Roland E. Barden

Oil-continuous microemulsions composed of hexane, water, and 2-propanol

Abstract On the basis of conductivity and centrifugation data and visual examination, ternary phase diagrams were prepared for systems composed of hexane, water, and 2-propanol. Certain ternary compositions were shown to be oil-continuous microemulsions, even though a soap or detergent was not present. Of a number of short-chain alcohols tested, only 2-propanol and 1-propanol could stabilize a microemulsified ternary solution. Addition of a small amount of hexadecyltrimethylammonium perchlorate to the ternary solutions produced only modest changes in the ternary phase diagram. However, in the presence of hexadecyltrimethylammonium salts, most short-chain alcohols containing less than eight carbons could stabilize an oil-continuous microemulsion.

Detergentless water/oil microemulsions. III. Effect of KOH on phase diagram and effect of solvent composition on base hydrolysis of esters

Abstract A ternary phase diagram was prepared for certain compositions of hexane, 2-propanol, and 5.0 mM KOH. The presence of KOH produced significant, though not major, changes in the ternary diagram as compared to the ternary diagram for the hexane/water/2-propanol system [see Smith et al., J. Colloid Interface Sci. 60, 488 (1977)]. The hydrolysis of p-nitrophenyl esters by hydroxide was investigated in solutions from different regions of the ternary diagram. Ternary solutions characterized by the presence of water-rich droplets (i.e., detergentless microemulsions) or by the presence of aggregates of water, propanol, and ions were found to be good solvents for the hydrolytic reaction. Ternary solutions with these characteristics appear to be quite suitable as solvents for reactions involving an organic, water-insoluble reactant and an ionic, water-soluble reactant.

S-(4-Bromo-2,3-dioxobutyl)-CoA: An affinity label for certain enzymes that bind acetyl-CoA

Abstract S -(4-Bromo-2,3-dioxobutyl)-CoA, a potential affinity label for enzymes possessing a receptor site( s ) for short-chain acyl-CoA, was synthesized by condensing CoA and 1,4-dibromo-2,3-butanedione in acidified methanol. The new reagent was tested as an active site-directed irreversible inhibitor with four enzymes that accept a short-chain acyl-CoA as substrate. With citrate synthase (pig heart) and acetyl-CoA hydrolase (beef kidney) irreversible inhibition was observed, and the rate of inactivation obeyed first-order kinetics. Benzoyl-CoA, a reversible competitive inhibitor versus acetyl-CoA with both citrate synthase and acetyl-CoA hydrolase, protected the active site of both enzymes against the irreversible inhibitor. The new reagent was an exceptionally potent irreversible inhibitor of acetoacetyl-CoA thiolase (beef liver). Relatively low concentrations of the reagent (≥1 μ m ) completely inhibited the thiolase in less than 2 min. Preincubation of thiolase with acetoacetyl-CoA protected the enzyme against inhibition by S -(4-bromo-2,3-dioxobutyl)-CoA. In contrast, irreversible inhibition of l -3-hydroxyacyl-CoA dehydrogenase (pig heart) was not observed. Instead, the new reagent appeared to be a weak alternate substrate for this dehydrogenase. In all cases, the new reagent exhibited tight reversible binding at the active site since the measured K i s (and K m ) were in the range, 30 to 120 μ m . It is anticipated that the new reagent will be suitable for investigating a number of acyl-CoA using enzymes by affinity labeling techniques.

Affinity labeling of fatty acid synthetase from lactating rat mammary gland with S-(4-bromo-2,3-dioxobutyl)-CoA: evidence for a "half-of-the-sites" catalytic mechanism.

Abstract The classical affinity label, S-(4-bromo-2,3-dioxobutyl)-CoA, rapidly and irreversibly inhibits fatty acid synthetase from lactating rat mammary gland. The limit stoichiometry of incorporated label and the kinetics of inactivation indicate that two sites can be labeled per enzyme dimer. Strong evidence of site-site interaction (weak negative cooperativity) was observed. At relatively low concentrations, the affinity label inhibits acetyl transacylase whereas the malonyl transacylase activity is enhanced. We propose that fatty acid synthetase from lactating rat mammary gland catalyses a “half-of-the-sites” mechanism.

PROPERTIES OF METAL COMPLEXES IN THE INTERPHASE OF AN OIL CONTINUOUS MICROEMULSION. 3. INTERACTION OF COPPER(II) WITH THE SIDE CHAIN OF TRYPTOPHAN12

Abstract In earlier work we have demonstrated the utility of oil continuous microemulsions for investigating ligand-metal interactions in cases where the ligand is an organic molecule sparingly soluble in water. Thus far, the interaction of several amino acid sidechains (histidine, lysine, glutamine, methionine) with Cu(II) has been investigated.2,4 The oil continuous microemulsion used in these studies is composed of hexane, water, 2-propanol and hexadecyltrimethylammonium perchlorate.5 The side chain is added to the system as a surface active derivative, i.e. an Nα-dodecanoylamino alcohol, which ensures that the side chain is located at the interface of the water-rich dispersed droplets. To date, the primary concern of our research has been to investigate the properties of metal complexes located in an interfacial environment. Our selection of ligands for these studies reflects an additional interest in bioinorganic chemistry. (Fendler and co-workers have previously argued that a host solution of the ty...

The average molecular weight and shape of the “polymeric acids” found in Black Trona Water from the Green River Basin

Abstract Macromolecular organic material, called “polymeric acids”, has been isolated from Black Trona Water by exhaustive dialysis and characterized as the sodium salt in 0.10 M sodium carbonate, pH 10, by several physico-chemical methods. Analysis by gel filtration chromatography on Sepharose-CL 6B indicates that the “polymeric acids” are polydisperse and composed of species of relatively high molecular weight (∼ 4 × 105, using proteins as standards). With this method, the range of molecular weights appears to be rather narrow. If “polymeric acids” are transferred from sodium carbonate, pH 10, into distilled water, selfassociation occurs and all species elute in the void volume. The weight-average molecular weight determined in 0.10 M sodium carbonate, pH 10, by the light scattering method is 1.7 × 105. Sedimentation velocity analysis at 20°C with the analytical ultracentrifuge gives a value for S20,w of 5.4 and the shape of the Schlieren patterns suggest a polydisperse sample with a relatively narrow range of sizes. Analysis of the molecular weight distribution by a sedimentation equilibrium method indicates that the range of molecular weights is 8 × 104 to 2.1 × 105. The partial specific volume ( v ) of “polymeric acids” is 0.874 ml/g. Viscosity measurements yield a value for [η] of 2.5 ml/g, which indicates that the “polymeric acids” are compact (spherical or ellipsoidal) in shape.

13C-NMR, i.r. and fluorescence spectroscopic studies of the polymeric acids found in Black Trona Water from the Green River Basin

Abstract A polymeric acid fraction has been isolated from Black Trona Water, a fossil water from the Green River Formation of Wyoming, by means of exhaustive dialysis. The polymeric acid is apparently of large molecular weight (>20,000) and constitutes 40–45% of the organic material in Black Trona Water from the DOE/LETC Black Water 1-A well. The fraction was investigated by means of 13 C-NMR, FTIR, and fluorescence spectroscopy. Most of the carbon content is aliphatic (∼63%), with some aliphatic chains greater than 4 carbons in length. A substantial aromatic fraction exists consisting of condensed structures having 4–6 rings. The chemical nature of the polymeric acid fraction is compared to that of kerogen from the same formation.

The 8-azidoadenine analog of S-benzoyl (3′-dephospho)coenzyme A — A photoaffinity label for acyl CoA:glycine N-acyltransferase

Abstract S-benzoyl (3′-dephospho-8-azido)-CoA was synthesized by condensing 8-azido-AMP and S-benzoyl-4′-phosphopantetheine. The reagent was shown to be photosensitive. Studies with acyl CoA:glycine N-acyltransferase from beef liver show that the reagent is an alternate substrate and that photolysis of a mixture of reagent and enzyme causes irreversible inhibition. Thus, S-benzoyl (3′-dephospho-8-azido)-CoA is a photogenerated active-site-directed irreversible inhibitor (i.e. a photoaffinity label) for this N-acyltransferase.

Some physico-chemical properties of aqueous solutions of Nα-acyl-L-histidine

Abstract The critical micelle concentrations of aqueous solutions of N α -acyl-L-histidine have been determined by the spectral shift method with Rhodamine 6G and by the light scattering method. With the spectral shift method critical micelle concentrations of 40, 9.0, 1.0, 0.11, and 0.012 mM were obtained for N α -acyl-L-histidine containing saturated acids of 8, 10, 12, 14, and 16 carbons respectively, at 45°C and pH 8.6 in the absence of added salt. For the homologs containing 10, 12, and 14 carbon acids, critical micelle concentration of 9.0, 1.0, and 0.11 mM were determined by the light scattering method. The light scattering studies yield micelle weights of 60, 66, and 84 thousand for the C-10, C-12, and C-14 homologs, respectively. N α -acyl-L-histidine is an unusual surfactant in that the hydrophilic portion of the molecule is relatively large and contains both an ionic group (carboxylate group) and a nonionic group (imidazole side-chain). The bulky hydrophilic group of N α -acyl-L-histidine causes this molecule to exhibit physico-chemical behavior which is not typical of that exhibited by most ionic surfactants. In particular, the dependence of the critical micelle concentration on the acyl chain length and on the concentration of added salt is atypical. Chemical shift measurements (by NMR) on the C-2 and C-5 protons of imidazole in micellar N α -dodecanoyl-L-histidine indicate that the imidazole group is, indeed, positioned at the water-micelle interface.

The synthesis and biological activity of thiolcarnitine and its thiolesters

Acetyl-D,L-thiolcarnitine was synthesized by the acid-catalyzed addition of thiolacetic acid to 4-trimethylammonio-2-butenoic acid. Acetyl-D,L-thiolcarnitine was the precursor of D,L-thilcarnitine, which was prepared by base hydrolysis. Thiolcarnitine significantly enriched in the L-isomer was prepared from acetyl-D,L-thiolcarnitine using carnitine acetyltransferase as the resolving agent. The C2, C8 and C16 carnitine thiolesters were obtained by acylating thiolcarnitine with the corresponding N-hydroxysuccinimide esters. As a substrate for carnitine acetyltransferase, acetylthiolcarnitine gave the same kinetic constants as did acetylcarnitine; on the other hand, thiolcarnitine and carnitine gave the same Km but the Vmax with thiolcarnitine was less than 5% of the value obtained with carnitine. With thiolcarnitine and acetylthiolcarnitine as reactants, the measured Keq, at 30 C and pH 7.0, for the reaction catalyzed by carnitine acetyltransferase (see below) was 4.6±.1. acetylthiolcarnitine+CoA—acetyl-CoA+thiolcarnitine