Total internal reflection-fluorescence recovery after photobleaching (TIR-FRAP) was applied to measure solute translational diffusion in the aqueous phase of membrane-adjacent cytoplasm. TIR fluorescence excitation in aqueous solutions and fluorescently labeled cells was produced by laser illumination at a subcritical angle utilizing a quartz prism; microsecond-resolution FRAP was accomplished by acousto-optic modulators and electronic photomultiplier gating. A mathematical model was developed to determine solute diffusion coefficient from the time course of photobleaching recovery, bleach time, bleach intensity, and evanescent field penetration depth; the model included irreversible and reversible photobleaching processes, with triplet state diffusion. The validity and accuracy of TIR-FRAP measurements were first examined in aqueous fluorophore solutions. Diffusion coefficients for fluorescein isothiocyanate-dextrans (10-2000 kDa) determined by TIR-FRAP (recovery t1/2 0.5-2.2 ms) agreed with values measured by conventional spot photobleaching. Model predictions for the dependence of recovery curve shape on solution viscosity, bleach time, and bleach depth were validated experimentally using aqueous fluorescein solutions. To study solute diffusion in cytosol, MDCK epithelial cells were fluorescently labeled with the small solute 2',7'-bis-2-carboxyethyl-5-carboxyfluorescein-acetoxymethyl-ester (BCECF). A reversible photobleaching process (t1/2 approximately 0.5 ms) was identified that involved triplet-state relaxation and could be eliminated by triplet-state quenching with 100% oxygen. TIR-FRAP t1/2 values for irreversible BCECF bleaching, representing BCECF translational diffusion in the evanescent field, were in the range 2.2-4.8 ms (0.2-1 ms bleach times), yielding a BCECF diffusion coefficient 6-10-fold less than that in water. These results establish the theory and the first experimental application of TIR-FRAP to measure aqueous-phase solute diffusion, and indicate slowed translational diffusion of a small solute in membrane-adjacent cytosol.

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The integral membrane protein CHIP28 is an important water channel in erythrocytes and kidney tubule epithelia and is a member of a family of channel/pore proteins including the lens protein MIP26. The purposes of this study were to purify functional, delipidated CHIP28 to homogeneity and to determine secondary structure by circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR). CHIP28 was initially purified and delipidated by anion-exchange chromatography following solubilization of N-lauroylsarcosine-stripped erythrocyte membranes with beta-octylglucoside (OG); MIP26 was initially purified and delipidated by anion-exchange chromatography following solubilization of urea-stripped bovine lens membranes by monomyristoylphosphatidylcholine. CHIP28 (glycosylated and nonglycosylated) and MIP26 were purified further by high-performance size-exclusion chromatography, eluting in OG as apparent dimers and tetramers, respectively. Proteoliposomes reconstituted with purified CHIP28 were highly water-permeable, with an osmotic water permeability Pf of 0.04 cm/s at 10 degrees C that was inhibited by 0.1 mM HgCl2. Proteoliposomes reconstituted with MIP26 had a low Pf of 0.005 cm/s. CD spectra of CHIP28 in OG or in reconstituted proteoliposomes gave a maximum at 193 nm and minima at 208 and 222 nm. Spectral decomposition using protein basis spectra gave 40 +/- 5% alpha-helix and 43 +/- 3% beta-sheet and -turn. HgCl2 did not affect the CD spectrum of CHIP28. Attenuated total reflectance FTIR of air-dried, membrane-associated CHIP28 gave 38 +/- 5% alpha-helix and 40 +/- 4% beta-sheet and -turn by spectral decomposition of the amide I resonance. For comparison, CD of MIP26 in OG gave 49 +/- 7% alpha-helix and 32 +/- 12% beta-sheet and -turn; FTIR gave 32 +/- 8% alpha-helix and 45 +/- 6% beta-sheet and -turn. Analysis of CHIP28 and MIP26 sequence data by the generalized hydropathy method of Jähnig [Jähnig, F. (1990) Trends Biochem. Sci. 15, 93-95] predicted 39-47% alpha-helix and 15-20% beta-structures. These results establish procedures to obtain large quantities of pure CHIP28 and MIP26 in functional forms and provide evidence for multiple membrane-spanning alpha-helices or mixed alpha/beta-domains.

The purpose of this study was to determine whether the unique physical milieu just beneath the cell plasma membrane influences the rheology of fluid-phase cytoplasm. Cytoplasmic viscosity was evaluated from the picosecond rotation of the small fluorophore 2',7'-bis-(2-carboxyethyl)-5-carboxyfluorescein (BCECF) by parallel-acquisition Fourier transform microfluorimetry (Fushimi and Verkman, 1991). Information about viscosity within < 200 nm of cell plasma membranes was obtained by selective excitation of fluorophores in an evanescent field created by total internal reflection (TIR) of impulse-modulated s-plane-polarized laser illumination (488 nm) at a glass-aqueous interface. Measurements of fluorescence lifetime and time-resolved anisotropy were carried out in solutions containing fluorescein or BCECF at known viscosities, and monolayers of BCECF-labeled Swiss 3T3 fibroblasts and Madin-Darby canine kidney (MDCK) cells. Specific concerns associated with time-resolved fluorescence measurements in the evanescent field were examined theoretically and/or experimentally, including variations in lifetime due to fluorophore proximity to the interface, and the use of the s and p polarized excitation. In fluorescein solutions excited with s-plane polarized light, there was a 5-10% decrease in fluorescein lifetime with TIR compared to trans (subcritical) illumination, but no change in rotational correlation time (approximately 98 ps/cP). Intracellular BCECF had a single lifetime of 3.7 +/- 0.1 ns near the cell plasma membrane. Apparent fluid-phase viscosity near the cell plasma membrane was 1.1 +/- 0.2 cP (fibroblast) and 1.0 +/- 0.2 cP (MDCK), not significantly different from the viscosity measured in bulk cytoplasm far from the plasma membrane. The results establish the methodology for time-resolved microfluorimetric measurement of polarization in the evanescent field and demonstrate that the cell plasma membrane has little effect on the fluid-phase viscosity of adjacent cytoplasm.

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