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Stopped-flow Light ...
Stopped-flow Light Scattering Analysis of Red Blood Cell Glycerol Permeability
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- Gena, Patrizia (author)
- University of Bari Aldo Moro
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- Portincasa, Piero (author)
- Bari University Hospital
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- Matera, Sabino (author)
- University of Bari Aldo Moro
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- Sonntag, Yonathan (author)
- Lund University,Lunds universitet,Biokemi och Strukturbiologi,Centrum för Molekylär Proteinvetenskap,Kemiska institutionen,Institutioner vid LTH,Lunds Tekniska Högskola,Biochemistry and Structural Biology,Center for Molecular Protein Science,Department of Chemistry,Departments at LTH,Faculty of Engineering, LTH
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- Rützler, Michael (author)
- Lund University,Lunds universitet,Biokemi och Strukturbiologi,Centrum för Molekylär Proteinvetenskap,Kemiska institutionen,Institutioner vid LTH,Lunds Tekniska Högskola,Biochemistry and Structural Biology,Center for Molecular Protein Science,Department of Chemistry,Departments at LTH,Faculty of Engineering, LTH,Apoglyx AB
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- Calamita, Giuseppe (author)
- University of Bari Aldo Moro
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(creator_code:org_t)
- 2020
- 2020
- English.
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In: Bio-protocol. - 2331-8325. ; 10:16
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Abstract
Subject headings
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- 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 (Pgly, cm/s) of RBCs is calculated with the following equation: Pgly = 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 Pgly values resulting after the exposure to serial concentrations of the blockers.
Subject headings
- NATURVETENSKAP -- Kemi -- Fysikalisk kemi (hsv//swe)
- NATURAL SCIENCES -- Chemical Sciences -- Physical Chemistry (hsv//eng)
Keyword
- Aquaglyceroporins
- Aquaporin inhibitors
- Erythrocytes
- Glycerol membrane permeability
- Stopped-flow light scattering
Publication and Content Type
- art (subject category)
- ref (subject category)
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