Keith R. Thulborn

Properties and the locations of a set of fluorescent probes sensitive to the fluidity gradient of the lipid bilayer.

The synthesis and properties of a set of four fluorescent probes (n-(9-anthroyloxy) fatty acids, n = 2, 6, 9, 12) sensitive to the fluidity gradient of the lipid bilayer are described. Fluorescent quenching experiments show that the probes locate at a graded series of depths in the bilayer. A fifth probe, methyl-9-anthroate, locates near the bilayer centre. As an example of their application, the probes are used to study the phase transitions of dipalmitoyl phosphatidyl-choline. Changes in the rotational relaxation times of the probes across the transitions are more pronounced at the centre of the bilayer than at the surface.

A microviscosity barrier in the lipid bilayer due to the presence of phospholipids containing unsaturated acyl chains

Abstract A series of fluorescent probes which locate a graded series of depths from the surface to the centre of the lipid bilayer have been used to measure the fluidity gradient in liposomes and natural membranes. In dioleoyl phosphatidylcholine liposomes and in cells which have a high content of unsaturated phospholipids, a region of high microviscosity is detected near the cis double bond/s. The significance of this phenomenon is discussed in terms of the penetration and lateral movement of membrane protein.

Perturbations to lipid bilayers by spectroscopic probes as determined by dielectric measurements.

Dielectric measurements on lecithin/cholesterol bimolecular lipid membranes have indicated that the series of extrinsic fluorescent probe molecules, the n-(9-anthroyloxy) fatty acids, cause significant perturbation to the bilayer structure at concentrations equivalent to those used in fluorescence experiments (0.1 mol%). Perturbations were observed in the capacitance and conductance of the electrically distinct substructural regions of the bilayer that were consistent with the putative location of the probe molecules. Inclusion of stearic acid decreased the thickness of the hydrocarbon region of the membrane, presumably by expanding the average surface area per unit membrane mass, and also significantly disrupted the surface regions. The attachment of the anthroyloxy moiety to position 2 of a fatty acid accentuated both these effects. Attachment at position 12 had the reverse effect by increasing the volume of the hydrocarbon region without further disturbance of the surface organisation. The 9-positioned probe had an intermediate effect. The degree of perturbation by the 2-positioned probe was dependent on the probe concentration within the range (probe:lipid) 1:1000 to 1:10 000. The technique therefore detects perturbation of structure at probe levels which are lower than those commonly used in fluorescence-labelling experiments.

Resolution of partition coefficients in the transverse plane of the lipid bilayer

Abstract The distribution of a small lipid soluble molecule across a lipid bilayer has been determined using fluorescence quenching techniques. The neutral form of the amine, N , N -dimethylaniline (DMA) quenches the fluorescence of a series of n -(9-anthroyloxy) fatty acids ( n = 2,6,9,12,16) which place a fluorophore at a graded series of positions from the surface to the centre of the lipid bilayer. A method is described for determining the partition coefficient of a quencher at each transverse position. The results show that DMA is located at all depths within the bilayer leaflet but that it is concentrated at the bilayer centre and to a lesser extent at the bilayer surface.

13C NMR studies on fluorescent probes: 13C chemical shifts and longitudinal relaxation times of n-hydroxy-fatty (n=2,6,9,12) acids and n-(9-anthroyloxy)-stearic (n=6,12) acids

Abstract 13C NMR has been used to confirm the structure of two fluorescent probes, n-(9-anthroyloxy)-stearic acids (n=6,12), and the series of n-hydroxy-fatty acids (n=2,6,9,12) from which the set of fluorescent fatty acids may be synthesised. 13C longitudinal relaxation times and correlation times of the individual carbon atoms in 12-hydroxy- and 6- and 12-(9-anthroyloxy)-stearic acids show differences in motional properties between these derivatives and the parent stearic acid in chloroform(d) solution. The correlation times of the substituted carbons in 6-, 9-, and 12-hydroxy-stearic acids are longer than the corresponding carbons in stearic acid. The change in correlation times at the substituted carbons reflects the increase in motion along the acyl chain. Attachment of the bulky anthracene ring causes greater restriction of motion at the substituted carbon atom but the gradient of motion along the chain is preserved. These results are discussed in terms of the types of motion which lead to fluorescence depolarization when the fluorescent fatty acids are used as fluidity probes in biomembranes.

Leghemoglobins: Immunochemistry and phylogenetic relationships

Comparative immunological analyses of proteins from different phylogenetic groups of animals and plants have been useful in assessing taxonomic relationships and measuring genetic homologies among species, especially in the absence of amino acid sequence data [1,2]. Among some isofunctional, homologous globular proteins antigenic cross-reactivity is seen if the amino acid sequences differ by less than 40% [3,4], the antigenic effects of substitutions being approximately equal and additive. Previously [5] we have shown that the 5 leghemoglobins isolated from the root nodules of the soybean plant (Glycine max. c.v. Lincoln) completely crossreact immunologically with each other and with the main leghemoglobin from serradella (Ornithopus sativus Brot.). The major leghemoglobin from snake bean (Vigna sinensis L.) only partially cross-reacts with antisera to the soybean leghemoglobin while no cross-reaction at all is seen between the lupin (Lupin luteus) and soybean leghemoglobins. These studies have now been extended to leghemoglobins from clover (Trifolium subterraneum c.v. Woogenellup) and broad bean (Viciafaba c.v. Triple White) using a radioimmunoassay procedure with antisera to soybean and lupin leghemoglobins. A comparison of immunological cross-reactivities between leghemoglobins from plants of different

Leghaemoglobin from Trifolium subterraneum. Purification and characterization.

Leghaemoglobin from the subclover, Trifolium subterraneum cultivar Woogenellup, has been fractionated into at least four electrophoretically distinct components using the ion-exchange chromatographic procedure described by Appleby et al. (Appebly, C.A., Nicola, N.A., Hurrell, J.G.R. and Leach, S.J. (1975) Biochemistry 14, 4444--4450) for soybean leghaemoglobins. Unlike those of soybean, the subclover leghaemoglobins showed no evidence of autoxidation under identical isolation procedures, implying that these proteins have an unusually stable ferrous oxidation state. Circular dichroism in the far-ultraviolet (200--240 nm) indicated a high helicity (approx. 70%) as has been reported for other species of leghaemoglobins. However, circular dichroism in the near-ultraviolet region (240--300 nm) indicated that the haem-protein interactions may be considerably different in the subclover leghaemoglobins and this may explain their atypical resistance to autoxidation and the absence of nicotinate binding in these proteins.