Detergent Polyoxyethylene Research Articles

Selective Extraction and Quantitation of Polyoxyethylene Detergents and its Application in Protein Determination

Abstract A simple, rapid and selective method for the nearly quantitative extraction of high amounts (10% w/v) of polyoxyethylene nonionic detergents like Triton X-100R is presented based on solvent extraction with 1, 2-dichloroethane. Protein determination after extraction of protein containing solutions can be accomplished using the Bradford assay1 or other standard methods. High protein recoveries can be obtained. The detergent analysis by HPLC using reversed-phases is not disturbed in the presence of proteins and therefore provides a fast and elegant method for the exact quantitation of nonionic detergents.

Solubilization and partial purification of 3-((+-)-2-carboxypiperazine-4-yl)-[1,2-3H]propyl-1-phosphonic acid recognition proteins from rat brain synaptic membranes.

The receptors on neuronal membranes for N-methyl-D-aspartate (NMDA), an analog of L-glutamic acid, are the focus of intensive study because of their importance in many neurophysiological and neuropathological states. Since there is very little knowledge of the molecular characteristics of the NMDA receptors, we undertook the development of methods for the solubilization and purification of proteins that form the receptor complex. Optimal conditions for solubilization of NMDA receptors from isolated synaptic plasma membranes involved the use of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) together with NH4SCN, 10% glycerol, and the nonionic detergent polyoxyethylene 10 tridecyl ether. The presence of NMDA receptors was monitored as the binding activity for the specific NMDA receptor ligand 3-((+-)-2-carboxypiperazine-4-yl)-[1,2-3H]propyl-1-phosphonic acid ([3H]CPP). Approximately 50% of membrane proteins were solubilized, and an equal quantitative recovery of [3H]CPP-binding proteins was achieved. The selectivity of [3H] CPP-binding proteins for excitatory amino acid agonists and aminophosphonocarboxylic acid antagonists remained essentially unchanged following solubilization. The effect of the NMDA receptor modulator, glycine, and of the ion channel-blocking cation Mg2+ on [3H]CPP-binding proteins was drastically altered by solubilization. Both became activators of [3H]CPP-binding sites. The NMDA receptor agonist ibotenic acid was used to develop an affinity matrix for the isolation of the NMDA receptor complex. The [3H]CPP-binding proteins were selectively eluted by the introduction of 2 mM Mg2+ in the elution buffers. This fraction was highly enriched in CPP-binding entities and in a protein of 58-60-kDa molecular size. The CPP binding activity of the proteins in this fraction was enriched by a factor of approximately 20,000 over that of brain homogenate. There was no L-[3H]glutamate binding activity associated with this fraction. Proteins interacting with glutamate, NMDA, and ibotenate were recovered in the 1 M KCl-eluted fraction. We propose that the 58-60-kDa protein is the aminophosphonocarboxylic acid antagonist-binding subunit of the NMDA receptor complex.

Application of nonionic polyoxyethylene detergents in studies of phosphatidylinositol kinase

Four commercial nonionic polyoxyethylene detergents were compared with Triton X-100 in terms of physicochemical properties and interaction with an integral membrane protein, phosphatidylinositol kinase. Tergitol 15-S-9, Tergitol TMN-10, polyoxyethylene 10 lauryl ether, and polyoxyethylene 10 tridecyl ether possess aliphatic hydrophobic moieties and therefore do not absorb significantly in the ultraviolet region. Like Triton X-100, these detergents have cloud points well above the physiological temperature range and partial specific volumes greater than 0.9 cm 3/g. Critical micelle concentrations range from 0.02 to 0.03% with the exception of Tergitol TMN-10, for which the value is about 0.15%. All five detergents support activity of phosphatidylinositol kinase from rat liver in a mixed micellar assay and show similar ionic strength dependences for solubilization of the enzyme. Enzyme/detergent mixed micelles are somewhat smaller with Triton X-100 than for the other detergents. The data presented suggest that these detergents may be generally substituted for Triton X-100 in applications where uv invisibility is important.

Caffeine interaction with the Ca-release channels of heavy sarcoplasmic reticulum. Evidence that 170 kD Ca-binding protein is a caffeine receptor of the Ca-channels

The study of Ca 2+- and caffeine-induced Ca 2+ release from heavy sarcoplasmic reticulum vesicles under the different conditions suggests that Ca 2+ and caffeine can interact with the common receptor of the Ca-release channels. The reticulum membranes were solubilized using nonionic detergent polyoxyethylene 9-lauryl ether, and affinity chromatography on reactive red 120-agarose was carried out. The 170 kD Ca-binding protein which is eluted by caffeine is the most probable candidate for the caffeine receptor of the Ca-channels.

Modification of human erythrocyte membrane potentials by the action of the polyoxyethylene detergent Triton X-100

Triton X-100 in sublytic concentrations causes a marked increase in human erythrocyte membrane permeability to cations with small selectivity to K + ions. This membrane permeabilization induces modification of membrane potential: hyperpolarization in low K + media and depolarization in high K + media. Since the specificity of the K +-ionophorous action of Triton X-100 is very low in comparison with valinomycin, the membrane potential changes are only in the range of −9 to +5 mV. It is suggested that they are determined by the generation of a diffusion potential across the membrane, although the influence of Triton X-100 on the potentials at cell surfaces cannot be neglected.

Kinetic properties of C 12E 8-solubilized (Na ++K +)-ATPase

(1) The properties of the rectal gland (Na + + K +)-ATPase (ATP phosphohydrolase, EC 3.6.1.8) solubilized in octaethyleneglycol dodecylmonoether (C 12E 8) have been investigated. (2) The Kinetic properties of the solubilized enzyme resemble those of the membrane-bound enzyme to a large extent. The main difference is that K m for ATP for the (Na + + K +)-ATPase is about 30 μM for the solubilized enzyme and about 100 μM for the membrane-bound enzyme. (3) The Na +-form (E 1) and the K +-form (E 2) can also be distinguished in the solubilized enzyme, as seen from tryptic digestion, the intrinsic fluorescence and cosin fluorescence responses to Na + and K +. (4) The number of vanadate-binding sites is unchanged upon solubilization, and it is shown that vanadate binding is much more resistant to detergent inactivation than the enzymatic activities. The number of phosphorylation sites on the 95–100% pure supernantant enzyme is about 3.8 nmol/mg, and is equal to the number of vanadate sites. (5) Inactivation of the enzyme by high concentrations of detergent can be shown to be related to the C 12E 8/protein ratio, with a weight ratio of about 4 being a threshold for the onset of inactivation at low ionic strength. At high ionic strength, more C 12E 8 is required both for solubilization and inactivation. (6) It is observed that the commercially available detergent polyoxyethylene 10-lauryl ether is much less deleterious than C 12E 8, and its advantages in the assay of detergent-solubilized (Na + + K +)-ATPase are discussed. (7) The results show that (Na + + K +)-ATPase can be solubilized in C 12E 8 in an active form, and that most of the kinetic and conformational properties of the membrane-bound enzyme are conserved upon solubilization. C 12E 8-solubilized (Na + + K +)-ATPase is therefore a good model system for a solubilized membrane protein.

Purification of (Ca2+-Mg2+)-ATPase from rat liver plasma membranes.

The Ca2+-stimulated, Mg2+-dependent ATPase from rat liver plasma membranes was solubilized using the detergent polyoxyethylene 9 lauryl ether and purified by column chromatography using Polybuffer Exchanger 94, concanavalin A-Sepharose 4B, and Sephadex G-200. The molecular weight of the enzyme, estimated by gel filtration in the presence of the detergent on a Sephadex G-200 column, was 200,000 +/- 15,000. The enzyme was purified at least 300-fold from rat liver plasma membranes and had a specific activity of 19.7 mumol/mg/min. Polyacrylamide gel electrophoresis under nondenaturing conditions of the purified enzyme indicated that the enzymatic activity correlated with the major protein band. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one major band in the molecular weight range of 70,000 +/- 5,000 was seen. The isoelectric point of the purified enzyme was 6.9 +/- 0.2 as determined by analytical isoelectric focusing. The enzyme was activated by Ca2+ with an apparent half-saturation constant of 87 +/- 2 nM for Ca2+. Calmodulin and trifluoperazine at the concentration of 1 microgram/ml and 100 microM, respectively, had no effect on the enzymatic activity.

Low-resolution structural studies of mitochondrial ubiquinol: Cytochrome c reductase in detergent solutions by neutron scattering

Mitochondrial ubiquinol:cytochrome c reductase (Mr approximately 600,000) was cleaved into a complex (Mr approximately 280,000) of the subunits III (cytochrome b), IV (cytochrome c1) and VI to IX, a complex (Mr approximately 300,000) of the subunits I and II, and the single subunit V (iron-sulphur subunit, Mr approximately 25,000). Neutron scattering was applied to the whole enzyme, the cytochrome bc1 complex, both in hydrogenated and deuterated alkyl (phenyl) polyoxyethylene detergents, and the complex of subunits I and II in detergent-free solution. The neutron parameters were compared with the structures of the enzyme and the cytochrome bc1 complex previously determined by electron microscopy. Using the method of hard spheres, comparison of the calculated and experimental radius of gyration implies that the length of the enzyme across the bilayer or the detergent micelle is between 150 and 175 A and of the cytochrome bc1 complex between 90 and 115 A. The subunit topography was confirmed. The cleavage plane between the cytochrome bc1 complex and the complex of subunits I and II lies at the centre of the enzyme and runs parallel to the membrane just outside the bilayer. The detergent uniformly surrounds the protein as a belt, which is displaced by 30 to 40 A from the protein centre of the enzyme and by about 20 A from the protein centre of the cytochrome bc1 complex. The low protein matchpoint of the whole enzyme as compared to the subunit complexes is accounted for in terms of the non-exchange of about 30 to 60% of the exchangeable protons within the intact enzyme. Polar residues are, on average, at the protein surface and non-polar residues and polar residues with non-exchanged protons are buried within the enzyme.

Synthesis of 14C-labeled alkyl polyoxyethylene detergents

Procedures for the synthesis and purification of two 14C-labeled alkyl polyoxyethylene detergent preparations are described. One of the preparations consisted mainly of [14C]dodecyloctaoxyethylene ether, the other consisted mainly of [14C] octylhexaoxyethylene ether, with critical micellar concentrations of approximately 50 microM and 10 mM, respectively. Condensation of alkyl bromides with fractionated polyethyleneglycol resulted in less polydisperse products than conventional ethylene oxide condensation. Highly purified detergent species could easily be obtained from the preparations by thin-layer chromatography. Their use as tracers greatly simplifies determination of detergent concentration during protein solubilization and fractionation.

Experimental pancreatitis in the rat. Light and electron microscopical observations on early pancreatic lesions induced by intraductal injection of trypsin, phospholipase A2, lysolecithin and non-ionic detergent

Trypsin, phospholipase A2, lysolecithin or non-ionic detergent polyoxyethylene p-t-octyl phenol solutions were injected into the rat biliopancreatic duct. Histological and ultrastructural changes in the gland were studied 15 min and 3 h after the injections. The rough surfaced endoplasmic reticulum disintegrated in two ways: (1) the endoplasmic reticulum in the cell periphery was vesiculated but ribosomes were well preserved at 15 min, and (2) large, round membranous structures appeared in apical cytoplasm at 3 h. Zymogen granules disintegrated in the second type, which possibly represents autodigestion. Both types of injury lead ultimately to structureless necrosis. Lesions induced by phospholipase A2 and lysolecithin were identical. Trypsin-induced damage developed slowly and the two phases of endoplasmic reticulum disintegration were not sharply separable. Lesions caused by polyoxyethylene p-t-octyl phenol were variable at 15 min, but at 3 h the type 2 injury described above was observed. It was concluded that although the initial damage in pancreatic acinar cells may vary, necrotic changes are similar despite the injected material at the later time interval. During acute pancreatitis, the acinar cell necrosis is most probably due to the action of lysolecithin produced by the activation of phospholipase A2.

Highly reactive impurities in Triton X-100 and Brij 35: Partial characterization and removal

Triton X-100 (from three different suppliers) and Brij 35, substituted ethers of polyoxyethylene alcohols, were found to contain variable amounts of powerful oxidizing impurities representing a range of 0.04-0.22% H 2O 2 equivalents. These detergents contain also a considerable quantity of carbonyl compounds (0.5-2%) originating from carboxylic acids and ketones or aldehydes. Tween 20, also a polyoxyethylene detergent, and sodium dodecyl sulfate were free from oxidizing contamination. Aqueous solutions of Triton X-100 and Brij 35 (1–4%) reacted readily with SH groups of protein and nonprotein molecules as well as with Fe 2+ ion. Both detergents were purified from the oxidizing impurities by treating aqueous solution of detergent with either NaHSO 3 or SnCl 2 followed by an extraction procedure. The present findings may clarify as well as complicate the interpretation of previous studies where these detergents were used for biological purposes, especially in enzyme and protein purifications, or when present in assay procedures that are based on the formation or consumption of reducing reagents.

Effects of polyoxyethylene detergent (Brij 58) on platelet aggregation, release and clotting activity

Low concentrations of a polyoxyethylene detergent, Brij 58, inhibited the secondary phase of platelet aggregation induced by ADP in human citrated platelet-rich but had no effect on primary aggregation. Thrombin-induced aggregation of washed human platelets suspended in Tyrode's buffer was inhibited after incubation of cells with 4 · 10 −6 M detergent. Efflux of [ 14C]serotonin, 45Ca 2+ and labile aorta contracting substance (thromboxane A 2) and development of prothrombin-converting activity (platelet factor 3) were abolished concomitantly. Aggregation of washed platelets either by sodium arachidonate or by collagen was also inhibited by the same concentration of Brij 58 which inhibited thrombin aggregation. This concentration did not itself produce any release of a cytoplasmic marker, lactate dehydrogenase, from platelets. Higher concentrations of Brij 58, exceeding 4 · 10 −5 M, lysed the cells liberating lactate dehydrogenase, serotonin and Ca 2+. When albumin was included as a platelet stabilizer in the suspending medium the concentration of detergent required for the inhibitory effects was increased ten-fold. This could be attributed to competitive binding of the detergent to albumin, demonstrated with [ 14C]acetylated Brij 58. A variety of other polyoxyethylene detergents, at concentrations from 8 · 10 −4 to 5 · 10 −3 M, also inhibited platelet aggregation induced by thrombin. It is concluded that low concentrations of Brij 58 stabilize the platelets against the action of aggregation agents, while higher concentrations produce membrane destabilization and cell lysis.