Stopped-flow Light Scattering Analysis of Red Blood Cell Glycerol Permeability

Patrizia Gena; Piero Portincasa; Sabino Matera; Yonathan Sonntag; Michael Rützler; Giuseppe Calamita

doi:10.21769/BioProtoc.3723

Stopped-flow Light Scattering Analysis of Red Blood Cell Glycerol Permeability

Bio Protoc. 2020 Aug 20;10(16):e3723. doi: 10.21769/BioProtoc.3723.

Authors

Patrizia Gena¹, Piero Portincasa², Sabino Matera¹, Yonathan Sonntag³, Michael Rützler^{3

4}, Giuseppe Calamita¹

Affiliations

¹ Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari "Aldo Moro", Bari, Italy.
² Clinica Medica "A. Murri", Department of Biomedical Sciences and Human Oncology, Medical School, University of Bari "Aldo Moro", Bari, Italy.
³ Department of Biochemistry and Structural Biology, Lund University, Lund, Sweden.
⁴ ApoGlyx AB, Malmö, Sweden.

Abstract

Stopped-Flow Light Scattering (SFLS) is a method devised to analyze the kinetics of fast chemical reactions that result in a significant change of the average molecular weight and/or in the shape of the reaction substrates. Several modifications of the original stopped-flow system have been made leading to a significant extension of its technical applications. One of these modifications allows the biophysical characterization of the water and solute permeability of biological and artificial membranes. Here, we describe a protocol of SFLS to measure the glycerol permeability of isolated human red blood cells (RBCs) and evaluate the pharmacokinetics properties (selectivity and potency) of isoform-specific inhibitors of AQP3, AQP7 and AQP9, three mammalian aquaglyceroporins allowing transport of glycerol across membranes. Suspensions of RBCs (1% hematocrit) are exposed to an inwardly directed gradient of 100 mM glycerol in a SFLS apparatus at 20 °C and the resulting changes in scattered light intensity are recorded at a monochromatic wavelength of 530 nm for 120 s. The SFLS apparatus is set up to have a dead time of 1.6-ms and 99% mixing efficiency in less than 1 ms. Data are fitted to a single exponential function and the related time constant (τ, seconds) of the cell-swelling phase of light scattering corresponding to the osmotic movement of water that accompanies the entry of glycerol into erythrocytes is measured. The coefficient of glycerol permeability ( P_gly , cm/s) of RBCs is calculated with the following equation: P_gly = 1/[(S/V)τ] where τ (s) is the fitted exponential time constant and S/V is the surface-to-volume ratio (cm^-1) of the analyzed RBC specimen. Pharmacokinetics of the isoform-specific inhibitors of AQP3, AQP7 and AQP9 are assessed by evaluating the extent of RBC P_gly values resulting after the exposure to serial concentrations of the blockers.

Keywords: Aquaglyceroporins; Aquaporin inhibitors; Erythrocytes; Glycerol membrane permeability; Stopped-flow light scattering.