorescence spectroscopy

Massimo Sassaroli, Katsumi Uchiyama, Heikki Vaananen, Robert E. Dale, Peter L. A. Giesen, J. B. Alexander Ross

Proceedings Volume 3256, Advances in Optical Biophysics; (1998) https://doi.org/10.1117/12.307053
Event: BiOS '98 International Biomedical Optics Symposium, 1998, San Jose, CA, United States

Abstract

We wish to develop a biophysical understanding of the structure-function relationship of Tissue Factor (TF), the membrane-bound protein that triggers hemostasis and arterial thrombosis by essential activation of the enzyme Factor VIIa (VIIa). Catalysis by TF-bound VIIa occurs in the blood stream, and flow-dependent shear affects its enzyme kinetics. From the known structure of the TF:VIIa complex, the catalytic domain of VIIa could be as close as 1 nm or as far as 12 nm from the membrane surface depending on geometry under the influence of shear. As models of blood vessels, we use glass capillary tubes coated on the inside surface with a lipid bilayer containing TF. This setup permits two types of measurements as a function of flow: enzyme kinetics of TF:VIIa by a colorimetric assay; and analysis of the spatial and dynamic relationship of TF:VIIa with respect to the membrane surface. Time-resolved depolarization and resonance energy transfer are measured via a microscope using either direct or evanescent wave excitation. We demonstrate the feasibility of these experiments by using rhodamine-labelled phospholipids at probe densities down to approximately 300 molecules/micrometer² in the presence or absence of the acceptor malachite green and by the emission anisotropy decay of probes in glycerol.

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Massimo Sassaroli, Katsumi Uchiyama, Heikki Vaananen, Robert E. Dale, Peter L. A. Giesen, and J. B. Alexander Ross "Studying effects of capillary flow on membrane proteins by time-resolved fluorescence spectroscopy", Proc. SPIE 3256, Advances in Optical Biophysics, (1 May 1998); https://doi.org/10.1117/12.307053

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