Article

Relation of Detergent HLB Number to Solubilization and Stabilization of D-Alanine Carboxypeptidase from Bacillus subtilis Membranes

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Abstract

The ability of various nonionic detergents to solubilize D-alanine carboxypeptidase and other membrane-bound enzymes was correlated to a physical property of the detergent, the HLB number. Only a fairly narrow range of detergents were effective solubilizing agents. Purified carboxypeptidase required either a detergent or detergent plus a lipid fraction for stability. Only those detergents effective in solubilizing the enzyme were effective in stabilizing it.

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... The larger the molecular weight of a detergent head compared to its tail, the larger the HLB value. Detergents that are good in solubilizing membranes commonly have a HLB between 12 and 14, such as DDM (13.3) and C8E4 (12.6) (Table S1 and S3) [25]. The hybrid Fig. 3. Investigating delipidation outcomes with native mass spectrometry. ...
... detergent has a larger head and is therefore more polar compared to DDM and C8E4, as indicated by a larger HLB (15.2). Considering this background, this indicates that increasing the polarity of detergents, as indicated by a larger HLB, decreases their ability to solubilize membranes [25]. Although the hybrid detergent can stabilize AqpZ in solution, it is less efficient in disrupting lipid membranes and lower protein yields are obtained. ...
Article
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Non-ionic detergents are important tools for the investigation of interactions between membrane proteins and lipid membranes. Recent studies led to the question whether the ability to capture protein-lipid interactions depends on the properties of detergents or their concentration in purification buffers. To address this question, we present the synthesis of an asymmetric, hybrid detergent that combines the head groups of detergents with opposing delipidating properties. We discuss detergent properties and protein purification outcomes to reveal whether the properties of detergent micelles or the detergent concentration in purification buffers drive membrane protein delipidation. We anticipate that our findings will enable the development of rationally design detergents for future applications in membrane protein research.
... It is nevertheless known that polyoxyethylene(20) oleyl ether is a rather "strong" surfactant that disintegrates lipid bilayers completely at an effective molar lipid/surfactant ratio R sat e , 1 (Heerklotz and Seelig 2000); Cn C EOn EO surfactants are similarly effective when used on biological membranes (Preté et al. 2002). This is due to relatively low water solubility of such surfactants, which for C 18:1 EO 20 is CMC ¼ 25 mM (Umbreit and Strominger 1973;Klammt et al. 2005), and to the correspondingly high molar partition coefficient, which for C 18:1 EO 20 in PC bilayers amounts to log K < 2 4.3 M 21 (Heerklotz and Seelig 2000). This exceeds partition coefficient of C 12 EO 8 (CMC ¼ 70 mM (Edwards and Almgren 1991); CMC ¼ 90 mM ) in egg yolk PC: log K < -2.1 M 21 (Edwards and Almgren 1991) to log K < 2 2.8 M 21 (Levy et al. 1990). ...
... With the molar partition coefficient value of log K , 4.3 M 21 (Heerklotz and Seelig 2000a, 2000b), we calculated the corresponding free, i.e. monomeric, surfactant concentration from Equation (8) to be c s,f ¼ 0-12 mM. This is reasonably close to the independently measured value of CMC ¼ 25 mM (Umbreit and Strominger 1973;Klammt et al. 2005). Lichtenberg (1993) and Lichtenberg et al. (2000) reported for most surfactants free monomer concentrations at the saturation limit below critical micelle concentration: c sat s;f , CMC. ...
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Novel potential carriers for non-invasive drug delivery were prepared from polyoxyethylene(20) oleyl ether (C(18:1)EO(20)) and soybean phosphatidylcholine (SPC) in different relative molar ratios, R(e); this produced stiff SPC liposomes (2r(ves) approximately 120 nm) at one end and much smaller (2r(mic) <or= 15 nm), comparably non-deformable, mixed micelles at the other end of aggregate spectrum. Deformability of the mixed amphiphat vesicles (2r(v) approximately 105 nm) existing in-between increases with R(e) non-linearly, up to a quasi-plateau at R(e) >or= R(e)(sat) = 0.25 in the bilayer. The surfactant-saturated bilayers exhibit bending rigidity of kappa(c) approximately 2.1 k(B)T, as determined with an improved vesicle adaptability assay involving analysis of normalised flux density through a nano-porous barrier as an activated transport process. Pore penetrability vs. driving pressure data measured with the mixed amphiphat vesicles resemble results of computer simulation of deformable vesicles penetrating a constriction [Gompper G, Kroll DM. 1995. Driven transport of fluid vesicles through narrow pores. Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Topics. 52:4198-4208], confirming basic similarity of both processes. The improved assay can reveal partial lipid solubilisation at R(e)>R(e)(sat), which is linearly proportional to R(e) - R(e)(sat). C(18:1)EO(20)-SPC mixed vesicles that can cross narrow pores are arguably suitable for targeted drug delivery across intact skin.
... B30 has a low critical micelle concentration (0.02-0.04 mM [18]), high aggregation number (90 [19]), low cloud temperature (4°C [20]), and low hydrophilic-lipophilic balance (9.7). By itself, it holds the ability to solubilize membrane proteins [21]. In contrast to B30, DPS is a mild zwitterionic surfactant which has been used as a component of buffers designed for solubilizing membranes and nuclear proteins for 2D electrophoresis [22]. ...
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Collection of cytosolic proteins from cells and tissues is the first and essential step in many bioanalytical assays that play a key role in medical applications such as diagnostics, theranostics, and regenerative medicine. Dissolution of cell and tissue constituents without deactivation of their constituents, especially proteins, is a challenging task. Here, we report on a recently identified cell and tissue lysis agent in terms of its ability to solubilize cells and tissues as well as preservation of cellular proteins, particularly enzymes. The lysis agent comprises a mixture of a zwitterionic surfactant, N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (DPS) and a non-ionic surfactant, Brij 30 (B30). Mixtures of DPS and B30 successfully solubilized keratinocytes and human vascular endothelial cells in vitro while preserving detectable quantities of cellular enzymes including glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase for at least 4 h. Mixtures of DPS and B30 were also effective in solubilizing tissues, especially tough tissues such as skin in vitro. Collectively, the mixture of DPS/B30 was effective in solubilizing cells and tissues while preserving its constituent proteins, which opens up its applications for use in studying the effect of environmental factors on tissue proteomics. As an example, the ability of DPS/B30 to detect alterations in skin proteins in response to UV exposure was assessed. These studies revealed that UV exposure induce upregulation of a number of inflammatory, apoptotic and stress-activated proteins as well as downregulation of cell cycle progression proteins.
... TX has been shown to release proteins associated with the cytoplasmic membrane of both gram-positive (44) and gram-negative (35, 36) bacterial species. Within a Triton X series of surfactants of varying hydrophobic carbon chain lengths, those with a chain length between 16 and 24 carbon atoms were most efficient for the extraction of the membrane-associated carboxypeptidase of Bacillus subtilis (44), for the extraction of rat liver mitochondrial proteins and phospho-lipids (15), and for inducing an autolytic response with growing cultures of S. faecalis (data not shown). ...
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Lysis of exponential-phase cultures of Streptococcus faecalis ATCC 9790 was induced by exposure to both anionic (sodium dodecyl sulfate) and nonionic (Triton X-100) surfactants. Lysis in response to sodium dodecyl sulfate was effective only over a limited range of concentrations, whereas Triton X-100-induced lysis occurred over a broad range of surfactant concentrations. The data presented indicate that the bacteriolytic response of growing cells to Triton X-100: (i) was related to the ratio of surfactant to cells and not the surfactant concentration per se; (ii) required the expression of the cellular autolytic enzyme system; and (iii) was most likely due to an effect of the surfactant on components of the autolytic system that are associated with the cytoplasmic membrane. The possibility that Triton X-100 may induce cellular lysis by releasing a lipid inhibitor of the cellular autolytic enzyme is discussed.
... Only a small fraction enters the detergents phase of Triton X-114. Less than half of the protein is extracted with non-ionic detergents (Fig. 2) and, using various detergents, there is not the sharp extraction optimum for detergent polarity (measured by the HLB — hyrophile/lipophile balance number, a commercial scale of 1– 20 with increasing hydrophilicity of the detergent) that is often seen with integral membrane proteins, e.g. [158]. It is possible that lack of extractability by neutral detergents could mean membrane attachment of a detergent resistant oligomer or aggregate because ionic detergents such as SDS and cetyl tetramethyl ammonium bromide completely solubilize all of this resistant tubulin. ...
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The association of tubulin with the plasma membrane comprises multiple levels of penetration into the bilayer: from integral membrane protein, to attachment via palmitoylation, to surface binding, and to microtubules attached by linker proteins to proteins in the membrane. Here we discuss the soundness and weaknesses of the chemical and biochemical evidence marshaled to support these associations, as well as the mechanisms by which tubulin or microtubules may regulate functions at the plasma membrane.
... Furthermore, the incentive of solubilizing membrane protein in 2D gels has always been a driving force to test non-classical detergents in conjunction with urea as protein solubilizers for 2D gels. As Triton X- 100 is known to be one of the best solubilizers of the polyethylene glycol-based detergents [76], the interest has turned to other classes of uncharged detergents, such as glucosides [77], but also members of the sulfobetaine class. The first one to be used, and by far still the most popular, was CHAPS [78]. ...
Article
Electrophoretic separations of proteins are widely used in proteomic analyses, and rely heavily on SDS electrophoresis. This mode of separation is almost exclusively used when a single dimension separation is performed, and generally represents the second dimension of two-dimensional separations. Electrophoretic separations for proteomics use robust, well-established protocols. However, many variations in almost all possible parameters have been described in the literature over the years, and they may bring a decisive advantage when the limits of the classical protocols are reached. The purpose of this article is to review the most important of these variations, so that the readers can be aware of how they can improve or tune protein separations according to their needs. The chemical variations reviewed in this paper encompass gel structure, buffer systems and detergents for SDS electrophoresis, two-dimensional electrophoresis based on isoelectric focusing and two-dimensional electrophoresis based on cationic zone electrophoresis.
... The reason why the encapsulation efficiency is sensitive to the ratio of PEG-b-PCL and Pluronic F-108 is that PEG-b-PCL and Pluronic F-108 have different hydrophilic-lipophilic balance (HLB) numbers. According to Griffin's formula [22,23], the expression pertaining to PEG-b-PCL HLB numbers is 20 × W PEG /W copolymer . Here W PEG is the weight of the hydrophilic portion and W copolymer is the weight of main chain polymer. ...
Article
7-Ethyl-10-hydroxycamptothecin (SN-38) loaded poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (Pluronic F-108) and poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-b-PCL) nanoparticles were successfully prepared by a modified film hydration method and characterized by scanning electric microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). Satisfactory drug loading of 20.73 ± 0.66% and a high encapsulation efficiency of 83.83 ± 1.32% were achieved. The SN-38 nanoparticles (SN-38 NPs) can completely disperse into a phosphate buffered saline (PBS) medium to produce a clear aqueous suspension that remains stable for up to three days. Total drug releases were 67.91% and 91.09% after 24 h in a PBS or fetal bovine serum (FBS) medium. Half maximal inhibitory concentration (IC(50)) tests of SN-38 and SN-38 NPs on A549 lung cells produced results of 200.0 ± 14.9 ng ml(-1) and 80.0 ± 4.6 ng ml(-1), respectively. Similarly, IC(50) tests of SN-38 and SN-38 NPs on MCF-7 breast cells yielded results of 16.0 ± 0.7 ng ml(-1) and 8.0 ± 0.5 ng ml(-1), respectively. These in vitro IC(50) studies show significant (p < 0.01) enhancement of the SN-38 NP drug efficiency in killing cancer cells in comparison to the free drug SN-38 control. All the materials used for this nanoformulation are approved by the US FDA, with the virtue of extremely low toxicity to normal cells.
... 28 The HLB values of BA-PEGs are expected to be similar to that of sodium cholate (HLB = 18) 34 or those of Tween surfactants (HLB = 11−17 for Tweens with 20 EG units). 34,35 Fluorescence Studies. Time-Resolved Fluorescence. ...
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Bile acids are amphiphilic endogenous steroids that act as anionic surfactants in the digestive tract and aggregate in aqueous solutions. Nonionic surfactants were synthesized by grafting poly(ethylene glycol) chains of various lengths (pegylation) to three bile acids (lithocholic, deoxycholic, and cholic acid) using anionic polymerization. The aggregation properties of the derivatives were studied with viscosity measurements and light scattering as well as with steady-state and time-resolved fluorescence techniques, and the aggregates were visualized by transmission electron microscopy to elucidate the effect of pegylation on the aggregation process. The fluorescence results showed a good correlation with the capacity of the bile acid derivatives to solubilize a hydrophobic drug molecule. The solubilization of ibuprofen depends on the length and the number of grafted PEG chains, and the solubilization efficiency increases with fewer PEG chains on the bile acid. The results indicate their potential for use in the design of new bile acid-based drug-delivery systems.
... The critical micelle concentration for Triton X-100 depends on the medium, but is approximately 0.02% (20,47). Inhibition of growth of adapted cells became severe only at concentrations approaching the critical micelle concentration (Fig. 2b). ...
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Variants of Bacillus subtilis resistant to the detergent Triton X-100 may exhibit: (i) normal cell morphology, (ii) reduced cell diameter, or (iii) helical cell shape. One variant of type ii was studied in some detail. Triton resistance, cell diameter reduction, and poor sporulation all may have resulted from a single mutation. High concentrations of Triton caused rapid lysis of wild-type cells. B. subtilis adapted to low Triton concentrations such that, upon subsequent exposure to higher concentrations, growth continued, although it bacame inhibited at very high concentrations. The variant studied retained its sensitivity to Triton-induced lysis but, after adaptation, grew at very high Triton levels. In this strain, cell diameter and cross-sectional area were reduced to about 73 and 50%, respectively, of those of wild type, yet the cells grew at normal rates, and DNA/protein/RNA ratios were largely unaltered. Peptidoglycan content per unit of cell surface area was higher in the variant than in the wild type under at least certain growth conditions.
... This non-ionic detergent has a total of 20 ethylene oxide units (x + y + w + z) and the lauric acid as the fatty acid tail. 15 Supporting Figure 3. The structure of Brij 35. ...
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NADPH-cytochrome P450 oxidoreductase (POR) is a membrane protein in the endoplasmic reticulum of eukaryotic cells. POR is as a key reducing partner for a number of cytochrome P450 proteins involved in different metabolic degradation and signaling pathways. Preparation of the full-length recombinant POR expressed in bacteria has been reported and, typically, involved the use of Triton X-100 detergent for extraction of the overexpressed POR from bacterial membranes. However, extraction efficiency is always relatively low hindering structural studies, particularly—the NMR spectroscopy requiring isotopic enrichment. In this paper, we assessed the effect of a variety of detergents and additives on the efficiency of the membrane-extraction step in POR preparation protocol. We evaluated non-ionic detergents with the variable hydrophobicity (Triton X-100, X-114, and X-405) and structure (Triton X-100, TWEEN-20, Brij-35), a zwitterionic/non-ionic detergent combination (Triton X-100 and CHAPS), as well as a range of alkylamines and polyamines as additives to the conventional extraction buffer containing Triton X-100. None of the detergents or detergent-additive combinations yielded better extraction efficiency than the conventional protocol with the Triton X-100. Lack of variation of the extraction yield allows to hypothesize that the conventional protocol extracts all of the available natively-folded monomeric POR while the remaining fraction is possibly an unfolded aggregated POR, which did not insert in the membranes during expression. We propose that the yield of soluble POR may be increased by a careful optimization of expression conditions while monitoring the distribution of POR between soluble and insoluble fractions in the detergent extraction step.
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HLA antigens have been purified to homogeneity after detergent solubilization from RPMI 4265, a human lymphoblastoid line. The inhibition of cytotoxicity assay for HLA antigen was modified, using preincubation with bovine serum albumin of antigen samples containing detergent to prevent lysis of target cells by detergent. Solubilization was tested with many types of detergents. A polyethyleneglycol oleyl ether nonionic detergent mixture, Brij 99:Brij 97 (2:1) was selected for solubilization, since it selectively solubilized HLA antigens, had a low absorbance at 280 nm and was uncharded. HLA antigens were then purified by Lens culinaris lectin affinity chromatography and Bio-Gel A-5m filtration. The antigen specifity HLA-A2 was separated from specificities HLA-B7,12 by isoelectric focusing. Purified HLA antigens contained a subunit of Mr=44,000 with NH2-terminal glycine, and a subunit of Mr=12,000, beta2-microglobulin, with NH2-terminal isoleucine.
Article
Separation/purification of nuclei from cells is a critical process required for medical and biochemical research applications. Here, we report a flow-through microfluidic device for isolating cell nuclei by selectively digesting the cell membrane by using the concept of hydrodynamic filtration (HDF). When a cell suspension is continuously introduced into a microchannel (main channel) possessing multiple side channels, cells flow through the main channel, whereas the carrier medium of the cells is drained through the side channels. Introductions of a cell treatment solution containing a surfactant and a washing buffer enable the two-step exchange of the carrier-medium and the cell treatment by the surfactant for a short span of time. The precise control of the treatment time by changing the flow rate and/or the size of the microchannel enables the selective digestion of cell membranes, resulting in the isolation of cell nuclei after separation from membrane debris and cytoplasmic components according to size. We examined several surfactant molecules and demonstrated that Triton X-100 exhibited high efficiency regarding nucleus isolation for both adherent (HeLa) and nonadherent (JM) cells, with a recovery ratio of ~80 %. In addition, the isolation efficiency was evaluated by western blotting. The presented flow-through microfluidic cell-nucleus separator may be a useful tool for general biological applications, because of its simplicity in operation, high reproducibility, and accuracy.
Article
Hemolytic activity of nonionic surfactants, polyoxyethylene cholesteryl ethers, C27H45O(CH2CH2O)nH (Chol-En, n=, 25, 30, 50) and polyoxyethylene dihydrocholeseryl ethers, C27H47O(CH2CH2O)nH (DHChol-En, n=15, 30 50) were measured, changing the concentration of surfactant and erythrocyte at 37 C. Maximum hemolytic activity was observed in these cholesteryl derivatives with 25–30 oxyethylene units. The time course of hemolysis was also measured as a function of the concentrations of surfactant and erythrocyte. Hemolysis started after a certain induction period,, and then apparently proceeded as a first-order reaction with respect to the erythrocyte concentration. The surfactant inducing 50% hemolysis at low concentration had a small value and large rate constant. The maximum amount of adsorption without inducing hemolysis,a0, decreased with increasing polyoxyethylene chain length. Chol-E25 has the maximum activity for the solubilization of egg yolk lecithin at 37 C. Based on these results, the mechanism of hemolysis by these surfactants was quantitatively discussed.
Article
This paper investigates the effects of a macromolecular amphiphile poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO–PPO–PEO) on a photoresponsive membraneprotein, bacteriorhodopsin (BR). After incubation of BR in EO23–PO65–EO23 (P123) solution, BR maintained its function as a light-driven proton pump; however, the rate of proton uptake and lifetime of the M intermediate in the photocycle of BR upon illumination were, under appropriate conditions, prolonged by about three orders of magnitude compared with that of native BR, even at neutral pH. Measurements using circular dichroism spectroscopy and dynamic light scattering indicated that BR molecules were still in a trimer state after treatment with the copolymers. This is quite different to BR, which showed a much slower photoresponse during drying. The BR–P123 assemblies did not exhibit significantly different photoresponsive behavior with changes to the water content, which implied that in the case of dried BR films or dried BR–polymer films, the elongation of the M decay may be caused not by lack of water molecules necessary for proton transfer, but by protein immobilization. Determination of the critical micelle concentration of P123 with and without BR revealed that this prolongation effect is closely related to the formation of micelles. The above phenomenon was also observed with 6 other Pluronic copolymers. In solutions of small molecular detergents, such as Triton X-100, the photoresponse of BR was prolonged as well; the extent of prolongation was, however, much less than in solutions of macromolecular amphiphiles. The formation of a local polymer coating due to self assembly of the copolymer and protein molecules might be responsible for this very significant prolongation effect, which is beneficial for the potential application of BR as an information material.
Article
Nonionic surfactants of the Triton X-series find various applications in extraction processes and as solubilizing agents for the purification of membrane proteins. However, so far no optimized parameters are available to perform molecular simulations with a biomolecular force field. Therefore, we have determined the first optimized set of CHARMM parameters for the Triton X-series, enabling all-atom molecular dynamics (MD) simulations. In order to validate the new parameters, micellar sizes (aggregation numbers) of Triton X-114 and Triton X-100 have been investigated as a function of temperature and surfactant concentration. These results are comparable with experimental results. Furthermore, we have introduced a new algorithm to obtain micelle structures from self-assembly MD simulations for the COSMOmic method. This model allows efficient partition behavior predictions once a representative micelle structure is available. The predicted partition coefficients for the systems Triton X-114/water and Triton X-100/water are in excellent agreement with experimental results. Therefore, this method can be applied as screening tool to find optimal solute-surfactant combinations or suitable surfactant systems for a specific application.
Article
Bioremediation of hydrophobic organic compounds (HOCs) contaminated soils involves several physicochemical and microbiological interfacial processes among the soil-water-microorganism interfaces. The participation of surfactants facilitates the mass transport of HOCs in both the physicochemical and microbiological interfaces by reducing the interfacial tension. The effects and underlying mechanisms of surfactants on the physicochemical desorption of soil-sorbed HOCs have been widely studied. This paper reviewed the progress made in understanding the effects of surfactant on microbiological interfacial transport of HOCs and the underlying mechanisms, which is vital for a better understanding and control of the mass transfer of HOCs in the biodegradation process. In summary, surfactants affect the microbiological interfacial behaviors of HOCs during three consecutive processes: the soil solution-microorganism sorption, the transmembrane process, and the intracellular metabolism. Surfactant could promote cell sorption of HOCs depending on the compatibility of surfactant hydrophile hydrophilic balance (HLB) with cell surface properties; while the dose ratio between surfactant and biologic mass (membrane lipids) determined the transmembrane processes. Although surfactants cannot easily directly affect the intracellular enzymatic metabolism of HOCs due to the steric hindrace, the presence of surfactants can indirectly enhanced the metabolism by increasing the substrate concentrations.
Article
Microfluidic paper-based analytical devices (μPADs) fabricated by wax-printing are suitable platforms for the development of simple and affordable molecular diagnostic assays for infectious diseases, especially in resource-limited settings. Paper devices can be modified for biological assays by adding appropriate reagents to the test areas. For this purpose, the use of affinity immobilization strategies can be a good solution for bioactive paper fabrication. This paper describes a methodology to capture labeled-DNA strands and hybrids on paper via the anchoring of antibodies with a fusion protein that combines a family 3 carbohydrate binding module (CBM) from Clostridium thermocellum, with high affinity to cellulose, and the ZZ fragment of the staphyloccocal protein A, which recognizes IgG antibodies via their Fc portion. Antibodies immobilized via CBM-ZZ were able to capture appropriately labeled (biotin, fluorescein) DNA strands and DNA hybrids. The ability of an antibody specific to biotin to discriminate complementary from non-complementary, biotin-labeled targets was demonstrated in both spot and microchannel assays. Hybridization was detected by fluorescence emission of the fluorescein-labeled DNA probe. The efficiency of the capture of labeled-DNA by antibodies immobilized on paper via CBM-ZZ construct was significantly higher when compared with a physical adsorption method where antibodies were simply spotted on paper without the intermediation of other molecules. The experimental proof of concept of wax-printed μPADs functionalized with CBM-ZZ for DNA detection at room temperature presented in this study constitutes an important step towards the development of easy to use and affordable molecular diagnostic tests.
Chapter
This chapter focuses on membrane proteins residing in the phospholipid bilayer and when exposed to a hydrophobic environment, it possesses one or more lipophilic domains. By applying two-dimensional gel analyses to membrane proteins, it is possible to acquire information about the protein and glycoprotein composition of a particular membrane, and in combination with other methods and strategies, it identifies proteins spanning the membrane, localize the proteins within a cell, and elucidates the whole pathway of epigenetic modifications that eventually produce the native membrane glycoproteins. Electrophoresis of complex mixtures of membrane proteins in polyacrylamide gels in the presence of detergents other than sodium dodecyl sulphate is characterized by poor resolution. The non-glycosylated membrane proteins exhibit no, or minor charge hetero–geneities. The “solubilized'” protein is subjected to electrophoresis in a large–pore polyacrylamide gel under conditions that preserve the protein complexes.
Article
The interaction of two non-ionic detergents Triton X-100 (TX-100) and Tween 20 (Tw20) with lipid bilayers was studied at the nanoscale with atomic force microscopy (AFM). The real-time interaction of these two detergents with biomembranes was followed by using a mixed dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) 1 : 1 (mol/mol) bilayer. For the two detergents, the solubilising activity was tested both below and above their critical micelle concentration (CMC) on the DOPC/DPPC bilayers. Below the CMC, Tw20 was able to solubilise the fluid DOPC leaving the gel phase unaltered on the mica while TX-100 had almost no effect on the bilayer. Above the CMC, the two detergents fully solubilised the DOPC fluid phase within the first minutes. These different behaviours were interpreted on the basis of their Hydrophile-Lipophile Balance (HLB). Another type of model membrane was also prepared to image the organisation of detergent-enriched domains at high resolution. To this end, liposomes of pre-mixed DPPC-detergent were fused on the mica substrate. It was shown that both detergents produced ordered patterns very similar to the striated domains described with helical peptides. Indeed, line-type depressions and elevated lines were observed. The depressions seemed to be provoked by the insertion of detergent molecules within the gel DPPC. This work highlights the uniqueness of AFM to evidence yet unexpected lipid-detergent interactions at the nanoscale.
Article
A particulate D-alanine carboxypeptidase that can cleave the terminal residue of D-alanine from UDPMurNAc-L-ala-D-isoglu-L-lys-D-ala-D-ala was isolated from . The enzyme was inhibited by penicillin G non-competitively with a Ki of 0.8 μM.
Chapter
For any proteomic study involving various control and experimental specimens, several factors need to be in place. A critical one is the extraction and solubilization of all components, regardless of whether a chromatographic (1,2) or two-dimensional (2-D) gel electrophoretic fractionation (3–6) is performed prior to analysis of proteins of interest by mass spectrometry of protein digests. All proteins must not only be extracted, but they must also be completely soluble, free from interacting partners (such as protein-RNA/DNA and protein-protein interactions, metabolites, and so on), and, in the case of 2-D gel electrophoresis, they must remain soluble as they approach their isoelectric points. The solubilization process should extract all classes of proteins reproducibly, such that statistically significant quantitative data can be obtained and correlated with experimental perturbations and the resulting biological responses.
Article
Five nonionic detergents enhanced the activity of L-glutamic acid dehydrogenase [L-glutamate:nicotinamide adenine dinucleotide phosphate oxidoreductase (deaminating) (EC 1.4.1.3)]. These detergents activated the enzyme toward α-ketoglutaric acid reduction, causing a decrease in the sensitivity of the enzyme to allosteric regulation by guanosine 5-triphosphate. There was also a diminution of the enhancing effect of the modifier adenosine 5-diphosphate on the enzyme's L-glutamic acid dehydrogenase activity. These detergents may cause a conformational change in the enzyme, and this change could lead to an increase in the binding of the substrates for the α-ketoglutaric acid reduction. Accompanied with this conformational change would be a decrease in the binding of the modifier guanosine 5′-triphosphate, with no concomitant change in the binding of the adenosine 5′-diphosphate modifier.
Article
Sulfhydryl oxidase of bovine milk was prepared by gel chromatography of crude skim milk membranes obtained by ammonium sulfate fractionation of whey. A variety of detergents were screened for their potential effectiveness as enzyme-solubilizing agents. Anionic detergents, such as sodium dodecyl sulfate and sodium octyl sulfate, inactivated the enzyme concomitantly with solubilization. Nonionic detergents, however, were generally effective in solubilizing sulfhydryl oxidase near their critical micelle concentrations. Polyoxyethylene-9-lauryl ether and β-octyl-D-glucoside afforded the best solubilization of sulfhydryl oxidase among the detergents tested; enzymic activity was retained fully at detergent concentrations producing over 80% solubilization.
Article
A versatile method to prepare non-covalently crosslinked polyHIPEs hydrogels from oil-in-water high internal phase emulsions (HIPEs) whose aqueous phase contained thermo-responsive linear polymers is described. The interconnected pore structure of the polyHIPEs is maintained by reversible physical aggregation of thermo-responsive polymer chains in an aqueous environment. This method to prepare interconnected porous hydrogels using a thermal trigger in the guise of thermo-responsive polymers by emulsion templating requires no chemical reaction during solidification of the template. This particular feature could provide a safer route to injectable scaffolds as issues of polymerisation/crosslinking chemistry and residual initiator fragments or monomers do not arise.
Chapter
To understand, in detail how adenylate cyclase works, we must understand its structure. One approach to this problem is to solubilize the enzyme in an active state, isolate and identify its components and reassemble the whole in a functional form. The first step of this process requires the use of detergents to solubilize adenylate cyclase since the enzyme is firmly membrane bound in all eukaryotic cells, except those from the mature rat testis1–4. Adenylate cyclase occurs in membranes in very small amounts, probably making up about 0.01–0.005% of the membrane protein4, therefore it is impossible to identify the enzyme by protein determination. This limits the kinds of detergents which one may use for solubilization because it is essential to retain enzymatic activity.
Article
Block copolymers were prepared by the direct polycondensation of an aqueous lactic acid solution on monomethoxy or dihydroxyl poly(ethylene glycol) (PEG) in the absence of a catalyst. The resulting poly(lactic acid) (PLA)–PEG diblock and PLA–PEG–PLA triblock copolymers were characterized by various analytical techniques, including matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), gel permeation chromatography, and 1H-NMR. The molecular structure between PLA–PEG and PLA–PEG–PLA could be distinguished after the calculation of the repeat unit masses and end-group masses through the MALDI-TOF MS spectra. Interestingly, both copolymers could serve as a hydrophilic emulsifier to stabilize the squalene/water interfaces and yield narrowly distributed oil-in-water nanoparticles. In contrast, the prepolymer PEG failed to stabilize the squalene/water interface under the same homogenization conditions. These features are of great interest for applications as bioactive agent delivery, especially for candidate vaccine antigens and lipophilic anticancer drugs. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009
Article
Full-text available
Conditions were developed for the long-term stabilization of Ca2+-ATPase in detergent-solubilized sarcoplasmic reticulum, purified Ca2+-ATPase, and purified-delipidated Ca2+-ATPase preparations. The standard storage medium contains 0.1 M KCl, 10 mM K-3-(N-morpholino)propanesulfonate, pH 6.0, 3 mM MgCl2, 20 mM CaCl2, 20% glycerol, 3 mM NaN3, 5 mM dithiothreitol, 25 IU/ml Trasylol, 2 micrograms/ml 1,6-di-tert-butyl-p-cresol, 2 mg/ml protein, and 2-4 mg of detergent/mg of protein. Preparations stored under these conditions at 2 degrees C in a nitrogen atmosphere retain significant Ca2+-stimulated ATPase activity for periods of 5-6 months or longer when assayed in the presence of asolectin. The same conditions are also conducive for the formation of three-dimensional microcrystals of Ca2+-ATPase. Of the 49 detergents tested for solubilization, optimal crystallization of Ca2+-ATPase was obtained in sarcoplasmic reticulum solubilized with octaethylene glycol dodecyl ether at a detergent/protein weight ratio of 2, and with Brij 36T, Brij 56, and Brij 96 at a detergent/protein ratio of 4. Similar Ca2+-induced crystals of Ca2+-ATPase were obtained with purified or purified delipidated ATPase preparations at lower detergent/protein ratios. The stabilization of the ATPase activity in the presence of detergents is the combined effect of high Ca2+ (20 mM) and a relatively high glycerol concentration (20%). Ethylene glycol, glucose, sucrose, or myoinositol can substitute for glycerol with preservation of ATPase activity for several weeks in the presence of 20 mM Ca2+.Ca2+-induced association between ATPase molecules may be an essential requirement for preservation of enzymatic activity, both in intact sarcoplasmic reticulum and in solubilized preparations.
Article
In the present paper, we report an analysis of acetylcholinesterase molecular forms in the bovine caudate nucleus and superior cervical ganglion. We show that: (1) The superior cervical ganglion contains a significant proportion (∼ 15%) of collagen-tailed forms (mostly A12 and A8), but these molecules are found only as traces (ca. 0.002%) in the caudate nucleus, even in favorable extraction conditions (i.e., in the presence of 1 m-NaCl, 5 mm-EDTA, 1% Triton X-100). (2) The bulk of acetylcholinesterase corresponds to globular forms, mostly the tetrameric G4 and the monomeric G1 forms, with a smaller proportion of the dimeric G2 form. (3) The tetrameric enzyme exists as a minor soluble component (GS4) that does not interact with Triton X-100, and a major hydrophobic component (GH4) that is partially solubilized in the absence of detergent in the caudate nucleus, but not in the superior cervical ganglion. (4) The monomeric G1 form presents a marked hydrophobic character, as indicated by its interaction with Triton X-100, although it may be solubilized in large part in the absence of detergent in both tissues. (5) The detergentsolubilized forms aggregate upon removal of detergent. This property disappears after partial purification of G4) that does not interact with Triton X-100, and a major hydrophobic component (GH4, but is restored upon addition of an inactivated crude extract, indicating that it is attributable to interactions with other hydrophobic components. (6) The proportions of molecular forms solubilized in detergent-free buffers vary with the ionic composition of the medium. Repeated extractions of caudate nucleus in Tris-HCl buffer produce a larger overall yield of G1 form (e.g., 40%) than appears in a single quantitative detergent solubilization (<15%). This G1 form apparently derives in part from a pool of GH4 form. (7) However, detergents that allow a quantitative solubilization of acetylcholinesterase yield the same proportions of forms (about 85% G4) independently of the ionic conditions. (8) Modifications of the molecular forms occur spontaneously during purification, or storage of the crude aqueous ex-tracts, in a manner that depends on the ionic conditions. In Tris-HCl buffer, G1 is converted into a well-defined 7.5S form. In Ringer, polydisperse components are formed. The effects observed in Ringer cannot be reproduced by addition of 5 mm-Ca2- to the Tris buffer either during or after extraction. (9) Proteases, such as pronase, convert the hydrophobic forms into molecules that do not appear to interact with Triton X-100, and do not aggregate in its absence. These results raise fundamental questions regarding the status of acetylcholinesterase in situ, the structure and interactions of its molecular forms. They are discussed with reference to previous publications.
Article
The effects of 17 non-ionic detergents on pig kidney plasma membrane adenylate cyclase were tested (digitonin; detergents from the Triton series: X45, X100, X102, X114, X165, X305, X405, N101; the Brij series: 35, 56, 96 and the Tween series: 20, 40, 60, 80, 85). Membranes (2.66 mg protein/ml) were treated with increasing amounts of detergent for 150 min at 0°C. The adenylate cyclase activities present in treated membranes and in their corresponding non-sedimentable fractions were measured at 30°C. Only detergents having hydrophilic lipophilic balance values from 12 to 14 were effective solubilizing agents. For these detergents, the solubilization yield was dependent on their structure. When solubilized adenylate cyclase kept at 0°C was incubated at 30°C, the enzyme catalytic activity exponentially decreased towards an equilibrium value with a half-time of 5 min. This temperature-dependent adjustment of enzyme activity was reversible. Its magnitude was dependent on the detergent used. A more progressive but less pronounced effect was observed with non-solubilizing detergents. The micellar form of detergent in the incubation medium was found to be responsible for the appearance of microheterogeneity in the population of solubilized enzyme molecules differing by the substrate accessibility. This effect was suppressed when reducing the detergent concentration in the incubation medium.
Article
The plasma membrane of erythrocytes, as of other cells, is thought to act as the barrier responsible for maintaining intracellular gradients of most ions and small molecular species between the cell and its environment. Controlled application of the nonionic detergent Brij 58 effectively opened the erythrocyte plasma membrane, as judged by electron microscopy and lipid mobilization, but the cytoplasm maintained much of its integrity for about 30 min. Release of K+ correlated well with release of protein into the surrounding medium. The results demonstrate that permeabilization of the erythrocyte plasma membrane does not result in an instantaneous equilibration of small ions, such as K+, between the cell and its environment. A comparison was made between erythrocytes treated with Brij 58 and Triton X-100. The lipid and protein solubilizing actions of Triton X-100 were not as easily separable in time as those of Brij 58. The results of treatment of the erythrocytes with different types of nonionic detergents suggest that the membranolytic and cytoplasmic protein destabilizing actions of nonionic detergents correspond with their hydrophilic-lipophilic balance numbers (HLB values). © 1994 wiley-Liss, Inc.
Article
The specific activity of all membrane-bound enzymes studied decreased during infection of Pseudomonas BAL-31 with the lipid bacteriophage PM2. NADH oxidase, nitro blue tetrazolium chloride reductase, succinic dehydrogenase, and Mg2+-dependent ATPase, all involved in oxidative phosphorylation, decreased in specific activity from the start of infection. Glucose phosphotransferase started to decrease rapidly in specific activity only at 10 min after infection (p.i.). Alkaline phosphatase, which presumably lies outside the cell membrane, decreased in a manner similar to the enzymes of oxidative phosphorylation, but not as much. In contrast, two cytoplasmic enzymes, hexokinase and glucose-6-phosphate dehydrogenase, increased in specific activity after infection, reaching a peak of twice the specific activity of uninfected cells at 30 min p.i.The overall rate of protein synthesis in control and infected cells was the same, at least until the commencement of cell lysis at about 60 min p.i. Whereas the bulk of cellular proteins labeled before infection was stable until about 60 min p.i., the specific radioactivity of prelabeled membrane-bound proteins decreased after infection. The latter observation led to the present working hypothesis: namely, that virus-specific proteins are inserted into the cellular membrane; this may or may not be accompanied by the displacement of some normal cellular membrane proteins. The altered cellular membranes, containing virus-specific proteins, may then form the outer shell of the virion.
Article
Phospho-MurNAc-pentapeptide translocase activity in the membrane of M. luteus was lost upon addition of the detergent, Triton X-100, but could be restored by addition of lipid fractions to the assay. By assay in the presence of lipid, the activity of the Triton-solubilized enzyme could be measured. The synthesis of C(55)-isoprenyl-P-P-MurNAc-pentapeptide from UDP-MurNAc-pentapeptide required C(55)-isoprenyl-P, and was stimulated by a neutral lipid. The exchange reaction of UDP-MurNAc-pentapeptide with UMP required a polar lipid fraction, but the reaction was not affected by C(55)-isoprenyl-P or the neutral lipid. Thus, measurement of activity of the detergent-solubilized enzyme requires addition of three lipids, the lipid substrate (C(55)-isoprenyl-P), the neutral lipid, and a polar lipid.
Article
Lipid decomposition studies in frozen fish have led to the development of a simple and rapid method for the extraction and purification of lipids from biological materials. The entire procedure can be carried out in approximately 10 minutes; it is efficient, reproducible, and free from deleterious manipulations. The wet tissue is homogenized with a mixture of chloroform and methanol in such proportions that a miscible system is formed with the water in the tissue. Dilution with chloroform and water separates the homogenate into two layers, the chloroform layer containing all the lipids and the methanolic layer containing all the non-lipids. A purified lipid extract is obtained merely by isolating the chloroform layer. The method has been applied to fish muscle and may easily be adapted to use with other tissues.Lipid decomposition studies in frozen fish have led to the development of a simple and rapid method for the extraction and purification of lipids from biological materials. The entire procedure can be carried out in approximately 10 minutes; it is efficient, reproducible, and free from deleterious manipulations. The wet tissue is homogenized with a mixture of chloroform and methanol in such proportions that a miscible system is formed with the water in the tissue. Dilution with chloroform and water separates the homogenate into two layers, the chloroform layer containing all the lipids and the methanolic layer containing all the non-lipids. A purified lipid extract is obtained merely by isolating the chloroform layer. The method has been applied to fish muscle and may easily be adapted to use with other tissues.
Nonionic Surfactants (Marcel Dekker
  • M J Schick
Schick, M. J.; ed. (1967) Nonionic Surfactants (Marcel Dekker, New York).
  • W G Griffin
Griffin, W. G. (1949) J. Soc. Cosmet. Chem. 1, 311.