Robert B. Cundall

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.

Pulse radiolysis of aqueous solutions of sodium azide: Reactions of azide radical with tryptophan and tyrosine☆☆☆

Abstract Azide radicals (N.3) are formed on reactions of axide anions (N-3aq) and hydroxyl radicals in aqueous solutions. Mechanisms of formation of N.3 and its reactions with the amino acids tryptophan (trpH) and tyrosine(tyrH), which gave the radicals trp and tyr, respectively, and with some inorganic transients, have been studied by use of the pulse radiolysis technique. Variation of pH has no significant effect on the formation or decay of the azide radical. Its decay rate increases with the concentration of N⨪3aq; this is consistent with the formation of the diazide radical anion ((N3)-2aq). Electron transfer reactions of N-3aq with the isopropyl radical and the dithiocyanate radical anion have been studied. The formation of the radicals trp and tyr most likely occurs by hydrogen atom transfer to N.3 from the amino acids. The efficiencies of reactions of N.3 and (N3)⨪2aq, with the two amino acids appear to be different.

Free Radical Reactions with Alpha-Tocopherol and N-Stearoyl Tryptophan Methyl Ester in Micellar Solutions

The rate constants have been measured for one-electron oxidation by N3. and Br2-. of N-stearoyl tryptophan methyl ester (STME) and alpha-tocopherol (alpha-T) in micelles of sodium dodecyl sulphate (SDS) or tetradecyl trimethyl ammonium bromide (TTAB). Compared with analogous reactions of tryptophan and Trolox C in aqueous solution, the rate constants for oxidation in micellar solution by N3. are reduced by 30-70%. The micellar charge increased the rate of oxidation of STME by Br2-. in TTAB micelles by almost an order of magnitude, compared with the reaction of Br2-. with tryptophan in aqueous solution. In SDS micelles the rate of oxidation of STME by Br2-. was reduced more than 30-fold. Quenching of fluorescence from STME in micelles by acrylamide confirmed the accessibility of the indole ring to the aqueous solvent. The rate of repair of the neutral STME radical by alpha-T in TTAB micelles was found to be accelerated by a factor of at least 27, compared with the similar reaction between Trolox C and tryptophan radicals in aqueous solution.

Factors influencing the photosensitizing properties and photoluminescence of thioflavin T

Abstract The fluorescence intensity of aqueous solutions of thioflavin T greatly increases when the dye is bound to ribonucleic acid and to single-stranded poly-nucleotides containing purine bases. Binding is due to both ionic and hydrophobic interactions. Thioflavin is an inefficient photosensitizer for the oxidation of histidine but the photosensitizing power of thioflavin increases considerably when the dye is bound to polynucleotides. These effects are interpreted in terms of a stabilization of the excited singlet and triplet states of the dye in the bound state.

A simple purpose-built fluorimeter for the titrimetric assay of glycosaminoglycans

Abstract A purpose-built fluorimeter for the assay of glycosaminoglycans has been constructed and extensively used. The technique uses the fluorescence of the cationic dye acridine orange and the changes that accompany its interaction with the anionic sites of glycosaminoglycans. The operator titrates a volume of glycosaminoglycan solution from a burette into the cell containing acridine orange and the fluorescent changes are recorded on a digital multimeter. The data obtained are plotted and the equivalence point of the titration is calculated. In minutes, information can be obtained about the concentration of glycosaminoglycans, the degree of ionization of the anionic sites, the availability of anionic sites in glycosaminoglycan-protein mixtures, and the strength of the dye-polyanion interaction. This fluorimeter will prove to be a valuable addition to biochemical, medical, and food science laboratories.

Polyelectrolyte complexes. 2. Interaction between collagen and polyanions

Abstract The electrostatic interactions that occur in connective tissue between polyanions and proteins have been studied in model systems by a technique involving a fluorescent probe, acridine orange. It was found that collagen bound more strongly than bovine serum albumin to the polyanions studied. At pH 3.0, collagen formed strong complexes of definite stoichiometry with chondroitin-4-sulphate, chondroitin-6-sulphate, heparin and polystyrene sulphonate that were stable in sodium chloride solution of 0.1 M. The complexes of collagen with hyaluronic acid, or carboxymethylcellulose were less stable. The effect of pH variations (3.0–9.0) on the binding was investigated. Critical electrolyte concentrations (NaCl) were determined for complexes of collagen with glycosaminoglycans that dissociated at salt concentrations below that at which collagen precipitates. The values obtained were, 0.1 M for hyaluronic acid, and ∼0.5 M for chondroitin sulphate.

Photochemistry of pyrenylmethyltriphenyl-phosphonium salts and related compounds

Abstract Pyrenylmethyltriphenylphosphonium salts in alcoholic solutions undergo photochemical solvolysis to form the corresponding alkoxymethylpyrene and triphenylphosphine compounds in high yield. The analogous naphthylmethyl compounds also undergo similar reactions. The corresponding tri-n-butylphosphonium salts, however, are photostable under the same conditions. The first excited singlet is probably involved in the reaction. The photophysical properties of the pyrenyl compounds have been measured and the triplet states have been recorded.

Free radical reactions with proteins and enzymes: The inactivation of bovine carbonic anhydrase B

A comparison of the inactivation of bovine carbonic anhydrase B (carbonate hydro-lyase, EC 4.2.1.1) by OH, (SCN)(2) and Br(2) shows that the enzyme contains one or more essential tryptophan residues. Direct oxidation of histidine and tyrosine residues by the radicals is less important in causing inactivation of the enzyme. The effectiveness of all these radicals in inactivating carbonic anhydrase decreases with increasing pH in the region where the activity-linked ionizable group dissociates. Differences between the rates of reaction of Br(2) and SCN(2) with the holo- and apo-enzyme and between the resulting transient product spectra indicate that access to the reactive tyrosine and tryptophan residues is diminished by the presence of Zn2+ in the active site region.

Oxidation photosensitized by 2-chlorothioxanthone: the role of singlet oxygen☆

2-chlorothioxanthone, solubilized in sodium dodecylsulphate micelles, photosensitized the oxidation of methionine. The reaction was quenched by azide ion and 1,4-diazabicylo [2.2.2] octane in accordance with a mechanism involving O2(1Δg). 2-chlorothioxanthone was incorporated in the bilayer membrane of lipid vesicles where it performed a dual role of photosensitizer and fluorescent probe. The rate of photsensitized oxidation increased with increasing egg phosphatidylcholine in the membrane. The process was quenched efficiently by azide ion showing that O2(1Δg) plays an important part. On irradiation of a mixture of dipalmitoyl-l-α-phosphatidyl-choline vesicles containing membrane-bound chlorothioxanthone and egg phosphatidylcholine vesicles containing no chlorothioxanthone, oxygen absorption occurred. It is concluded that O2(1Δg) diffused from its site of formation in the membranes of the dipalmitoylphosphatidylcholine vesicles, through the intervening aqueous phase, into the egg phosphatidylcholine vesicles where it oxidized the unsaturated fatty acid side-chains.

Interaction of acridine orange and polyanions: fluorimetric determination of binding strengths and the influence of simple electrolytes

Binding affinities of Acridine Orange and six polyanions have been determined by measuring association constants in essentially salt-free solution, and also by observing competition between the dye and a simple salt for the polyanion site. Differences were found in the dye-binding order for the six polyanions using the two techniques. These differences were related to the breakdown in the uniform binding behaviour of carboxy and sulphate groups bound to the same polymer backbone as the salt concentration is increased. A mechanism is proposed which fits the thermodynamic parameters calculated by the application of Schwarz treatment and the Langmuir isotherm. The difference in the free-energy of binding between the strongest binding polymer, polystyrene sulphonate (ΔG°=–41 kJ mol–1) and the weakest binding, hyaluronic acid (ΔG°=–29 kJ mol–1) could be accounted for by considering coulombic site-dye interactions which destabilise the dye aggregates. The main influence promoting dye binding appears to be the free-energy of dye aggregation.The degree of co-operativity of dye binding, q, has also been determined. An attempt has been made to relate this parameter to the physical properties of the polyanion chain. It appears unlikely that q has the significance initially attached to it.