Edward Blatt

THE TRANSVERSE LOCATION OF FLUOROPHORES IN LIPID BILAYERS AND MICELLES AS DETERMINED BY FLUORESCENCE QUENCHING TECHNIQUES

Abstract— A fluorescence quenching technique has been used to determine the absolute partition coefficients of a set of n‐doxyl stearates which partition between the aqueous and lipid phases of a phospholipid dispersion, and it is shown that the values of the coefficients vary in a systematic way depending on the position of the doxyl group on the acyl chain of the fatty acid. The n‐doxyl stearates quench the fluorescence of n‐(9‐anthroyloxy) fatty acids in lipid bilayers, and consideration of the absolute partition coefficients of the quencher and of the microviscosity of the bilayer is necessary to extract information about the proximity of the fluorophore and quencher. In addition, quenching of the fluorescent fatty acids with a quencher which is small relative to the width of the lipid bilayer allows determination of both the absolute and local partition coefficients, the latter referring to a subvolume of the bilayer within which quenching occurs. The relationship between absolute and local partition coefficients is defined for micelles and bilayers and a method for determining the relative transverse positions of the anthroyloxy rings in these structures is described.

Partition and binding constants in micelles and vesicles from fluorescence quenching data

Abstract A method of analysing steady-state fluorescence quenching in compartmentalized systems is proposed which allows the evaluation of partition and binding constants where both association processes occur simultaneosly. The number of binding sites is shown to be dependent on the nature of the quencher.

The association of acrylamide with proteins. The interpretation of fluorescence quenching experiments

The association properties of acrylamide with a number of proteins in aqueous solution have been investigated by a fluorescence-quenching method previously used in micelles and lipid bilayers (Blatt, E., Chatelier, R.C. and Sawyer, W.H. (1984) Chem. Phys. Lett. 108, 397-400). At pH 7.0, acrylamide partitions between the bulk aqueous phase and the proteins, human serum albumin, monellin and ovalbumin. Comparison with an earlier method of analysis (Sikaris, K.A., Thulborn, K.A. and Sawyer, W.H. (1981) Chem. Phys. Lipids 29, 23-36) confirms the data quantitatively. For human serum albumin at pH 2.2, acrylamide associates according to both partition and binding processes. Equilibrium dialysis experiments performed for the latter system verify that acrylamide associates with proteins.

Fluorescence anisotropy studies of n-(9-anthroyloxy) fatty acids in Triton X-100 micelles

Abstract The molecular motion of a set of n -(9-anthroyloxy) fatty acids ( n = 2, 6, 9, 12) solubilized within the non-ionic micelle Triton X-100 has been investigated using fluorescence anisotropy measurements. The anthroyloxy fluorophores occupy a graded series of positions within the micelle and rotational relaxation time data indicate a uniform microviscosity of approximately 215 cP.

A novel means of investigating the polarity gradient in the micelle sodium lauryl sulphate using a series of n-(9-anthroyloxy) fatty acids as fluorescent probes

The fluorescence behaviour of a series of n-(9-anthroyloxy) fatty acids has been studied in saturated hydrocarbon solvents of increasing viscosity and solvents of increasing polarity. Fluorescence quenching experiments in these solvents and in micelles of sodium lauryl sulphate show that the probes locate at a graded series of depths in the micelle. The spectral characteristics of emission from the probes indicate an increasing polarity gradient from the core to the surface of the micelle.

Fluorescence quenching in lipid micelles and bilayers: Evaluation of bimolecular rate constants

Abstract Dynamic quenching of fluorophores and quenchers in lipid micelles and bilayers can yield information about the bimolecular rate constant for the quenching reaction, and hence information about the microviscosity of the fluorophore-quencher environment. When the fluorophore and quencher have relatively fixed transverse positions in the bilayer, the analysis of Sikaris et al. (Chem. Phys. Lipids. 29 (1981) 23) can be used to separate the microviscosity and proximity contributions to quenching. We now extend this method to show explicitly the effect of static quenching on the analysis. We show by simulation and experiment that a correction factor must be included when static quenching contributes to the observed quenching efficiency.

Time-Resolved Phosphorescence Anisotropy For Measuring Slow Rotational Diffusion In The Erythrocyte Cytoskeleton

The cytoskeletal architecture of a cell controls many cell processes and characteristics (cell shape, motility, endocytosis, cell division, organelle position and movement). Many of these processes involve the assembly and disassembly of cytoskeletal elements, but the highly cross-linked polymer system must, of necessity, possess flexibility and motional freedom. Components of the erythrocyte cytoskeleton (actin, spectrin and band 4.1) may be reconstituted into a ternary complex which forms a viscous cross-linked gel. It is unlikely that this structure is identical to that existing in vivo, however, it does provide a convenient experimental model system in which the rotational motion of the individual components may be studied. We have examined this system using time-resolved phosphorescence anisotropy which measures rotational diffusion in the microsecond to millisecond time window.