Microviscosity and enzyme-ligand dissociation

The effect of viscosity on the rate constant for the dissociation of an enzyme-ligand complex has been calculated from a microscopic theory. Using the Kubo representation for the rate constant and the second quantization formalism a Stokes-Einstein-Debye expression is obtained. A microscopic expression for the viscosity is derived. The equation for the microviscosity shows how important information on the enzyme-ligand complex can be obtained from studies on the viscosity dependence of the dissociation rate constant.     

Bose-Einstein condensation in biological systems

A theoretical model is presented for describing a previously untreated effect of viscosity on the apparent decomposition rate of enzyme-ligand complexes.Since the translational diffusion is hindered by the viscosity, its increased value results in an enlarged portion of ligands which can be rebound by the enzyme immediately after the dissociation of the complex.The model accounts for the experimentally observed decrease in maximal velocity of enzymic reactions at high viscosity. At the same time, it serves as a tool to obtain new information about the energetic processes of enzyme action.     

Protein fluctuation and enzyme activity

The possible participation of protein fluctuation in enzyme activity is considered in this paper. Fluctuation is defined as vibrations, involving collective motion of a large number of atoms in protein molecules. Kinetic and thermodynamic aspects of protein fluctuation and enzyme-ligand interactions are discussed, paying special attention to the influence of electric fields and environmental microviscosity. A brief analysis of available experimental and theoretical data strongly suggests an interrelationship between protein fluctuation and enzyme function.     

Comparison between internal microviscosity of low-density erythrocytes and the microviscosity of hemoglobin solutions: an electron paramagnetic resonance study.

The hypothesis that the internal viscosity of erythrocytes is governed by the intracellular hemoglobin (Hb) concentration is examined. Here viscosity is determined by labeling of the cytoplasmic reduced glutathione with the spin label maleimido-Tempo. Erythrocyte populations with different Hb concentrations in isosmotic conditions were obtained through incomplete lysis, followed by cell resealing, and discontinuous density gradient separation. This procedure maintains normal cell shape and volume. Microviscosity of membrane-free Hb solutions was measured by addition of spin labeled glutathione. It was found that microviscosity values are similar for the erythrocyte cytoplasm and for Hb solutions of equivalent concentrations, showing that the erythrocyte membrane does not have any influence on internal microviscosity. The dependence of the microviscosity on the concentration of Hb solutions was compared with results of macroscopic viscosity obtained by other authors. It is concluded that microviscosity is sensitive to individual properties of the Hb molecule (intrinsic viscosity), but that it is not sensitive to intermolecular interactions. As the microviscosity behavior as a function of Hb concentration is the same in Hb solutions as in the erythrocyte cytoplasm, the inferences regarding macroscopic viscosity in Hb solutions could be translated to the rheological properties of the erythrocyte cytoplasm. Thus, these properties could be predicted from the values of the mean corpuscular Hb concentration.     

Membrane-potential-dependent changes of the lipid microviscosity of mitochondria and phospholipid vesicles.

The effects of a transmembrane potential difference upon the lipid microviscosity of cytochrome oxidase vesicles (COVs) and rat liver mitochondria (RLM) were investigated. COVs and RLM were labelled with the fluorescent probe 1,6-diphenylhexa-1,3,5-triene (DPH). The fluorescence polarization of the probe was then measured when potentials of different magnitudes were induced across the membranes of these particles. It was shown that the absolute value of the microviscosity changes to quite a significant extent, owing to the imposition of large membrane potentials. On relaxation of the membrane potential the lipid microviscosity was also shown to return to the value before the induction of the potential. The largest change in lipid microviscosity was observed when coupled respiration was initiated. This occurred in both the COV system and the RLM system. The absolute value of the lipid microviscosity was shown to change by as much as 22% with the induction of membrane potentials, owing to respiration. To confirm the viscosity measurements made with DPH, lipid microviscosity was also measured with the spin-labelled fatty acid 5-doxyl stearate. Measurements of the order parameters indicated that, in agreement with the results of fluorescence experiments, viscosity changes occurred that were due to the induction of a membrane potential. The significance of these findings to the regulation of metabolism is briefly discussed, the main conclusion being that, although there is certainly a significant variation of lipid microviscosity with electric field, mechanistic interpretations will require further studies.     

Gel--sol transition in kappa-carrageenan systems: microviscosity of hydrophobic microdomains, dynamic rheology and molecular conformation.

The effect of gel-sol transition in kappa-carrageenan systems on the microviscosity of hydrophobic microdomains, as well as its relation to macroscopic rheology and molecular conformation, was studied in kappa-carrageenan systems. The microdomains were probed by 1,3-di(-1-pyrenyl)propane (P3P) for which the excimer intensity (Ie) provides relative measures of the microviscosity in the immediate probe surroundings. In particular the applicability of P3P to monitor the gel--sol transition was proved, the results showing a dramatic decrease in microviscosity in the vicinity of the transition point. The corresponding changes in rheological properties and carrageenan conformation were investigated by dynamic viscometry (DV) and optical rotation (OR), respectively. The temperature of onset of the transition as indicated by the microviscosity data (T0) was found to correlate well with the OR and DV-results. The application of microviscosity and OR-measurements allowed an estimation of the helical content at T0 to be determined. P3P-data indicate a microenvironment viscosity for the probe sites in the kappa-carrageenan system comparable to that found in SDS micelles.     

Altered cell-averaged microviscosity of murine peritoneal macrophages undergoing activation in vivo or in vitro

The cell-averaged microviscosity of intact murine peritoneal mononuclear phagocytes in various stages of activation was assessed by quantifying fluorescent depolarization of 1,6-diphenyl-1,3,5-hexatriene. Macrophages activated in vivo with Mycobacterium bovis, strain BCG, were significantly more fluid than resident peritoneal macrophages, responsive macrophages elicited with thioglycollate broth, proteose peptone broth, or fetal bovine serum, or primed macrophages elicited with pyran copolymer, MVE-2. Specifically, the cell-averaged microviscosity decreased from a mean of 3.47 +/- .07 eta 25 degrees C (poise) (range of 3.32 to 3.67 p) to 2.62 eta 25 degrees C. Exposure of responsive macrophages in vitro to bacterial endotoxin plus hybridoma supernatants containing macrophage-activating factor or purified recombinant interferon gamma resulted in decreased microviscosity; the largest effect was seen after 24 hr. Macrophages primed in vivo with MVE-2 and treated in vitro with endotoxin also developed decreased microviscosity. Similar changes in microviscosity were observed in a plasma membrane-enriched fraction isolated from macrophages activated in vitro with interferon gamma and endotoxin, thus suggesting that the cell-averaged measurements reflected changes in membrane viscosity. The optimum concentration of MAF-inducing decreased overall microviscosity was identical to that for inducing tumoricidal capacity. Taken together, the data indicate activation of lytic capacity in murine macrophages is closely associated with decreased cell-averaged microviscosity and that this change reflects, at least in part, decreased microviscosity of the plasma membrane of these cells.     

The influence of oxidative stress on microviscosity of hemoglobin-containing liposomes

Encapsulation of hemoglobin (Hb) within a liposome is one of the strategies in the development of artificial oxygen carriers. In this study the effects of oxygen radical generating system (xantine/xantine oxidase) on the internal microviscosity and protein degradation of hemoglobin-containing liposomes ('hemosomes') prepared from dipalmitoylphosphatidylcholine (DPPC) and different amounts of cholesterol (Ch) (0-0.5 mol/mol) were investigated. The results demonstrated a direct relationship between increasing oxidant stress and microviscosity of Hb vesicles and also showed clearly that the increase in internal viscosity was caused mainly by globin degradation. It was shown that the higher content of Ch, the lower Hb degradation and smaller increase in internal viscosity were observed. The significant protection effect against oxygen radicals was observed only for liposomes with the addition of 0.3 mol/mol or more of Ch. It seems that Ch concentration in liposomes is of prime importance for stabilizing of Hb in 'hemosomes'.     

The fluorescence studies of the sol-gel transition by styrylpyridine derivative

The gelation process of tetraethylorthosilanes in acid environment was monitored with the trans-4-(p-N,N-dimethylaminostyryl)-N-vinylbenzylopyridinium chloride (vbDMASP) fluorescent probe. The fluorescence steady-state and anisotropy measurements of material during sol-gel transition are reported. The results are compared with fluorescence studies of the probe in a modeled viscous system of water-glycerol mixtures. A strong increase of anisotropy, from 0.1 to 0.9, with gelation time as well with wavelength, was observed. Although the increase of anisotropy with wavelength is due to specificity of the compounds exhibiting charge transfer properties, the increase of the anisotropy with gelation time is due to an increase of microviscosity. On this basis, suitability of the applied fluorophore in recording of viscosity changes during sol-gel transition is discussed. The molecular structure of vbDMASP in the excited states in dependence on environmental polarity was optimized using the HyperChem and Amsol program. The dynamics of torsional angle C35-C34-N31-C28 of the multichromophore dye in correlation with micropolarity and microviscosity of the network formation during the sol-gel transition is discussed.     

A new spin label method for the measurement of erythrocyte internal microviscosity

A new spin-label method for the measurement of the internal microviscosity of erythrocyte is presented. The spin label used is 2,2',5,5'-tetramethyl-3-maleimidopyrrolidinyl-N-oxyl (MAL-5) which penetrates inside the red blood cell and binds covalently on cytoplasmic glutathione. After washing off the external label, 98% of the electron paramagnetic signal is due to the labelled glutathione. This signal allows one to measure the rotational correlation time of the label. A calibration curve established with spin-labelled glutathione in sucrose solutions of increasing viscosity is used to convert the measured rotation times into viscosity units. This method avoids the use of unphysiological salts like potassium ferricyanide, and permits the study of red blood cells in various suspension media. In normal human subjects, the mean value of microviscosity is 4.45 +/- 0.16 mPa . s at 20 degrees C in isotonic saline (25 subjects) and 6 +/- 0.25 mPa . s in plasma. The variations of microviscosity as a function of the osmolarity of the medium are explained according to a theoretical model taking into account the variations of the red blood cell volume and the viscometric properties of haemoglobin.     

Microviscosity of mucosal cellular membranes in toad urinary bladder: Relation to antidiuretic hormone action on water permeability

Summary The microviscosity of cellular membranes (or membrane fluidity) was measured in suspensions of single mucosal cells isolated from the urinary bladder of the toad,Bufo marinus, by the technique of polarized fluorescence emission spectroscopy utilizing the hydrophobic fluorescent probe, perylene. At 23°C, 5mm dibutyryl cyclic 3,5-AMP decreased the apparent microviscosity of the cell membranes from 3.31 to 3.07 P, a minimum decrease of 7.3% (P<0.001) with a physiological time course. Direct visualization of the cell suspension indicated that 98% of the cells were viable, as indicated by Trypan Blue dye exclusion. The fluorescent perylene could be seen only in plasma membranes, suggesting that the measured viscosity was that of plasma membrane with little contribution from the membranes of cellular organelles. Addition of antidiuretic hormone to intact hemibladders stained with perylene produced changes in fluorescence consistent with a similar 7% decrease in apparent microviscosity with a physiological time course. However, finite interpretation of the findings in intact tissue cannot be made because the location and the fluorescent lifetime of the probe could only be conducted on the isolated cells. Comparison with previously determined relationships between water permeability and microviscosity in artificial bilayers suggests that the 7% (a lower limit) decrease in microviscosity would produce only a 6.5% increase in water permeability.     

Effects of native and oxidized apolipoprotein A-I on lipid bilayer microviscosity of erythrocyte plasma membrane

Using a pyrene as a fluorescent probe, we investigated the influence of native and oxidized apolipoprotein A-I (apo A-I) and their complexes with tetrahydrocortisol (THC) on the microviscosity of the erythrocyte plasma membrane. The addition of THC to isolated membranes led to a 17% increase in the membrane microviscosity. In contrast, native apo A-I reduced the microviscosity (i.e., increased the fluidity) of the membranes by 15%. A more pronounced increase (by 25%) in the membrane fluidity was found in the presence of the complex of apo A-I with THC. Unlike native apo A-I, oxidized apo A-I and its complex with THC did not change the membrane viscosity. In view of the fact that apo A-I plays an important role in the binding of membrane cholesterol we suggest that the observed increase in the membrane fluidity under the influence of the native apo A-I is associated with the cholesterol efflux from plasma membrane. Oxidative modification of apo A-I likely disturbs the mechanisms of the cholesterol efflux and prevents the decrease in the membrane microviscosity.     

A method for measuring membrane microviscosity using pyrene excimer formation. Application to human erythrocyte ghosts.

In order to determine the microviscosity of human erythrocyte membrane suspensions, a method has been developed which is based on pyrene excimer formation. First, measurements of partitioning of pyrene into membranes, in conjunction with known values for the volume of the lipid compartment of erythrocyte ghosts are used to determine the concentration of pyrene in the membrane lipid. Secondly, reported measurements of the diffusion constants of aromatic hydrocarbons similar in structure to pyrene, are used to derive an empirical equation relating solvent viscosity and the diffusion constant of pyrene. Then, measurements of pyrene excimer formation in a series of solvents ranging up to several poise in viscosity are used to determine that the interaction diameter of the excimer formation reaction is 3 +/- 1 A. Finally all these data are brought together in order to conclude that the viscosity of the lipid in the human erythrocyte ghost is 8.0, 4.0 and 1.6 P at 10, 25 and 40 degrees C, respectively.     

Fluidity of natural membranes and phosphatidylserine and ganglioside dispersions. Effect of local anesthetics, cholesterol and protein.

The microviscosity of artificial lipid membranes and natural membranes was measured by the fluorescence polarization technique employing perylene as the probe. Lipid dispersions composed of brain gangliosides exhibited greater microviscosity than phosphatidylserine (268 cP vs 173 cP, at 25 degrees C). Incorporation of cholesterol (30-50%) increased the microviscosity of lipid phases by 200-500 cP. Cholesterol's effect on membrane fluidity was completely reversed by digitonin but not by amphotericin B. Incorporation of membrane proteins into lipid vesicles gave varying results. Cytochrome b5 did not alter membrane fluidity. However, myelin proteolipid produced an apparent increase in microviscosity, but this effect might be due to partitioning of perylene between lipid and protein binding sites since tha latter have a higher fluorescence anisotropy than the lipid. The local anesthetics tetracain and butacaine increased the fluidity of lipid dispersions, natural membranes and intact ascites tumor cell membranes. The effect of anesthetics appears to be due to an increased disordering of lipid structure. The fluidity of natural membranes at 25 degrees C varied as follows: polymorphonuclear leukocytes, 335 cP; bovine brain myelin, 270 cP; human erythrocyte, 180 cP; rat liver microsomes, 95 cP; rat liver mitochondria, 90 cP. In most cases the microviscosity of natural membranes reflects their cholesterol: phospholipid ratio. The natural variations in fluidity of cellular membranes probably reflect important functional requirements. Similarly, the effects of some drugs which alter membrane permeability may be the result of their effects on membrane fluidity.     

The effect of some alkyl derivatives of cholesterol on the permeability properties and microviscosities of model membranes

The properties of lecithin liposomes or vesicles containing a variety of sterols have been studied by measuring either the release of entrapped glucose or determining microviscosity by fluorescence depolarization of the probe diphenylhexatriene. Sterols containing alkyl substituents at C₃, C₄, or C₁₄ were less effective in reducing glucose permeability or increasing microviscosity than cholesterol. 19-Norcholesterol was also less effective than cholesterol in raising membrane viscosity. These results support the hypothesis that the selective biological demethylation of lanosterol to a planar (α-face) structure optimizes the ability of the sterol molecule to condense the lipid phase of the membrane bilayer. Removal of an angular methyl group (C₁₉), a rare event in biological systems, has the opposite effect.     

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.^1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.^4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.^6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the Förster Hoffman equation.⁹ This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.^6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.     

Quantification of lipid bilayer effective microviscosity and fluidity effect induced by propofol

Electron spin resonance (ESR) spectroscopy with nitroxide spin probes was used as a method to probe the liposome microenvironments. The effective microviscosities have been determined from the calibration of the ESR spectra of the probes in solvent mixtures of known viscosities. In the first time, by measuring ESR order parameter (S) and correlation time (tau(c)) of stearic spin probes, we have been able to quantify the value of effective microviscosity at different depths inside the liposome membrane. At room temperature, local microviscosities measured in dimyristoyl-l-alpha phosphatidylcholine (DMPC) liposome membrane at the different depths of 7.8, 16.95, and 27.7 A were 222.53, 64.09, and 62.56 cP, respectively. In the gel state (10 degrees C), those microviscosity values increased to 472.56, 370.61, and 243.37 cP. In a second time, we have applied this technique to determine the modifications in membrane microviscosity induced by 2,6-diisopropyl phenol (propofol; PPF), an anaesthetic agent extensively used in clinical practice. Propofol is characterized by a unique phenolic structure, absent in the other conventional anaesthetics. Indeed, given its lipophilic property, propofol is presumed to penetrate into and interact with membrane lipids and hence to induce changes in membrane fluidity. Incorporation of propofol into dimyristoyl-l-alpha phosphatidylcholine liposomes above the phase-transition temperature (23.9 degrees C) did not change microviscosity. At 10 degrees C, an increase of propofol concentration from 0 to 1.0 x 10(-2) M for a constant lipid concentration mainly induced a decrease in microviscosity. This fluidity effect of propofol has been qualitatively confirmed using merocyanine 540 (MC540) as lipid packing probe. Above 10(-2) M propofol, no further decrease in microviscosity was observed, and the microviscosity at the studied depths (7.8, 16.95, and 27.7 A) amounted 260.21, 123.87, and 102.27 cP, respectively. The concentration 10(-2) M was identified as the saturation limit of propofol in dimyristoyl-l-alpha phosphatidylcholine liposomes.     

Fluorescence depolarization studies and phase transition in human apoprotein . phospholipid complexes.

The microviscosity of unilamellar vesicles of dimyristoyl-3-sn-phosphatidylcholine and that of phosphatidylcholine . apoprotein complexes was followed by fluorescence depolarization after labeling with 1,6-diphenyl-1,3,5-hexatriene. The transition temperature from gel-crystalline to liquid-crystalline phase in 24 degrees C for the dimyristoyl-phosphatidylcholine vesicles and is shifted to around 30 degrees C in the complexes between phosphatidylcholine and apoA-I, apoA-II, apoC-I, apoC-III proteins while the cooperativity of the transition is decreased. At temperatures below the transition of the phospholipid, the microviscosity of the complexes of phosphatidylcholine with apoA-I, apoA-II and apoC-I proteins is lower than that of the phosphatidylcholine, while the opposite effect is observed above 30 degrees C. The phosphatidylcholine . apoprotein complexes isolated on a Sepharose 6B column have a molecular weight around 100 000 and a phosphatidylcholine/apoprotein ratio of 2--2.6 (w/w). The microviscosity measurments at 35 degrees C performed after elution of the column enable the complex to be detected. The size and microviscosity of the apoprotein . phosphatidylcholine complex is compatible with a model where the vesicular structure has disappeared and the amino acid side chains present hydrophobic interaction with the phosphatidylcholine acyl chains.     

Effect of nitric oxide on viscosity of nerve cell membranes

The influence of nitric oxide on the microviscosity of nerve cell membranes was investigated by resonance Raman (RR) spectroscopy. Changes in membrane viscosity were estimated from the resonance Raman-spectra of carotenoids localized in the axon plasmatic membrane and membranes of subcellular vesicles (cytosomes). For the nerve fibre, the extracellular addition of nitric oxide donor, sodium nitroprusside (0.5 mM), caused an increase in the 1526 cm(-1) band relative half-width and the modification of 1160 cm-1 band structure. Moreover, sodium nitroprusside led to an increase in the I1526/I1160 ratio by 13% in 25 min and a decrease in this ratio by 10% in 50 min. In the case of cytosomes, sodium nitroprusside (0.5 mM) resulted in the reduction of the I1526/I1160 ratio by 8% in 25 and 50 min. It was shown that the neuron rhythmic activity correlated with the I1526/I1160 ratio and cytosome membrane microviscosity. We suppose that nitric oxide causes a conformational transition of carotenoids in the axon plasmatic membrane and the membranes of cytosomes. This process can be due to nitric oxide-induced changes of the membrane microviscosity or potential.     

Function of the membrane lipids of the heart sarcoplasmic network in izadrin myocarditis studied by using a pyrene probe

The fluorescent hydrophobic pyrene probe was employed to study the viscosity of membrane lipids of rat heart sarcoplasmic reticulum in isoproterenol myocarditis. During pyrene incorporation into the reticulum obtained from the affected myocardium, the increase in the microviscosity occurred at lower temperatures and more rapidly both in "bound" and "free" membrane lipids as compared with normal. The increase of the viscosity of the reticulum membranes in isoproterenol myocarditis was accompanied by a lowering of the activity of Ca, Mg-ATPase of the sarcoplasmic reticulum coupled with an elevation of the content of lipid peroxidation products.