Emrys W. Thomas

Fluorescence lifetime and quenching studies on some interesting diphenylhexatriene membrane probes

Abstract The fluorescence lifetimes of a number of membrane probes based on the 1,6-diphenylhexatriene (DPH) chromophore have been measured in small unilamellar phospholipid vesicles and found to be multiphasic. These probes were quenched by sodium iodide with different efficiencies in vesicles and this has been attributed to the depth of the particular probe in the bilayer. The distribution of the probe between the outer and inner monolayer has been determined for those probes with fixed positions in the bilayer. The iodide ion permeability of the bilayer was found to be immeasurably small over a 3 h period.

Impermeant auxin analogues have auxin activity

Protein conjugates of 5-aminonaphthalene-1-acetic acid and of 5-azido-naphthalene-1-acetic acid have been prepared and evaluated for auxin activity in two types of assay. In standard elongation tests with pea (Pisum sativum L.) epicotyl sections the conjugates are inactive. However, if the epicotyls are abraded to perforate the cuticle, auxin activity is observed provided that the conjugates are not too large to traverse the cell wall. In a system lacking a cell wall — tobacco (Nicotiana tabacum L.) protoplasts — conjugates of widely differing size are able to induce membrane hyperpolarization. These results support other recent evidence that auxin receptors are exposed at the exterior face of the plasma membrane and indicate that auxins can produce both rapid and longer-term responses without entering the cell.

The use of a phospholipid analogue of diphenyl-1,3,5-hexatriene to study melittin-induced fusion of small unilamellar phospholipid vesicles

A phospholipid analogue incorporating the diphenyl-1,3,5-hexatriene (DPH) chromophore has been synthesized. The compound has been shown to have similar fluorescence properties to DPH itself but, unlike DPH, is unable to exchange freely through solution when incorporated as probe in a subset of phospholipid vesicles of given composition. The non-exchangeability of this probe has been exploited to study the fusion of phospholipid vesicles to form larger structures. The peptide melittin was used to initiate fusion, and it was shown that vesicles which had been induced to fuse by heating in the presence of melittin would not fuse with subsequently added vesicles.

Photophysical properties of DPH derivatives

Abstract The absorption spectra, fluorescence emission spectra, quantum yields and fluorescence lifetimes have been measured for a range of DPH derivatives. Difference in the photophysical properties as compared with those of DPH are noted and discussed.

Incorporation of a novel photochromic phospholipid molecule into vesicles of dipalmitoylphosphatidylcholine

Abstract A phospholipid molecule has been synthesised, bearing an azobenzene linkage within one acyl chain. The lipid has been incorporated into vesicles of dipalmitoylphosphatidylcholine, and the effects of photoisomerisation on the vesicle permeability and phase behaviour studied by light scattering and fluorescence spectroscopy. The phase transition temperature of DPPC is reduced, and the transition is broadened by the trans Azo-lipid. After photolysis the transition temperature is further reduced, and non-equilibrium effects are evident. Vesicles containing the Azo-lipid can sustain a pH gradient before photolysis, but pH slowly equilibrates following irradiation. Results indicate that photoisomerisation causes no loss of bilayer integrity. Electron microscopy shows that the Azo-lipid alone forms vesicles on dispersion using an ethanol injection technique.

Fluorescence solvatochromism of nitrodiphenylhexatrienes

Examination of the absorption and fluorescence spectra of symmetrically and asymmetrically substituted 4′-nitro derivatives of 1,6-diphenyl-1,3,5,-hexatriene reveals fundamental transformations of photophysical behaviour relative to the parent molecule. The manifestations of this divergence are solvent dependent. In non-polar solvents the primary effect is a reduction of the fluorescence quantum yield to negligible levels. This condition is superseded as solvent polarity rises by the appearance of extensively red-shifted fluorescence. The correlation of this spectral shift (solvatochromism) with macroscopic solvent polarity is evaluated. These distinctive fluorescence properties, together with the complementary but less dramatic variations of absorption spectra, are interpreted in terms of the involvement of substituent associated intramolecular charge-transfer excited states.

Inhibition of (Ca2++Mg2+)-ATPase by carbodiimides. A structure-activity study

Nine hydrophobic carbodiimides were synthesized and their chemical reactivities (towards acetic acid) and inhibitory capacities (towards the (Ca2+ + Mg2+)-ATPase were measured. No correlation between chemical reactivity and inhibitory efficacy emerges, but a significant effect of molecular bulk on reactivity towards the calcium-protectable carboxyl groups of the ATPase is noted: methyl-substituted compounds inhibit the enzyme in the presence of Ca2+, while aryl- or cyclohexyl-substituted compounds do not inactivate in the presence of Ca2+.

Temperature-dependences of the kinetics of reactions of papain and actinidin with a series of reactivity probes differing in key molecular recognition features

The temperature-dependences of the second-order rate constants (k) of the reactions of the catalytic site thiol groups of two cysteine peptidases papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) with a series of seven 2-pyridyl disulphide reactivity probes (R-S-S-2-Py, in which R provides variation in recognition features) were determined at pH 6.7 at temperatures in the range 4-30 degrees C by stopped-flow methodology and were used to calculate values of DeltaS++, DeltaH++ and DeltaG++. The marked changes in DeltaS++ from negative to positive in the papain reactions consequent on provision of increase in the opportunities for key non-covalent recognition interactions may implicate microsite desolvation in binding site-catalytic site signalling to provide a catalytically relevant transition state. The substantially different behaviour of actinidin including apparent masking of changes in DeltaH++ by an endothermic conformational change suggests a difference in mechanism involving kinetically significant conformational change.

Evidence for ‘lock and key’ character in an anti-phosphonate hydrolytic antibody catalytic site augmented by non-reaction centre recognition: variation in substrate selectivity between an anti-phosphonate antibody, an anti-phosphate antibody and two hydrolytic enzymes

The substrate selectivities of an anti-phosphonate and an anti-phosphate kinetically homogeneous polyclonal catalytic antibody preparation and two hydrolytic enzymes were compared by using hapten-analogous and truncated carbonate and ester substrates each containing a 4-nitrophenolate leaving group. Syntheses of the truncated substrates devoid of recognition features in the non-leaving group parts of the substrates are reported. The relatively high kinetic selectivity of the more active anti-phosphonate antibody preparation is considered to depend on a relatively rigid catalytic site with substantial reaction centre specificity together with other important recognition interactions with the extended non-leaving group part of the substrate. In contrast, the less catalytically active, more flexible anti-phosphate antibody exhibits much lower kinetic selectivity for the substrate reaction centre comparable with that of the hydrolytic enzymes with activity much less dependent on recognition interactions with the non-leaving group part of the substrate. The ways in which haptenic flexibility and IgG architecture might contribute to the differential kinetic selectivities are indicated.

Improvement in hydrolytic antibody activity by change in haptenic structure from phosphate to phosphonate with retention of a common leaving-group determinant: evidence for the 'flexibility' hypothesis.

To investigate the hypothesis that decreased hapten flexibility may lead to increased catalytic antibody activity, we used two closely related immunogens differing only in the flexibility of the atomic framework around the structural motif of the haptens, analogous to the reaction centre of the corresponding substrates. Identical leaving-group determinants in the haptens and identical leaving groups in the substrates removed the ambiguity inherent in some data reported in the literature. Anti-phosphate and anti-phosphonate kinetically homogeneous polyclonal catalytic antibody preparations were compared by using carbonate and ester substrates respectively, each containing a 4-nitrophenolate leaving group. Synthetic routes to a new phosphonate hapten and new ester substrate were developed. The kinetic advantage of the more rigid anti-phosphonate/ester system was demonstrated at pH 8.0 by a 13-fold advantage in k(cat)/k(non-cat) and a 100-fold advantage in the proficiency constant, k(cat)/k (non-cat) x K(m). Despite these differences, the pH-dependences of the kinetic and binding characteristics and the results of chemical modification studies suggest closely similar catalytic mechanisms. The possible origin of the kinetic advantage of the more rigid hapten/substrate system is discussed.