Measurement of facilitated calcium diffusion by a soluble calcium-binding protein

The flux of calcium through an aqueous compartment was determined in a flow-dialysis cell in which two dialysis membranes separated the middle aqueous compartment from two outer compartments. The contribution of convection to the total calcium flux was large but could be removed by addition of 1% agar. The flux of calcium through the gelled aqueous compartment agreed with theoretical expectations. The self-diffusion coefficient for calcium from these results was calculated to be 0.81 X 10(-5) cm2 X s-1. Carp parvalbumin significantly enhanced the calcium flux at 2.3 X 10(-6)M free calcium. The calcium flux increased linearly with parvalbumin concentration. These observations are consistent with the hypothesis that the overall unidirectional calcium flux J is the sum of free calcium diffusion and protein-calcium diffusion: J = D[Ca] + D'[CaPr]. The value of D', the self-diffusion coefficient for parvalbumin, was calculated from the flux data to be 13.7 X 10(-7) cm2 X s-1.     

Calcium diffusion in transient and steady states in muscle.

Rates of diffusion through the extracellular space of thin sheets of myocardium from the right ventricular outflow tract of kittens were estimated at 23 degrees C for 45Ca2+ and an inert reference tracer, [14C]sucrose. The myocardial sheets were mounted in an Ussing chamber and equilibrated with Tyrode solution with varied calcium concentrations, Cao. The tracers were added to one side and their concentrations on the other side measured at 5-15-min intervals for 6 h. The apparent tracer diffusion coefficient for sucrose was 1.11 +/- 0.06 X 10(-6) cm2s-1 (mean +/- SEM, n = 74), 22% of the free diffusion coefficient; the lag time before reaching a steady state provided estimates of the intratissue volume of distribution or diffusion space of 0.41 +/- 0.15 ml/ml tissue (n = 74), a value compatible with expectations for extracellular fluid space. Over the range of Cao from 0.02 to 9.0 mM, the intratissue apparent diffusion coefficient for Ca, DCa, averaged 1.65 +/- 0.10 X 10(-6) cm2s-1, n = 74, which is 21% of the free DoCa, and was not influenced by Cao. Because transsarcolemmal Ca permeation is slow, DCa is the diffusion coefficient in the extracellular region. The paired ratios DCa/Ds averaged 1.32 +/- 0.05 (n = 67) for all levels of Cao but at physiologic or higher Cao averaged 1.45 +/- 0.07 (n = 39), close to the ratio of free diffusion coefficients, 1.53. Equations distinguishing transient from steady state diffusion were fitted to the data, showing that the apparent distribution volume of "binding sites" external to the diffusion pathway diminished at higher Cao in a fashion suggesting that a least two different Ca2+ binding sites were present.     

Intracellular diffusion, binding, and compartmentalization of the fluorescent calcium indicators indo-1 and fura-2.

We studied intracellular binding and possible compartmentalization of the fluorescent Ca2+ indicators, indo-1 and fura-2, in single mammalian cardiac ventricular cells that had been loaded with indo-1 and fura-2 by exposure to the acetoxymethylester form of the indicators (indo-1/AM and fura-2/AM). Techniques similar to those used in experiments on fluorescence recovery after photobleaching (FRAP) were used. It was assumed that reversible binding in myoplasm would be evident as slowed recovery of fluorescence after photobleaching, and that irreversible binding of the indicators to immobile myoplasmic sites (or "compartmentalization" in organelles) would be evident as incomplete recovery. Through the use of a mask, one half of a cell was exposed to high-intensity ultraviolet (UV) light to bleach the indo-1 or fura-2 in only that part of the cell. Upon removal of the mask and termination of the high-intensity UV illumination, fluorescence recovered in the bleached half of the cell, indicating diffusion of indo-1 and fura-2. Mathematical modeling of the diffusional redistribution of the indicators indicated that in these cells the apparent diffusion coefficient for indo-1 is 1.57 x 10(-7) cm2 s-1 (SD 0.48 x 10(-7) cm2 s-1; n = 5 cells, 21 degrees C), and for fura-2 is 3.19 x 10(-7) cm2 s-1 (SD 1.85 x 10(-7) cm2 s-1; n = 6 cells, 21 degrees C). These values are approximately 6 and 3, respectively, times smaller than those expected for free diffusion in the myoplasm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microscopic versus macroscopic diffusion in model membranes by electron spin resonance spectral-spatial imaging.

The macroscopic and the microscopic diffusion coefficients of a phospholipid spin label (16-PC) in the model membrane 1-palmitoyl-2-oleoyl-sn-glycero-phosphatidylcholine have been measured simultaneously in the same sample utilizing the new technique of spectral-spatial electron spin resonance imaging. The macroscopic diffusion coefficient Dmacro for self-diffusion of 16-PC spin label is obtained from imaging the concentration profiles as a function of time, and it is (2.3 +/- 0.4) x 10(-8) cm2/s at 22 degrees C. The microscopic diffusion coefficient Dmicro for relative diffusion of the spin probes is obtained from the variation of the spectral line broadening with spin label concentration, which is due to spin-spin interactions. Dmicro is found to be substantially greater than Dmacro for the same sample at the same conditions, and is estimated to be at least (1.0 +/- 0.4) x 10(-7) cm2/s. Possible sources for their difference are briefly discussed in terms of the models used for Dmicro.     

A method for the determination of diffusion coefficients for small molecules in aqueous solution

A method is described for determining the diffusion coefficients of small solutes in limited volumes (approximately equal to 4-9 ml) of fluid. Diffusion is measured in a three-chamber diffusion cell across a central unstirred compartment. Compartments are separated by nitrocellulose membranes. The instantaneous concentration gradient and the instantaneous flux of solute into the dilute end compartment are derived from changes in the concentration of solute in the two stirred end compartments through time. The diffusion coefficient is calculated from the slope of the least-squares regression line relating the magnitude of the instantaneous solute flux to that of the instantaneous concentration gradient. The apparatus is calibrated with a solute of known diffusivity (KCl). Diffusion coefficients thus determined in water at 25 degrees C for CaCl2 (7.54 X 10(-6) cm2.s-1), Na2-ATP (7.01 X 10(-6) cm2.s-1), 2-deoxyglucose (5.31 X 10(-6) cm2.s-1), and D-Na-lactate (5.62 X 10(-6) cm2.s-1) differed by an average of 3.7% from literature values. The method described results in accurate estimates of diffusion coefficients by a simple and relatively rapid procedure.     

Rapid diffusion of spectrin bound to a lipid surface.

Human erythrocyte spectrin was labelled with the probe 5, 5'-disulfato-1-(6-hexanoic acid N-hydroxysuccinimide ester)-1'-ethyl-3,3,3',3'-tetramethylindocarbocyanine (Cy3). Cy3-spectrin was bound to the outer surface of dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles and its diffusion measured by fluorescence recovery after photobleaching (FRAP). It was found that at 30 degrees C, above the lipid gel to liquid-crystalline phase transition of the lipids, Cy3-spectrin had an unexpectedly high diffusion coefficient D=(2.1+/-0.6)x10(-7)) cm2/s. At the phase transition, diffusion of Cy3-spectrin was only slightly lower; D=(1.3+/-0.3)x10(-7) cm2/s, whereas at 14 degrees C, well below the lipid phase transition, diffusion was found to be much slower with D=(3.1+/-0.12)x10(-9) cm2/s. The fast diffusion of Cy3-spectrin on the lipid surface implies that the individual bonds which bind spectrin to the lipid surface must rapidly be made and broken. In the light of these results, spectrin-lipid interactions alone appear unlikely to have any significant role in supporting the cell membrane. Probably, the interactions serve only to localise the spectrin at the inner lipid surface in order to facilitate formation of the cytoskeleton.     

Diffusion coefficient of the cyclic GMP analog 8-(fluoresceinyl)thioguanosine 3'',5'' cyclic monophosphate in the salamander rod outer segment.

Cyclic GMP (cGMP) is the intracellular messenger mediating phototransduction in retinal rods, with its longitudinal diffusion in the rod outer segment (ROS) likely to be a factor in determining light sensitivity. From the kinetics of cGMP-activated currents in the truncated ROS of the salamander (Ambystoma tigrinum), the cGMP diffusion coefficient was previously estimated to be approximately 60 x 10(-8) cm2 s-1. On the other hand, fluorescence measurements in intact salamander ROS using 8-(fluoresceinyl)thioguanosine 3',5'-cyclic monophosphate (Fl-cGMP) led to a diffusion coefficient for this compound of 1 x 10(-8) cm2 s-1; after corrections for differences in size and in binding to cellular components between cGMP and Fl-cGMP, this gave an upper limit of 11 x 10(-8) cm2 s-1 for the cGMP diffusion coefficient. To properly compare the two sets of measurements, we have examined the diffusion of Fl-cGMP in the truncated ROS. From the kinetics of Fl-cGMP-activated currents, we have obtained a diffusion coefficient of 3 x 10(-8) cm2 s-1 for this analog; the cGMP diffusion coefficient measured from the same truncated ROSs was approximately 80 x 10(-8) cm2 s-1. Thus, a factor of 27 appears appropriate for correcting differences in size and intracellular binding between cGMP and Fl-cGMP. Application of this correction factor to the Fl-cGMP diffusion coefficient measurements by Olson and Pugh (1993) gives a cGMP diffusion coefficient of approximately 30 x 10(-8) cm2 s-1, in reasonable agreement with the value measured from the truncated ROS.     

Binding of ethidium to DNA measured using a 2D diffusion-modulated gradient COSY NMR experiment

The binding of ethidium bromide to a DNA hairpin (dU(5)-hairpin) was investigated using a novel 2D diffusion-modulated gradient correlation spectroscopy (DMG-COSY) experiment to evaluate the applicability of this technique for studying the binding of drugs to DNA. The DMG-COSY experiment includes a preparation period during which coherent magnetization is attenuated due to molecular self-diffusion. Magnetization then evolves due to scalar coupling during an evolution delay, and is detected using gradient pulses for coherence selection. The time-domain data are processed in an analogous manner as for gradient-selected COSY experiments. The diffusion coefficient for uridine in DMSO solution was determined from the H5-H6 crosspeak intensities for a series of 2D DMG-COSY experiments that differed in the magnitude of the gradient pulses applied during the preparation period of the DMG-COSY experiment. The diffusion coefficient for uridine calculated from the DMG-COSY experiments was identical (within experimental error) to that determined from 1D diffusion experiments (5.24x10(-6) cm(2)/s at 26 degrees C). The diffusion coefficients for ethidium bromide and for the dU(5)-hairpin were first measured separately using the DMG-COSY experiment, and then measured in the putative complex. The diffusion coefficient for free ethidium bromide (4.15x10(-6) cm(2)/s at 26 degrees C) was considerably larger than for the dU(5)-hairpin (1. 60x10(-6) cm(2)/s at 26 degrees C), as expected for the smaller molecule. The diffusion coefficient for ethidium was markedly decreased upon addition of the dU(5)-hairpin, consistent with complex formation (1.22x10(-6) cm(2)/s at 26 degrees C). Complex formation of 1:1 stoichiometry between ethidium and the stem of the dU(5)-hairpin was verified independently by fluorescence spectroscopy. These results demonstrate the utility of the DMG-COSY experiment for investigating the binding of drugs to DNA in aqueous solution.     

Diffusion of water in cat ventricular myocardium

The rates of diffusion of tritiated water (THO) and [14C]sucrose across cat right ventricular myocardium were studied at 23 degrees C in an Ussing-type diffusion cell, recording the time-course of increase in concentration of tracer in one chamber over 4--6 h after adding tracers to the other. Sucrose data were fitted with a model for a homogeneous sheet of uneven thickness in which the tissue is considered to be an array of parallel independent pathways (parallel pathway model) of varying length. The volume of the sucrose diffusion space, presumably a wholly extracellular pathway, was 23% of the tissue or 27.4 +/-1.7% (mean +/- SEM; n=11) of the tissue water. The effective intramyocardial sucrose diffusion coefficient, D8, was 1.51 +/- 0.19 X 10(-6)cm2.s-1 (n=11). Combining these data with earlier data, D8 was 22.6 +/- 1.1% (n=95) of the free diffusion coefficient in aqueous solution D degrees 8. The parallel pathway model and a dead-end pore model, which might have accounted for intracellular sequestration of water, gave estimates of DW/D degrees W (observed/free) of 15%. Because hindrance to water diffusion must be less than for sucrose (where D8/D degrees 8=22.6%), this showed the inadequacy of these models to account simultaneously for the diffusional resistance and the tissue water content. The third or cell-matrix model, a heterogeneous system of permeable cells arrayed in the extracellular matrix, allowed logical and geometrically reasonable interpretations of the steady-state data and implied estimates of DW in the cellular and extracellular fluid of approximately 25% of the aqueous diffusion coefficient.     

Anomalous diffusion of major histocompatibility complex class I molecules on HeLa cells determined by single particle tracking.

Single-particle tracking (SPT) was used to determine the mobility characteristics of MHC (major histocompatibility complex) class I molecules at the surface of HeLa cells at 22 degrees C and on different time scales. MHC class I was labeled using the Fab fragment of a monoclonal antibody (W6/32), covalently bound to either R-phycoerythrin or fluorescent microspheres, and the particles were tracked using high-sensitivity fluorescence imaging. Analysis of the data for a fixed time interval suggests a reasonable fit to a random diffusion model. The best fit values of the diffusion coefficient D decreased markedly, however, with increasing time interval, demonstrating the existence of anomalous diffusion. Further analysis of the data shows that the diffusion is anomalous over the complete time range investigated, 4-300 s. Fitting the results obtained with the R-phycoerythrin probe to D = D0talpha-1, where Do is a constant and t is the time, gave D0 = (6.7 +/- 4.5) x 10(-11) cm2 s-1 and alpha = 0.49 +/- 0.16. Experiments with fluorescent microspheres were less reproducible and gave slower anomalous diffusion. The R-phycoerythrin probe is considered more reliable for fluorescent SPT because it is small (11 x 8 nm) and monovalent. The type of motion exhibited by the class I molecules will greatly affect their ability to migrate in the plane of the membrane. Anomalous diffusion, in particular, greatly reduces the distance a class I molecule can travel on the time scale of minutes. The present data are discussed in relation to the possible role of diffusion and clustering in T-cell activation.     

Deoxygenation affects fluorescence photobleaching recovery measurements of red cell membrane protein lateral mobility.

We have used the fluorescence photobleaching recovery technique to study the dependence on oxygen tension of the lateral mobility of fluorescently labeled band 3, the phospholipid analogue fluorescein phosphatidylethanolamine, and glycophorins in normal red blood cell membranes. Band 3 protein and sialic acid moieties on glycophorins were labeled specifically with eosin maleimide and fluorescein thiosemicarbazide, respectively. The band 3 diffusion rate increased from 1.7 x 10(-11) cm2 s-1 to 6.0 x 10(-11) cm2 s-1 as oxygen tension was decreased from 156 to 2 torr, and a further increase to 17 x 10(-11) cm2 s-1 occurred as oxygen tension was decreased from 2 to 0 torr. The fractional mobility of band 3 decreased from 58 to 32% as oxygen tension was decreased from 156 to 0 torr. The phospholipid diffusion coefficient remained constant as oxygen tension was decreased from 156 to 20 torr, but increased from 2.3 x 10(-9) cm2 s-1 to 7.1 x 10(-9) cm2 s-1 as oxygen tension was decreased from 20 to 0 torr. Neither the diffusion coefficient nor the fractional mobility of glycophorins changed significantly at low oxygen tension. Under non-bleaching excitation conditions, intensities of fluorescence emission were identical for oxygenated and deoxygenated eosin-labeled RBCs. Deoxygenated eosin-labeled RBCs required 160-fold greater laser intensities than did oxygenated RBCs to achieve comparable extents of photobleaching, however. Oxygen seems to act as a facilitator of fluorophore photobleaching and may thereby protect the fluorescently labeled red cell membrane from photodamage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diffusion of a fluorescent probe in egg lecithin multilayers and the effect of a permanent magnetic field

A method is developed for determining the value of transverse diffusion coefficient through lipid multilayers. Using the absorption kinetics parameters of 1-aniline-8-sulphonate naphthalene (ANS) the value of diffusion coefficient was estimated as Dt = (3 +/- 1). X 10(-12) cm2 s-1 at (22 +/- 0.5) degrees C. The value of Dt was shown to increase under the action of the magnetic field parallel to the multilayer surface; a relative increase of Dt at field 1.3 T is +(40 +/- 15)%.     

Diffusion of lysozyme chloride in water and aqueous potassium chloride solutions.

The diffusion of lysozyme chloride in aqueous solution has been studied at 25 degrees C using the Goüy interferometric technique. The concentration dependence of the diffusion coefficient in water has been measured over the concentration range 1.1599-9.1556 gcm-3 and the results suggest a value of D 25, w at infinite dilution of 5.838 x 10(-6) cm2s-1. The variation in diffusion coefficient with ionic strength has also been considered by following the diffusion of 0.45% lysozyme chloride in a series of potassium chloride solutions. The value of D in 0.15 M KCl has been found to be approximately one quarter of that in water alone an the diffusion coefficient has been shown to increase markedly as the KCl concentration is reduced below 0.05 M. Interpretation of these observations involves consideration of solution electrostatic effects.     

Measurement of mass transport and reaction parameters in bulk solution using photobleaching. Reaction limited binding regime.

Fluorescence recovery after photobleaching (FRAP) has been used previously to investigate the kinetics of binding to biological surfaces. The present study adapts and further develops this technique for the quantification of mass transport and reaction parameters in bulk media. The technique's ability to obtain the bulk diffusion coefficient, concentration of binding sites, and equilibrium binding constant for ligand/receptor interactions in the reaction limited binding regime is assessed using the B72.3/TAG-72 monoclonal antibody/tumor associated antigen interaction as a model in vitro system. Measurements were independently verified using fluorometry. The bulk diffusion coefficient, concentration of binding sites and equilibrium binding constant for the system investigated were 6.1 +/- 1.1 x 10(-7) cm2/s, 4.4 +/- 0.6 x 10(-7) M, and 2.5 +/- 1.6 x 10(7) M-1, respectively. Model robustness and the applicability of the technique for in vivo quantification of mass transport and reaction parameters are addressed. With a suitable animal model, it is believed that this technique is capable of quantifying mass transport and reaction parameters in vivo.     

The diffusion coefficient of caffeine through agar gels containing a hyaluronic acid–protein complex. A model system for the study of the permeability of connective tissues

A hyaluronic acid-protein complex was embedded into agar gel. This gel complex resembles in some respects the physiological situation in connective tissue, but still permits precise physicochemical measurements to be made. The diffusion coefficient of caffeine into and from such gels has been measured as a function of both agar and hyaluronate concentration. The value for the diffusion coefficient of caffeine was also measured by using a Gouy type diffusiometer. From both types of measurement the value for D (Fick) for caffeine when extrapolated to zero caffeine and agar concentrations agreed at (6.79+/-0.01)x10(-6)cm(2).s(-1) at 25 degrees C. Although agar concentration had only a small effect on caffeine diffusion, hyaluronic acid caused a large decrease in caffeine diffusion co-efficient. The presence of the hyaluronic acid-protein complex within the gel tended to oppose gel syneresis, a concentration of 1.7mg/ml abolishing the effect and higher concentrations reversing it. The possible physiological implications of these results are discussed.     

Simultaneous diffusion of inositol and mannitol in the rat brain

The diffusion of both inositol and mannitol has been determined simultaneously by the integral bolus method in rat brain. The permeability constant (Kin) of inositol averaged 0.27 +/- 0.02 ml X (100 g)-1 X min-1 or 4 X 10(-7) cm X s-1 at a cerebral capillary surface area of 100 cm2 x g-1. The permeability of mannitol was 0.08 +/- 0.01 ml X (100 g)-1. min-1 or 1 X 10(-7) cm X s-1. Neither glucose nor galactose affected the inositol permeability. Hypoglycemia increased somewhat the Km value for mannitol. The basal ganglia showed an increase Km for both substrates as compared with those obtained for cortex, temporal and parietal tissues.     

Diffusion of water in the endosperm tissue of wheat grains as studied by pulsed field gradient nuclear magnetic resonance.

Pulsed field gradient nuclear magnetic resonance has been used to measure water self-diffusion coefficients in the endosperm tissue of wheat grains as a function of the tissue water content. A model that confines the water molecules to a randomly oriented array of capillaries with both transverse dimension less than 100 nm has been used to fit the data and give a unique diffusion coefficient at each water content. The diffusion rates vary from 1.8 x 10(-10) m2s-1 at the lowest to 1.2 x 10(-9) m2s-1 at the highest moisture content. This variation can be explained in terms of an increase in water film thickness from approximately 0.5 to approximately 2.5 nm over the moisture range investigated (200-360 mg g-1).     

Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples

We present a truly quantitative fluorescence recovery after photobleaching (FRAP) model for use with the confocal laser scanning microscope based on the photobleaching of a long line segment. The line FRAP method is developed to complement the disk FRAP method reported before. Although being more subject to the influence of noise, the line FRAP model has the advantage of a smaller bleach region, thus allowing for faster and more localized measurements of the diffusion coefficient and mobile fraction. The line FRAP model is also very well suited to examine directly the influence of the bleaching power on the effective bleaching resolution. We present the outline of the mathematical derivation, leading to a final analytical expression to calculate the fluorescence recovery. We examine the influence of the confocal aperture and the bleaching power on the measured diffusion coefficient to find the optimal experimental conditions for the line FRAP method. This will be done for R-phycoerythrin and FITC-dextrans of various molecular weights. The ability of the line FRAP method to measure correctly absolute diffusion coefficients in three-dimensional samples will be evaluated as well. Finally we show the application of the method to the simultaneous measurement of free green fluorescent protein diffusion in the cytoplasm and nucleus of living A549 cells.     

Lateral diffusion of wheat germ agglutinin-labeled glycoconjugates in the membrane of differentiating HL-60 and U-937 cells assessed with fluorescence recovery after photobleaching (FRAP)

The promyelocytic leukemia cell line HL-60 and the histiocytic cell line U-937 were grown in suspension culture. They were induced to differentiate during 5-d cultivation in the presence of dimethylsulfoxide (DMSO; 1.3% w/v) or phorbol-12-myristate-acetate (PMA; 10(-7) M), which yields granulocyte- and macrophage-like cells, respectively. Differentiation was evidenced by increased capacity to recognize and phagocytize IgG- or complement-coated yeast particles. Aliquots taken from the cultures with and without DMSO (or PMA) were spun down directly on glass microscope slides, washed, labeled with fluoresceinated wheat germ agglutinin (WGA), and directly examined at room temperature for the rate of fluorescence recovery after photobleaching (FRAP). It was found that cultivation of the HL-60 and the U-937 cells in the presence of DMSO, which yields granulocyte-like cells, reduced the average value of lateral diffusion coefficient D (X 10(10] from 1.72 +/- 0.13 cm2s-1 to 0.97 +/- 0.13 cm2s-1 and from 1.77 +/- 0.11 cm2s-1 to 0.82 +/- 0.13 cm2s-1, respectively. U-937 cells grown with PMA also showed a reduction of D(X 10(10] to 0.88 +/- 0.10 cm2s-1. There was a larger immobile fraction of fluorescence in the HL-60 cells than in the U-937 cells, viz., 70-80% compared to 10-50%. The total number of binding sites for WGA was not altered, but the surface density changed, since the HL-60 and the U-937 cells became smaller and larger, respectively, when grown in the presence of DMSO. It is concluded that differentiation reduces the average lateral mobility of the WGA-binding membrane component by a factor around 2.