Fluorimetric determination of critical micelle concentration avoiding interference from detergent charge

Upon mixing detergent solutions with the neutral fluorescent molecule 1,6-diphenyl-1,3,5-hexatriene a large increase in fluorescence is observed if detergent exceeds the critical micelle concentration. This property has been used to determine the critical micelle concentration of anionic, uncharged, zwitterionic, and cationic detergents. Regardless of detergent charge, the critical micelle concentrations obtained agree with the values obtained by other methods. This fluorescence assay is both sensitive and rapid, and should provide a simple and general method for determination of critical micelle concentration of any detergent.     

Preparation and properties of crystalline biliverdin IX alpha. Simple methods for preparing isomerically homogeneous biliverdin and [14C[biliverdin by using 2,3-dichloro-5,6-dicyanobenzoquinone.

Amorphous isomerically pure biliverdin IX alpha is readily prepared in more than 70% yield by dehydrogenation of bilirubin with 2,3-dichloro-5,6-dicyanobenzoquinone in dimethyl sulphoxide under carefully controlled conditions. Crystalline biliverdin IX alpha and amorphous [14C]biliverdin can be obtained similarly in more than 40+ yield. The pure crystalline pigment was characterized by elemental analysis, methylation, chemical and enzymic reduction to bilirubin, i.r.- and u.v.-visible-absorption spectroscopy, n.m.r. spectroscopy and field-desorption mass spectrometry, and its solubility was determined. Under certain conditions, dehydrogenation, gave biliverdin contaminated with III alpha and XIII alpha isomers as a result of disproporationation of bilirubin. Formation of non-IX alpha isomers depends on the concentrations of the reagents and the order in which they are mixed, and occurs under neutral anaerobic conditions. Free-radical reactions probably are responsible, suggesting that the first step in the deydrogenation of bilirubin with 2,3-dichloro-5,6-dicyanobenzoquinone in dimethyl sulphoxide is formation of a bilirubin cation radical, rather than hydride ion abstraction.     

Antimalarial drugs and heme in detergent micelles: An NMR study

Proton nuclear magnetic resonance relaxation times were measured for the protons of micelles formed by the detergents sodium dodecyl sulfate, dodecyltrimethyl ammonium bromide, and polyethylene glycol sorbitan monolaureate in the presence of ferriprotoporphyrin IX and the antimalarial drugs chloroquine, 7-chloro-4-quinolyl 4-N,N-diethylaminobutyl sulfide, and primaquine. Diffusion coefficients were extracted from pulsed gradient NMR experiments to evaluate the degree of association of these drugs with the detergent micelles. Results indicate that at low or neutral pH when the quinolyl N is protonated, chloroquine does not associate with neutral or cationic detergent micelles. For this reason, chloroquine’s interaction with heme perturbs the partitioning of heme between the aqueous medium and detergent micelles.     

Flurorometric study of the partition of bilirubin among blood components: basis for rapid microassays of bilirubin and bilirubin binding capacity in whole blood

Bilirubin binds to many sites in blood, the strongest binding being to a single site on albumin. Secondary sites on albumin, most sites on other plasma proteins, and sites on erythrocyte membranes have affinities for bilirubin that are at most one-hundredth as great. Bilirubin binds to hemoglobin in red cells with an effective affinity that is less than one-thousandth that of the primary albumin site. Essentially the only bilirubin present in blood which fluoresces is that bound to the primary albumin site. Almost all the other bilirubin in blood fluoresces with a yield no more than one-fiftieth as large. Quantitative fluorometry of whole blood is possible using the “front-face” technique. The concentration of bilirubin bound to the primary albumin site can be determined in this way. The albumin binding capacity of a blood specimen can be similarly assayed upon titration of the specimen with bilirubin. The nonionic detergent dodecyldimethylamine oxide (DDAO) scavenges bilirubin from all sites in blood, and, since bilirubin is fluorescent in DDAO micelles, the total blood bilirubin can be assayed fluorometrically after addition of DDAO to the specimen. This detergent method also allows facile assay of red-cell-bound bilirubin. These fluorometric assays for total blood bilirubin, albumin-bound bilirubin, and albumin binding capacity are simple and rapid and use very small volumes of blood. They should be of great value in the research on neonatal jaundice and in its clinical management.     

Monomer of sodium and potassium ion activated adenosinetriphosphatase displays complete enzymatic function

The distribution of sodium and potassium ion activated adenosinetriphosphatase [(Na+ + K+)-ATPase] among the various oligomeric forms present in a given solution is assessed unambiguously by cross-linking with glutaraldehyde. Purified enzyme dissolved in a solution of a nonionic detergent, octaethylene glycol dodecyl ether, remains dispersed and unaggregated after removal of the bulk of the detergent. Increases in the aggregation of the enzyme, which have been previously observed upon the addition of substrates to such a solution, are found to be due to changes in ionic strength rather than a consequence of the initiation of turnover. Furthermore, conditions are described that produce solutions containing stable, enzymatically active mixtures of the smaller oligomers of the asymmetric unit, alpha beta. Cross-linking by glutaraldehyde while the enzyme is turning over demonstrates that at least one of these oligomers is responsible for the observed enzymatic activity. A determination of which oligomers are present in each fraction from a glycerol gradient demonstrates that the profiles of the enzymatic activity and the concentration of monomer coincide. In addition, the monomer can form the sodium-dependent, phosphorylated intermediate of the mechanism for the enzyme. Finally, a preparation of (Na+ + K+)-ATPase, dissolved in solutions of the same nonionic detergent, can be prepared in which the predominant species (greater than 85%) is the monomer. The enzyme in this solution exhibits high specific activity, and its apparent Michaelis constants for the cationic substrates are very similar to those of the purified, membrane-bound enzyme. It is concluded from these results that a monomer of the alpha beta asymmetric unit is fully capable of catalyzing (Na+ + K+)-ATPase activity, and hence active transport, in the native enzyme. A reassessment of proposed molecular mechanisms for active transport is made in light of these discoveries.     

Phospholipids stabilize the interaction between the alpha and beta subunits of the solubilized receptor for immunoglobulin E

The cell-surface component (alpha) which binds monomeric immunoglobulin E with high affinity is associated with a second polypeptide (beta) in the plasma membrane. The latter component tends to dissociate during purification of the alpha chain from detergent extracts of cells, even at neutral pHs and physiological ionic strengths. We now report that the interaction of alpha and beta can be stabilized by maintaining an appropriate phospholipid to detergent ratio. Under such conditions, other discrete components reproducibly copurify with the alpha and beta chains. These results suggest that the subunits of this membrane protein--or the interaction of it with other constituents in the cell--may be stabilized in ways not observed with ordinary soluble proteins.     

Complex formation between endogenous toxin bilirubin and polyamidoamine dendrimers: a spectroscopic study

The effects of 4th and 5th generation cationic, neutral and anionic polyamidoamine (PAMAM) dendrimers on bilirubin absorbance and fluorescence were studied. Cationic and neutral PAMAM dendrimers shifted the bilirubin absorption maximum from 435 to 442-455 nm, increased the peak absorbance 1.5-fold, shifted the bilirubin fluorescence excitation and emission maxima, increased the fluorescence emission several-fold and significantly protected bilirubin against photodestruction. Using double fluorescence titration technique allowed to receive such constant of binding and the number of binding centers at 20 degrees C: for PAMAM G4 dendrimer, (2.4+/-1.4) x 10(6) (mol/l)(-1) and 0.07+/-0.012; for PAMAM G4-OH dendrimer, (3.1+/-1.3) x 10(6) (mol/l)(-1) and 0.08+/-0.014; for PAMAM G5 dendrimer, (7.6+/-3.6) x 10(6) (mol/l)(-1) and 0.09+/-0.02; and for PAMAM G5-OH dendrimer, (8.5+/-3.2) x 10(6) (mol/l)(-1) and 0.09+/-0.02. These effects can be explained by the formation of bilirubin-PAMAM dendrimer complexes and the formation of bilirubin monomers from tetramers. The formation of complexes sharply increased bilirubin solubility. We conclude that cationic and neutral PAMAM dendrimers bind bilirubin effectively and suggest that such dendrimers may serve as detoxication agents for hydrophobic endogenous toxins.     

Resonance Raman spectroscopy of bilirubins: band assignments and application to bilirubin/lipid complexation

Resonance Raman spectra of bilirubins IX alpha, III alpha, and XIII alpha and mesobilirubin XIII alpha in alkaline aqueous and chloroform solutions are reported. Partial band assignments of bilirubin IX alpha are proposed. The model compounds confirm assignments of bands of the Raman spectrum of bilirubin IX alpha to each of the two different pyrromethenones. Resonance Raman spectra of mesobilirubin IV alpha, vinylneoxanthobilirubinic acid, and vinylisoneoxanthobilirubinic acid in alkaline aqueous solution and of the tetra-n-butylammonium salt of bilirubin IX alpha are used to define markers for the presence or absence of internal hydrogen bonds. Interaction of bilirubin dianion and sphingomyelin liposomes is studied. The Raman evidence suggests that in the bilirubin dianion/liposome complex the intramolecular hydrogen bonds between the propionate groups and the lactam NH/CO are ruptured. It is proposed that in the complex the bilirubin propionates form ion pairs with the quaternary ammonium ion of the choline moiety of sphingomyelin.     

Molecular organization of pheophytin a in aqueous solutions of detergents

Absorption, fluorescence and excitation fluorescence spectra of pheophytin a have been measured in aqueous solutions of nonionic (Triton X-100), anionic (sodium lauryl sulphate) and cationic (Cetyl pyridinium chloride) detergents at different concentrations and pH after system relaxation. By measuring the second derivative and differential spectra, it has been shown, that if detergent concentrations are lower than critical micelles concentration, or if the detergent is completely absent, the pigment forms conglomerates containing both dimeric and monomeric forms with an efficient energy transfer between them. If detergent concentrations are higher than critical micelles concentration, pheophytin a localizes in detergent micelle in monomeric form at neutral and acidic pH, and allomerizes at alkaline pH. The spectral characteristics of pheophytin a dimers in the conglomerate and its monomers in micelles poorly (if at all) depend on the sign of the detergent molecule charge.     

An Evaluation of the Metachromasy of Anionic Dyes. II. Visual and Spectral Observations on Solutions

The reactions of 13 anionic dyes in solution with a basic protein (protamine), a cationic detergent, guanidine, histamine, procaine, quinine, and strychnine were examined visually and spectrophotometrically in order to distinguish metachromatic changes of the dyes. Disazo dyes (Congo red, benzopurpurin, but not trypan blue) were metachromatic; indigoid, triphenylmethane and xanthene dyes were not. The magnitude of metachromasy in this series of dyes was not great compared with cationic dyes, the shifts of absorbance maxima being only about 15 mμ against 90 mμ or more for some cationic metachromatic dyes. The most effective chromotropes were protamine and a cationic detergent. Agreement between visual observations on tissue sections, visual observations on solutions, and spectral observations on solutions was generally good.     

Aggregation, dissociation and denaturation of sesame (Sesamum indicum L.) alpha-globulin in cetyl trimethyl ammonium bromide solution.

The behaviour of the major protein of sesame seed (Sesamum indicum L.) alpha-globulin has been studied in a cationic detergent, cetyl trimethyl ammonium bromide solution. Up to a critical detergent concentration the protein is precipitated from solution, above which redissolution of the protein is observed. Sedimentation velocity patterns indicate the presence of higher aggregates in the detergent concentration range 5 X 10(-5)--1 X 10(-3) M. These are considered to be the soluble precursors of the insoluble aggregates. Fluorescence measurements show that tryptophanyl groups of the protein which are in contact with the aqueous phase are perturbed by the detergent. The difference spectra of the protein in higher concentration of detergent indicate considerable red shift in the spectrum. Spectrophotometric titration of phenolic groups in 1 X 10(-2) M CTAB indicate that a conformational change in the protein has taken place.     

Chemical modification of carboxyl groups of fibrinogen and its effect on the binding of cationic detergent.

Carboxyl groups of native human fibrinogen were modified with glycine methyl ester and 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. It seemed likely that the modification occurred stepwise. Approximately 26% of the carboxyl groups of fibrinogen was modified finally. The modified fibrinogen had no interaction with cationic detergent, and did not form any complex with the detergent. In dilute acid, fibrinogen was observed to show only a slight interaction with cationic detergent. It is probable that the exposed and ionized carboxyl groups are essential for the formation of a complex between fibrinogen and cationic detergent.     

Interactions in vitro and in vivo between anionic and cationic porcine trypsin and porcine plasma proteinase inhibitors

A method for purifying porcine anionic and cationic trypsin is presented. Reaction mixtures with increasing amounts of the two porcine trypsins and porcine serum were studied in vitro to evaluate the relative importance of alpha 1-macroglobulin and alpha 2-macroglobulin as well as alpha 1-proteinase inhibitor in the rapid binding of porcine anionic and cationic trypsin. Porcine cationic trypsin was preferentially bound to alpha 1-macroglobulin, while anionic trypsin exhibited equal binding to both alpha-macroglobulins. Both trypsins were also bound by the alpha 1-proteinase inhibitor but not until alpha 1-macroglobulin approached saturation. Trypsin-alpha-macroglobulin complexes were cleared from plasma with a half-life of 6 min. For trypsin-alpha 1-proteinase inhibitor-complexes the half-life was 120 min. These findings are in accordance with results for other mammalian species, including man.     

Complexes of bilirubin with proteins

Complexes of bilirubin with chymotrypsin, lysozyme and apomyoglobin in neutral aqueous solution are characterized by circular dichroism spectra in the visible region. These are analogous to previously investigated bilirubin-serum albumin complexes. Ferriprotoporphyrin IX displaces bilirubin from its apomyoglobin complex.     

Protein-cationic detergent interaction. Fourier transform infrared and laser Raman spectroscopic studies on the interaction between proteins and dodecylpyridinium bromide.

Fourier transform infrared and laser Raman spectroscopies were used to study the effects of dodecylpyridinium bromide on the conformation of haemoglobin, myoglobin, bovine serum albumin, ribonuclease, ovalbumin, lysozyme, trypsin and beta-lactoglobulin in aqueous solution. Addition of the cationic detergent caused a decrease in alpha-helix conformation in highly helical proteins. At low detergent concentrations stabilization of beta-sheet conformation was observed.     

Inhibition of the ATPase activity of the catalytic portion of ATP synthases by cationic amphiphiles

Melittin, a cationic, amphiphilic polypeptide, has been reported to inhibit the ATPase activity of the catalytic portions of the mitochondrial (MF1) and chloroplast (CF1) ATP synthases. Gledhill and Walker [J.R. Gledhill, J.E. Walker. Inhibition sites in F1-ATPase from bovine heart mitochondria, Biochem. J. 386 (2005) 591-598.] suggested that melittin bound to the same site on MF1 as IF1, the endogenous inhibitor polypeptide. We have studied the inhibition of the ATPase activity of CF1 and of F1 from Escherichia coli (ECF1) by melittin and the cationic detergent, cetyltrimethylammonium bromide (CTAB). The Ca2+- and Mg2+-ATPase activities of CF1 deficient in its inhibitory epsilon subunit (CF1-epsilon) are sensitive to inhibition by melittin and by CTAB. The inhibition of Ca2+-ATPase activity by CTAB is irreversible. The Ca2+-ATPase activity of F1 from E. coli (ECF1) is inhibited by melittin and the detergent, but Mg2+-ATPase activity is much less sensitive to both reagents. The addition of CTAB or melittin to a solution of CF1-epsilon or ECF1 caused a large increase in the fluorescence of the hydrophobic probe, N-phenyl-1-naphthylamine, indicating that the detergent and melittin cause at least partial dissociation of the enzymes. ATP partially protects CF1-epsilon from inhibition by CTAB. We also show that ATP can cause the aggregation of melittin. This result complicates the interpretation of experiments in which ATP is shown to protect enzyme activity from inhibition by melittin. It is concluded that melittin and CTAB cause at least partial dissociation of the alpha/beta heterohexamer.     

Concentrated,digestible DNA after hydroxylapatite chromatography with cetylpyridinium bromide precipitation

A method for the direct extraction of the DNA from the unfavorable phosphate eluant of hydroxylapatite chromatography is described. The DNA—reversibly precipitated with the cationic detergent cetylpyridinium bromide—can be subjected to further enzymatic manipulations within minutes. This method is applied to the rapid separation of pBR322 plasmid from the chromosomal DNA.     

Atomic force microscopy of supported lipid membranes and their complexes with polycations

The method of atomic force microscopy has been used to investigate the morphology of mica-supported bilayer lipid membranes and stability of their complexes with a cationic polymer, poly-(N-ethyl-4-vinylpyridinium bromide). Lipid bilayers with a minimum of defects were obtained by the fusion of monolamellar neutral or mixed anionic bilayer vesicles (liposomes) on the mica surface, followed by excessive solvent removal by means of rapid rotation of a plate in horizontal plane (spin-coating). It has been shown that the cationic polymer does not interact with the bilayers, where the outer leaflet (i.e., the monolayer exposed to the surrounding aqueous solution) is made of an electroneutral phosphatidylcholine (PC). At the same time, the polymer irreversibly binds to the bilayer containing an anionic lipid.     

Two-dimensional gel electrophoresis of membrane proteins using anionic and cationic detergents. Application to the study of mitochondrial F0-F1-ATPase

Polyacrylamide gel electrophoresis in the presence of a cationic detergent, tetradecyltrimethylammonium bromide (TDAB) has been compared to electrophoresis in the presence of an anionic detergent, sodium dodecyl sulfate (SDS). Although, in both systems, the peptides generally migrated as a function of their molecular weight, the TDAB electrophoresis permitted us to obtain a much better resolution of several peptides of the mitochondrial F0-F1-ATPase, especially for the alpha and beta subunits and for the oligomycin sensitivity conferring protein (OSCP). The differences between the two electrophoretic profiles have been used to devise a new technique of two-dimensional electrophoresis using successively anionic and cationic detergents. This method could be very useful in the case of membrane proteins, which are generally soluble only in the presence of powerful ionic detergents. It has been particularly successful in resolving the small peptides of the F0-F1-ATPase which were difficult to differentiate by other techniques in one- or two-dimensional polyacrylamide gel electrophoresis.