Lithium Dodecyl Sulfate Research Articles

Biochemical and Immunological Identification of Human Neutrophil Elastase on Nitrocellulose Membranes

Human neutrophil elastase (HNE) was analyzed for protein(s), antibody staining and activity staining, on lithium dodecyl sulfate (LDS) polyacrylamide gel electrophoresis followed by Western blotting. The HNE activity, which was identified with N-acetyl-D,L-alanine alpha-naphthyl ester as substrate, was well preserved in the presence of 0.1% LDS at 4 C during electrophoresis. As little as 0.1 microgram HNE was required for the activity staining. The HNE appeared to be three peptides having a major band at mass ratio 27,000, a second major band at mass ratio 28,000 with a minor protein band at mass ratio 29,000. On transfer to nitrocellulose, the mass ratio 28,000 band displayed poor immunoreactivity. This was the second most dense band with highest enzymatic staining. This procedure is a useful method and analytical tool to determine the correlation of enzymatically active proteins, subunits and immunoreactive protein(s) of elastase from various sources, including neutrophils.

Enhancement effects of dodecyl sulphates in flame atomic absorption spectrometry

Several enhancement models were used to explain the results obtained in the investigation of Al, Ca, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Rb, Sb, Sn, Sr and Zn using flame atomic absorption spectrometry with a pre-mix burner and six different micellar systems: cetyltrimethylammonium bromide, Triton X-100, ammonium dodecyl sulphate, lithium dodecyl sulphate, sodium dodecyl sulphate and potassium dodecyl sulphate. Similarly, 19 metal cations were used in an interference study with sodium dodecyl sulphate.

Photoionization in micelles: Addition of charged electron acceptors

The relative photoyield of the electron donor N, N, N′, N′-tetramethylbenzidine (TMB), solubilized in sodium and lithium dodecyl sulfate micelles with added charged electron acceptors was investigated. It was attempted to control the acceptor distance from a charged micellar interface by differently charged acceptors, cationic dimethyl viologen and anionic ferricyanide. However, back electron transfer from both cationic and anionic acceptors was found to be efficient. Thus simple electrostatic arguments for control of the photoyield do not seem applicable. Salt effects associated with the added ionic acceptors which partially neutralize the ionic micellar interface are suggested to be an important factor.

Low molecular weight urinary peptides in ceroid‐lipofuscinoses: Potential biochemical markers for the juvenile subtype

Urine from patients with classical and atypical forms of juvenile ceroid-lipofuscinosis (CL) was analyzed for the presence of disease-specific peptides. Two distinct peptide patterns were recognized on lithium dodecyl sulfate polyacrylamide gel electrophoresis in classical juvenile CL patients. Pattern 1 consisted of a single, intensely staining peptide of apparent Mr 2,000, and up to 4 heterogeneous, weakly staining peptides between 2,500 and 6,300 Mr. This peptide pattern was not seen in over 30 samples from patients with other neurodegenerative disorders, nor in normal control individuals. Reduced amounts of the 2,000 Mr peptide were seen in 2 of 3 female heterozygotes whose children had the peptide pattern 1. The presence of large amounts of the 2,000 Mr peptide in urine extracts made patient identification unequivocal. Pattern 2 had 2 to 3 intensely staining peptides of 3,800, 5,000 and 7,000 Mr, a variable number of minor bands, and diffuse staining above 7,000 and below 3,800 Mr. Parents had 2 to 3 weakly staining peptides with molecular weights similar to the major bands seen in the patients. No consistent peptide pattern was seen in 8 patients with atypical CL. Late infantile CL patients had no or very small amounts of low Mr urinary peptides. The urinary components stained well with silver, poorly with Coomassie Blue, and were digested by a nuclease-free protease, as expected for protein. They were distinctly different from the peptides isolated from ovine CL tissues. Amino acid composition analysis showed a predominantly normal spectrum of amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

The light-harvesting complex of Mamiella gilva: a character linking scaly and naked members of the Micromonadophyceae (Chlorophyta)

The major light-harvesting pigment-protein complex of Mamiella gilva (Parke et Rayns) Moestrup (Micromonadophyceae, Chlorophyta) was isolated by lithium dodecyl sulphate polyacrylamide gel electrophoresis at 4°C. This complex is similar to that of Mantoniella squamata (Manton et Parke) Desikachary and Micromonas pusilla (Butcher) Manton et Parke with regard to molecular weight, absorption spectrum, and immunochemical cross-reactivity. On the other hand, the light-harvesting complex of Chlamydomonas reinhardtii P. Dangeard is quite different. The distribution of this unusual light-harvesting complex, which has to date been reported only in Mamiella, Mantoniella, and Micromonas, suggests a line of evolution in the Micromonadophyceae separate from all other green algae. In addition, the inclusion of the naked Micromonas in this group suggests that Micromonas should be included with Mamiella and Mantoniella in the Mamiellales, an order which is presently defined by scale type.

Study of surfactant layer on alumina by ESR spin probe technique

The surfactant adsorbed layer formed on alumina was examined by using a nitroxide spin probe, 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO). The rotational correlation time of TEMPO changed significantly with the adsorbed amount of surfactant, such as lithium dodecyl sulfate (LiDS) or lithium perfluorooctane sulfonate (LiFOS). This change in the rotational correlation time was reflected by the change in the structure of surfactant aggregate. It was found that the rotational correlation time of TEMPO is considerably greater for the LiFOS layer than for the LiDS layer. Further, in the mixed bilayers of LiDS-polyoxyethylene nonylphenyl ether (NP10) and LiFOS-NP10 systems, the rotational correlation times of TEMPO changed, corresponding to the change of the amounts of NP10 adsorbed.

Comparison of cell-surface L-amino acid oxidases from several marine phytoplankton

Comparison of cell-surface L-amino acid oxidases from several marine phytoplankton

Femtosecond dynamics of energy transfer in B800-850 light-harvesting complexes of Rhodobacter sphaeroides.

We report femtosecond transient absorption studies of energy transfer dynamics in the B800-850 light-harvesting complex (LHC) of Rhodobacter sphaeroides 2.4.1. For complexes solubilized in lauryldimethylamine-N-oxide (LDAO), the carotenoid to bacteriochlorophyll (Bchl) B800 and carotenoid to Bchl B850 energy transfer times are 0.34 and 0.20 ps, respectively. The B800 to B850 energy transfer time is 2.5 ps. For complexes treated with lithium dodecyl sulfate (LDS), a carotenoid to B850 energy transfer time of less than or equal to 0.2 ps is seen, and a portion of the total carotenoid population is decoupled from Bchl. In both LDAO-solubilized and LDS-treated complexes an intensity-dependent picosecond decay component of the excited B850 population is ascribed to excitation annihilation within minimal units of the LHC.

Chlorophyll—protein complexes related to photosystem I in Chlamydomonas reinhardtii

A study of the photosystem I (PS I)-related chlorophyll (Chl)—protein complexes of the unicellular green alga Chlamydomonas reinhardtii was performed, using the wild type and six photosynthesis mutants. Three complexes have been previously described in the literature: CP Ia, undissociated PS I complex; CP 0, Chl a + b peripheral antenna of PS I; CP I, Chl a complex corresponding to the PS I reaction center and its core antenna. Another Chl a complex, CP 0a, has been more recently described by us. The electrophoretic behaviors of these complexes and of their polypeptide subunits have been studied using different solubilization mixtures and lithium dodecylsulfate polyacrylamide gels. It was determined that the non-denatured complex CP 0a shows an apparent relative molecular mass ( M r in the 80 000 range and that two polypeptides of M r = 50 000 and 38 000 resulted from its dissociation. Low temperature fluorescence emission spectra of cells of the wild type and of three mutants were measured both in oxidized and reduced plastoquinone pool conditions. It appeared obvious that, in a CP 0a-lacking mutant, the excitation energy transfer from the main light-harvesting antenna towards PS I was impaired and inefficient. This is interpreted as indicating that CP 0a plays the essential role of a connecting antenna between the peripheral antenna CP 0 and the core antenna (CP I) of PS I.

Fluorescence Probe Studies of Ionic Micelles in Concentrated Salt Solutions

AbstractWe have investigated the effect of salt concentration and temperature on the average aggregation number and micro‐polarity of the interior of micelles of sodium dodecyl sulfate (SDS). sodium tetradecyl sulfate (STDS) and lithium dodecyl sulfate (LiDS). The transient fluorescence decay of micelle‐solubilized pyrene has been measured and analyzed. An exponent weighted average aggregation number <n>e was obtained by this technique. For SDS and STDS in NaCl solution, <n>e increases as the temperature is lowered or salt concentration is increased <n>e increased from ∼ 50 to ∼ 250 over [NaCl] = 0 to 0.8 M. Due to the strong counterion binding of lithium in the micellar solution, the LiDS micelle is much, smaller and does not increase appreciabily even at [LiCl] =0.8 M. From the fluorescence spectrum fine structure of pyrene and the fluorescence decay of the monomer and excimer, we can understand the local polarity and the water penetration to the interior of the micelle upon addition of salts and with changing temperature. The interior of the micelle becomes more nonpolar as the salt concentration is increased and the temperature is lowered. A complete kinetic analysis of the time–dependence of the fluorescence is given. The kinetic analysis is in agreement with the results reached by fluorescence spectral analysis.

Adsolubilization Behavior of Dyes Caused by Mixed Surfactant Bilayers Formed on Alumina

Abstract The adsolubilization of dyes (yellow OB and azobenzene) was performed on mixed bilayers consisting of anionic and nonionic surfactants formed on α-alumina. The adsolubilization capacity of the two dyes increased with the increase in the concentration of the dyes solubilized in the supernatant. The mixed bilayer consisting of anionic hydrocarbon (lithium dodecyl sulfate) and nonionic hydrocarbon surfactants (polyoxyethylene nonylphenyl ether) showed a greater adsolubilization capacity than that consisting of anionic fluorocarbon (lithium perfluorooctanesulfonate) and nonionic hydrocarbon surfactants. Further, the adsolubilization capacity of the dyes was affected by the oxyethylene chain length of the nonionic hydrocarbon surfactant in the mixed bilayer.

The effect of alcohols on the properties of aqueous solutions of hydrocarbon and fluorocarbon surfactants

The effects of hydrocarbon alcohols (methanol to hexanol) and fluorocarbon alcohols (fluoroethanol to fluoropentanol) on the micellar properties (CMC, polarity of micelle interior) of two anionic surfactants, lithium dodecyl sulfate (LiDS) and lithium perfluorooctanesulfonate (LiFOS), have been investigated by means of electrical conductivity and fluoroescence of micelle-solubilized pyrene. Partition coefficients K of butanol, pentanol, hexanol, 2,2,3,3,3,-pentafluoropropanol, 2,2,3,3,4,4,4-heptafluorobutanol, and 2,2,3,3,4,4,5,5,5-nonafluoropentanol between aqueous phase and micellar pseudophase of LiDS and LiFOS have been determined using a saturation solubilization method. Moreover, aggregation numbers in surfactant-alcohol mixtures were obtained using time-resolved fluorescence. In all instances, apart from LiDS in the presence of MeOH, the effect of alcohol addition is to decrease the CMC. The decrease of CMC is larger for a surfactant-alcohol pair with alkyl chains of the same kind. From the values of the ratio of the fluorescence intensity of the first and third fluorescence emission peaks from monomeric pyrene, it appears that the solubilization of fluorocarbon alcohols into micelles causes a large decrease of the polarity of the micelle palisade layer than that of hydrocarbon alcohols. The standard free energy changes (Δ G°) for transfer of a methylene group from water to LiDS and LiFOS micelles, calculated from the partition coefficient, are −855 and −760 cal/mole, respectively. The values for the transfer of a perfluoromethylene group are −1090 cal/mole for LiDS and −1160 cal/mole of LiFOS micelles. These values are interpreted in terms of the favorable solubilization of alcohol into micelles of surfactant having the same kind of alkyl chain. From time-resolved dluorescence measurements, the surfactant aggregation number was found to be decrease with increasing alcohol concentration. Fluorocarbon alcohol solubilization into fluorocarbon micelles was found to increase the rate of exit of hydrocarbon solutes whereas hydrocarbon alcohols tended to decrease this rate.

Cytochromes of plant plasma membranes. Characterization by absorbance difference spectrophotometry and redox titration

The cytochrome composition of plasma membranes (PM) obtained by phase partitioning of microsomal fractions from spinach leaves (Spinacea oleracea L. cv. Medania), cauliflower inflorescences (Brassica oleracea L.), sugar beer leaves (Beta vulgaris L.) and barley (Hordeum vulgare L. cv. Kristina) roots and leaves was characterized by absorbance difference spectrophotometry at different reducing conditions at 20 and – 196°C, by redox titration, and by heme staining of polypeptide bands after lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS‐PAGE). The location of the α‐bands in the difference spectra and the loss of heme after treatment with LDS indicated that predominantly cytochromes of the b‐type were present in all species tested. The total concentration of cytochrome was ca 0.35 nmol (mg protein)−1. The main component (ca 70% of total) was completely reduced by ascorbate and partly by NADH and had a midpoint potential of ca 150 mV. At – 196°C, ascorbate reduction revealed a symmetrical α‐band at ca 557 nm with PM from spinach leaves, cauliflower and sugar beet leaves, but with barley root and leaf PM ascorbate reduction resulted in an asymmetrical α‐band (shoulder at 552, maximum at 559 nm). In the dithionite‐reduced minus ascorbate‐reduced spectrum at –196°C a split α‐band (552 + 558 nm) was seen with PM from all species. This minor component had a midpoint potential of ca – 50 mV and is probably identical to cytochrome b5, the presence of which would explain the relatively high NADH‐cytochrome c reductase activities observed with plant PM. With PM from cauliflower, CO‐difference spectra indicated that cytochromes P‐420 and P‐450 were present at concentrations up to 0.06 and 0.03 nmol (mg protein)−1, respectively. Visualization of cytochromes by heme staining after LDS‐PAGE was complicated by endogenous peroxidase activity and by loss of heme during solubilisation. A presumptive b‐cytochrome (heme‐stained band at 94 kDa) was only detected with barley leaf PM.

Characterization of the ndhC-psbG-ORF157/159 operon of maize plastid DNA and of the cyanobacterium Synechocystis sp. PCC6803

The ndhC and ORF159 genes of the maize plastid DNA (ptDNA) were sequenced and maize ORF159 was used to screen a library of genomic DNA of the blue-green alga Synechocystis sp. PCC 6803. The cyanobacterial gene homologous to ORF159 (ORF157) was isolated and sequenced. In sequencing the region upstream of ORF157, reading frames with homology to the ndhC and psbG genes of maize ptDNA were identified. The ndhC and psbG genes overlap in the ptDNAs of maize, tobacco and Marchantia polymorpha, but are separated by a noncoding spacer in Synechocystis. Northern blot analysis showed that the ndhC, psbG and ORF157/159 genes are cotranscribed in maize and Synechocystis. The three genes occur in the same order in ptDNA of maize, tobacco, and M. polymorpha as in Synechocystis 6803. The amino acid sequences of the NDH-C, PSII-G and the ORF157/159 proteins deduced from the maize genes are 65%, 52% and 53% homologous to those of Synechocystis. However, the cyanobacterial and higher plant NDH-C protein sequences are only 23% homologous to the mitochondrial NDH-3 protein. Protein products of in vitro transcription/translation of the Synechocystis transcription unit had apparent molecular masses of 6 kDa (NDH-C), 25 kDa (PSII-G) and 22 kDa (ORF157) on lithium dodecyl sulfate (LDS) polyacrylamide gel electrophoresis. If these are components of an NADH dehydrogenase, cyanobacteria appear to resemble mitochondria more than they do Escherichia coli and Rhodopseudomonas capsulata with regard to this enzyme complex.

The construction and properties of a mutant of Rhodobacter sphaeroides with the LH1 antenna as the sole pigment protein

Recent work on the spectroscopic properties of the B875 (LH1) antenna of Rb. sphaeroides has been performed on the complex purified from membranes of the wild-type solubilised in lithium dodecyl sulphate. In order to facilitate an examination of the properties of the membrane-bound antenna free from other complexes, mutant M2192 was constructed from mutant M21, which contains reaction centres and LH1 but lacks the B800–850 (LH2)complex. This was accomplished by insertion of transposon Tn5 into the puf L gene which encodes the L polypeptide of the reaction centre. This manipulation leaves B875 as the sole pigment protein, which has been confirmed by Southern and Northern hybridisation, gel electrophoresis and fluorescence and absorbance spectroscopy. Evidence from Gaussian deconvolution of the Qy absorbance region, and from fluorescence polarisation, suggests that the long-wavelength species BChl 896 is present, and may be an inherent property of the LH1 antenna.

Maturation-related gene expression of rat astroblasts in vitro studied by two-dimensional polyacrylamide gel electrophoresis

Maturation of rat astroblasts was induced in vitro by acidic fibroblast growth factor (aFGF), hydrocortisone and dibutyryl cyclic AMP. Cells grown for 20 days were treated for 48 h and labelled with [35S]methionine only during the last 18 h of treatment. Cell proteins solubilized in lithium dodecyl sulfate were submitted to two-dimensional polyacrylamide gel electrophoresis. About 300 radioactive proteins could be analysed visually and compared. All treatments induced visible quantitative and sometimes qualitative changes. A total of 81 proteins had their rate of biosynthesis modified. For some proteins, this rate was changed by only one treatment, while for others it was changed by two or even by the three treatments, mostly in the same way. These results are consistent with the hypothesis that the proteins involved in the maturation process are organized in sets, proteins belonging to one set always being regulated together under a common control. Some sets would be regulated by only one effector while others would be regulated by several effectors.

Electron spin resonance and electron spin echo modulation of n-doxylstearic acid and N,N,N',N'-tetramethylbenzidine photoionization in sodium versus lithium dodecylsulfate micellar solutions: effect of 15-crown-5 and 18-crown-6 ether addition

Electron spin echo modulation and electron spin resonance spectra of photogenerated N,N,N',N'-tetramethylbenzidine (TMB) cation radical and n-doxylstearic acids (n-DSA) in frozen micellar solutions of sodium and lithium dodecyl sulfate containing 15-crown-5 and 18-crown-6 ethers in D/sub 2/O have been studied as a function of crown ether concentration. Modulation effects due to N-DSA with water deuteriums give direct evidence that both crown ethers are mainly located at the micellar interface and that this causes a decrease of the hydration of the micellar interface. Crown ether complexation constants for sodium and lithium micellar counterions are reported and show that 18-crown-6 > 15-crown-5 for sodium counterion and 15-crown-5 > 18-crown-6 for lithium counterion. Modulation effects from TMB/sup +/ interaction with water deuteriums indicate that the TMB molecule moves toward the micelle interfacial region when sodium or lithium cations are complexed by crown ethers. The TMB/sup +/ yield upon TMB photoionization increases by about 10% with crown ether addition for SDS and LDS micellar systems, but it is greater if the absolute values for the LDS system are compared to those for the SDS micellar system. This behavior correlates with the strength of TMB/sup +/-water interactions and suggests that the main factor in themore » photoionization efficiency is the photocation-water interaction.« less

A resonance Raman investigation of the effect of lithium dodecyl sulfate on the B800–850 light-harvesting protein of Rhodopseudomonas acidophila 7750

The effect of the detergent lithium dodecyl sulfate (LDS) on the resonance Raman spectrum of the carotenoid and bacteriochlorophyll in the B800–850 light-harvesting complex Rhodopseudomonas acidophila 7750 was determined. Treatment of B800–850 complex with 0.1% LDS resulted in a 3 cm −1 downshift of the carotenoid v 3 Raman band, a 2 cm −1 upshift of the carotenoid v 1 band, with no change in the v 2. This indicates that LDS treatment induces a 7- cis-isomerization of the carotenoid and a supplementary torsion of the polyene chain around the C(8)–C(9) bond. Also, treatment of B800–850 complex with LDS resulted in several changes attributable to alterations in the nature of the binding to the protein of the acetyl and keto-carbonyls of one bacteriochlorophyll. No change was observed in the coordination of the bacteriochlorophyll central magnesium. These data indicate that the 800 nm-absorbing bacteriochlorophyll remains bound to the protein upon LDS treatment. The resonance Raman data indicate that the pronounced absorption spectral changes reported previously (Chadwick, B.W., Zhang, C., Cogdell, R.J. and Frank, H.A. (1987) Biochim. Biophys. Acta 893, 444–451) are attributable to a dramatic perturbation of the protein environment upon LDS treatment of the B800–850 complex.

Studies on the interaction of matrix-bound inhibitor with Band 3, the anion transport protein of human erythrocyte membranes

The effect of temperature and chemical modification on the interaction of the human erythrocyte Band 3 protein (the anion transport protein) with 4-acetamido-4′-isothiocyanostilbene 2,2′-disulfonate (SITS; K i = 10 μ M)-Affi-Gel 102 resin was studied. Band 3 binds to the affinity resin in two states; weakly bound, which is eluted by 1 mM 4-benzamido-4′-aminostilbene 2,2′-disulfonate (BADS; K i = 2 μ M ), and strongly bound, which is eluted only under denaturing conditions by 1% lithium dodecyl sulfate (LDS). At 4°C, most of band 3 was present initially in the weakly bound form and very little in the strongly bound form. With longer incubations at 4°C, the weakly bound form was slowly converted to the strongly bound form. At 37°C, most of Band 3 was rapidly converted to the strongly bound form, with some Band 3 still remaining in the weakly bound form. Band 3 dimers, labelled with 4,4′-diisothiocyanostilbene 2,2′-disulfonate (DIDS) in one monomer, did bind to immobilized SITS but did not become tightly bound upon incubation at 37° C. Since the covalent attachment of DIDS to one monomer prevented the adjacent monomer from becoming tightly bound to immobilized SITS ligand, this observation suggests that the inhibitor-binding sites of the two adjacent monomers must be interacting with each other. When the inhibitor site of Band 3 was selectively modified by citrate in the presence of 1-ethyl-3-(3-azonia-4,4-dimethylpentyl)carbodiimide (EAC), Band 3 bound to the resin was more easily eluted by BADS, suggesting reduced affinity for immobilized SITS. However, citrate-modified Band 3 did become tightly bound upon incubation at 37°C.

Immunobiological activities of a porin fraction isolated from Fusobacterium nucleatum ATCC 10953.

From Fusobacterium nucleatum ATCC 10953 cell envelope fraction whose inner membranes had been removed by treatment with sodium N-lauroyl sarcosinate, an outer membrane protein (37,000 Mr in a native state) was prepared by extraction with lithium dodecyl sulfate. The protein thus obtained showed distinct porin activity, namely, the ability to form hydrophilic diffusion pores by incorporation into the artificial liposome membrane. The porin fraction exhibited strong immunobiological activities in the in vitro assays: B-cell mitogenicity and polyclonal B-cell activation on murine splenocytes, stimulatory effects on guinea pig peritoneal macrophages, and enhancement of the migration of human blood monocytes. The porin fraction also exhibited immunoadjuvant activity to increase the antibody production against sheep erythrocytes in the spleen of mice that were immunized by sheep erythrocytes with porin. Although chemical analyses revealed that the test porin fraction contained a considerable amount of lipopolysaccharide (LPS) (around 12% of the fraction), the studies with LPS-nonresponding C3H/HeJ mice and on the inhibitory effects of polymyxin B strongly suggest that most of the above bioactivities are due to porin protein itself, not to coexistent LPS in the porin fraction.