Fluorescence Depolarization Technique Research Articles

Time-resolved spectroscopy of macromolecules: Effect of helical structure on the torsional dynamics of DNA and RNA

The torsional rigidity of DNA and RNA is measured via the fluorescence depolarization technique.(AIP)

Direct observation of the torsional dynamics of DNA and RNA by picosecond spectroscopy.

Picosecond time-dependent fluorescence depolarization techniques have been used to monitor the reorientation of ethidium bromide intercalated in DNA and RNA. The fluorescence polarization anisotropy reveals a nonexponential, exp(-at 1/2), torsional relaxation of the DNA double helix and provides an accurate value for its torsional rigidity, C = 1.3 +/- 0.2 X 10(-19) erg cm. Furthermore, from accurate measurements of the limiting anisotropy at zero time, we conclude that there is an additional fast (< 10 psec) internal motion that depends on the viscosity of the medium. Denatured DNA is considerably more flexible than the intact double helix, thus demonstrating the influence of secondary structure on internal motions.

Assessment of fetal lung maturity by a microviscosimeter.

A new method of rapid antenatal assessment of fetal lung maturity was evaluated in relation to the newborn outcome and two other accepted test. This method is based on fluorescence depolarization (FD) technique. The special instrumentation required for this method (the Microviscosimeter) was found to be simple and easy to handle even to nonprofessional personnel. Analysis of 47 samples of amniotic fluid received within 48 hours of delivery demonstrated that lung maturity threshold may be related to a numeric value (P value) measured by this technique. With a P value of less than 0.320 respiratory distress syndrome (RDS) is unlikely to develop. With a P value greater than 0.340, chances for RDS, usually severe, are high. With a P value of less than 0.340 but greater than 0.320, RDS may or may not develop. This method did not prove to be more reliable then the determination of L/S ratio by thin layer chromatography, but its advantage is that it supplies the results in less then an hour. The FD technique proved to be more reliable then the commonly used foam stability test.

Hydrocarbon phase transitions and lipid-protein interactions in the erythrocyte membrane. A 31P NMR and fluorescence study

1. 1. 31P NMR and other techniques have been employed to study various derivatives of the human erythrocyte ghost membrane, derived liposomes and liposomes composed of mixtures of synthetic phosphatidylcholines. It is shown that the previously reported (Cullis, P.R. (1976) FEBS Lett. 68, 173–176) phospholipid phase transition observed in ether extracted ghosts is also observable employing fluorescence depolarization techniques. This phase transition can be (reversibly) removed by re-addition of cholesterol. 2. 2. A new semi-biological model membrane system is described which may be obtained by proteolytic digestion of peripheral membrane protein and a subsequent ether extraction. Freeze-etch studies and other considerations indicate that these “pronase digested ether extracted ghosts” consist of segments of integral membrane protein surrounded by one or two boundary layers of lipid. 3. 3. Within the terms of the “fluid mosaic” model of biological membranes lipids may exist in at least four different environments. These include unperturbed bilayer regions, bilayer regions shielded by peripheral membrane protein, lipid experiencing strong polar interactions with membrane protein (bound lipid), and lipid associated with integral membrane protein (boundary lipid). The results obtained here are consistent with the following general conclusions. 3.1. (a). The ether extraction procedure removes most, if not all, of the unperturbed bilayer lipid. 3.2. (b). The amount of membrane phospholipid “bound” to membrane protein so as to cause immobilization in the phosphate group region (on the time scale of 10 −5 s) is at most 3% of the membrane phospholipid. 3.3. (c). Phospholipids in all three of the major classes of lipid (unperturbed bilayer, shielded bilayer and boundary lipid) exhibit 31P NMR spectra consistent with bilayer structure. 3.4. (d). The motion (on the time scale of 10 −5 s) in the phosphate group region of membrane phospholipids is not sensitive to the presence of peripheral membrane protein. 3.5. (e). The phase transition behaviour and 31P NMR lineshapes observed suggest that in the ether extracted ghost system phospholipids in bilayer regions (possibly consisting largely of sphingomyelin) enter the gel state below 20°C. Below 20°C “boundary” phospholipids also experience restricted motion. Above 20°C the results would be consistent with a model whereby boundary phospholipids experience fast exchange with lipids in liquid crystalline bilayer regions.

Changes in membrane microviscosity associated with phagocytosis: effects of colchicine.

The effects of phagocytosis on plasma membrane microviscosity were studied by fluorescence depolarization techniques. It was shown that lipophilic probes are accumulated in intracellular vesicles to a significant degree in fibroblasts and neutrophils. Microviscosity was thus determined from the behavior of probes in isolated membranes. Phagocytosis of oil emulsions or polystyrene beads by rabbit polymorphonuclear leukocytes induces a marked decrease in plasma membrane microviscosity that parallels the extent of phagocytosis. Liposomes made from extracts of membrane lipid show qualitatively the same changes, indicating that the alteration of microviscosity results at least in part from changes in lipid composition. The decrease in microviscosity is abolished when colchicine is present during phagocytosis. Addition of colchicine to membranes previously isolated from control or phagocytic cells has no effect on their microviscosity. The results suggest that phagocytosis is accompanied by a microtubule-dependent reorganization of membrane lipids.

Fluorescence polarization of .ALPHA.s1, .KAPPA.-caseins and .ALPHA.s1-.KAPPA.-casein complex.

Conjugates of αs1, κ-caseins and αs1-κ-casein complex were prepared with dimethyl-aminonaphthalenesulfonate and pyrenebutyrate. Their fluorescence lifetimes and the rotational relaxation times were measured by single photon counting technique and fluorescence depolarization technique, respectively. Both dimethylaminonaphthalenesul-fonate and pyrenebutyrate conjugates had more than two lifetimes and the longer lifetime of pyrenebutyrate conjugates was near 140 nsec. The rotational relaxation time of pyrenebutyrate αs1-κ-casein complex was smaller than that of pyrenebutyrate κ-casein polymer, which suggested that the complex formation of αs1- and κ-casein polymers led to dissociation of the κ-casein polymer. Changes of the rotational relaxation time as a function of weight ratio of αs1- and κ-casein polymers (αs1/κ) showed the specific variation and it was suggested that 4 moles of αs1-κ-casein complex were formed from one mole of κ-casein polymer.

Application of Fluorescence Depolarization Technique to Studies of Biopolymers, especially proteins

Application of Fluorescence Depolarization Technique to Studies of Biopolymers, especially proteins

Effect of the cation on micelle formation by sulfonates in benzene

The states of aggregation of dinonylnaphthalene sulfonates of ten cations (Li, Na, Cs, NH 4, Mg, Ca, Ba, Zn, Al, and H) have been studied in benzene by fluorescence depolarization, cryoscopy, viscometry, and densimetry. The micelles observed by fluorescence depolarization contained 9 to 14 acid residues each. Their aggregation number was usually independent of concentration and almost independent of the water content of the system, although moisture moderately increased the aggregation of the zinc salt. The relative insensitivity of the sulfonate micelle size to influences of the cation and moisture contrasts sharply with the behavior of the phenyl-stearate soaps, whose aggregation depends critically upon these variables. This indicates that the size of the dinonylnaphthalene sulfonate micelles depends primarily on the geometry of the acid residue. However, the apparent volume of the anion was inversely related to the coordinating tendency of the cation, suggesting that coordination forces are a factor in micelle stability. The acid is associated principally to the dimer, but in the presence of water some larger aggregates are formed. Viscosities of the sulfonate solutions exceeded those predicted by the Einstein relation for spherical particles. This anomaly corresponded to an asymmetry of the order of 2, or a solvation of 10% to 20%. It might also have resulted from surface irregularities of the micelles. Present evidence does not justify a choice among these explanations. Cesium dinonylnaphthalene sulfonate was shown cryoscopically to have an aggregation number of 6 or more. It differed from the other micelle-forming sulfonates, however, in not solubilizing enough Rhodamine B to permit determination of micelle size by the fluorescence depolarization technique. The micelle size found cryoscopically for the magnesium salt agreed with that derived from fluorescence depolarization measurements.