High Solvent Concentrations Research Articles

Lignin Peroxidase Oxidation of Aromatic Compounds in Systems Containing Organic Solvents

Lignin peroxidase from Phanerochaete chrysosporium was used to study the oxidation of aromatic compounds, including polycyclic aromatic hydrocarbons and heterocyclic compounds, that are models of moieties of asphaltene molecules. The oxidations were done in systems containing water-miscible organic solvents, including methanol, isopropanol, N, N-dimethylformamide, acetonitrile, and tetrahydrofuran. Of the 20 aromatic compounds tested, 9 were oxidized by lignin peroxidase in the presence of hydrogen peroxide. These included anthracene, 1-, 2-, and 9-methylanthracenes, acenaphthene, fluoranthene, pyrene, carbazole, and dibenzothiophene. Of the compounds studied, lignin peroxidase was able to oxidize those with ionization potentials of <8 eV (measured by electron impact). The reaction products contain hydroxyl and keto groups. In one case, carbon-carbon bond cleavage, yielding anthraquinone from 9-methylanthracene, was detected. Kinetic constants and stability characteristics of lignin peroxidase were determined by using pyrene as the substrate in systems containing different amounts of organic solvent. Benzyl alkylation of lignin peroxidase improved its activity in a system containing water-miscible organic solvent but did not increase its resistance to inactivation at high solvent concentrations.

A method to increase contaminant tolerance in protein matrix‐assisted laser desorption/ionization by the fabrication of thin protein‐doped polycrystalline films

AbstractThis paper describes the fabrication and use of thin polycrystalline films in matrix‐assisted laser desorption experiments. These films consist of microcrystals of protein‐doped matrix (crystal dimensions ⩽ 1 μm). The films produce intense protein‐ion currents and can be grown in the presence of high concentrations of involatile solvents (e.g., glycerol, 6 M urea) without any purification. They strongly adhere to the substrate, allowing easier washing of the film, compared to dried‐droplet deposits. The films are also more uniform than dried‐droplet deposits, with respect to ion production. It is suggested that matrix‐assisted laser desorption/ionization samples are composed of nonlinear optical devices that function as polymer ion‐current sources. These devices (protein‐doped matrix crystals) can be designed and fabricated in many forms to serve the special functions required by the analytical scientist.

Adaptation of Pseudomonas putida S12 to high concentrations of styrene and other organic solvents.

Pseudomonas putida S12 could adapt to grow on styrene in a two-phase styrene-water system. Acetate was toxic for P. putida S12, but cells were similarly able to adapt to higher acetate concentrations. Only by using these acetate-adapted cells was growth observed in the presence of supersaturating concentrations of toxic nonmetabolizable solvents such as toluene.

Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide.

Random mutagenesis has been used to engineer the protease subtilisin E to function in a highly nonnatural environment--high concentrations of a polar organic solvent. Sequential rounds of mutagenesis and screening have yielded a variant (PC3) that hydrolyzes a peptide substrate 256 times more efficiently than wild-type subtilisin in 60% dimethylformamide. PC3 subtilisin E and other variants containing different combinations of amino acid substitutions are effective catalysts for transesterification and peptide synthesis in dimethylformamide and other organic media. Starting with a variant containing four effective amino acid substitutions (D60N, D97G, Q103R, and N218S; where, for example, D60N represents Asp-60-->Asn), six additional mutations (G131D, E156G, N181S, S182G, S188P, and T255A) were generated during three sequential rounds of mutagenesis and screening. The 10 substitutions are clustered on one face of the enzyme, near the active site and substrate binding pocket, and all are located in loops that connect core secondary structure elements and exhibit considerable sequence variability in subtilisins from different sources. These variable surface loops are effective handles for "tuning" the activity of subtilisin. Seven of the 10 amino acid substitutions in PC3 are found in other natural subtilisins. Great variability is exhibited among naturally occurring sequences that code for similar three-dimensional structures--it is possible to make use of this sequence flexibility to engineer enzymes to exhibit features not previously developed (or required) for function in vivo.

Organic brain damage and occupational solvent exposure.

Three hundred and nine men with organic dementia, cerebral atrophy, or psycho-organic syndrome admitted for five nights or more to one of 18 Quebec hospitals were individually matched with patients admitted (1) with some other psychiatric diagnosis and (2) to a general hospital. Lifetime occupational histories were obtained by telephone. Occupational exposure to solvents was assessed blind to type of case by (1) individual ratings and (2) a job exposure matrix; men who worked in moderate or high solvent concentrations for at least 10 years were considered exposed. With the psychiatric referent series, an odds ratio of 1.4 (90% CI 1.0-2.0) was calculated by individual exposure ratings and 1.4 (90% CI 0.9-2.2) by job matrix. Increased risk was mainly in those with organic dementia or cerebral atrophy and an alcohol related diagnosis. The same pattern of risk was found against the general hospital referents. Adjustment for possible confounders did not alter the risk estimates appreciably. Also, lifetime job histories, compared in selected case-referent pairs, gave similar evidence of increased risk (odds ratio 2.3; 90% CI 1.0-5.5). It is concluded that the combined effect of occupational solvent exposure and alcohol intake is probably an important cause of organic brain damage.

Dog mastocytoma cells secrete a growth factor for fibroblasts.

It is suspected that mast cells play a part in the pathogenesis of fibrotic diseases, but the mediators that might be involved in induction of fibrosis have not been identified. We asked whether cultured dog mast cell lines produced growth factor(s) for fibroblasts. Three mastocytoma cell lines were found to secrete proliferative activity for human, hamster, and rabbit fibroblasts. Both mastocytoma cell-conditioned medium and cell extract served as competence factors for induction of DNA synthesis in confluent mouse Swiss 3T3 fibroblasts. The mitogenic activity in the conditioned medium was stable to heat, acid, and high concentrations of chaotropic agents or organic solvents but was decreased by treatment with proteases or reducing agents. The activity had an apparent molecular mass of 10 kD and did not bind to heparin. Activity eluted in a single peak from reverse-phase HPLC, and retention time differed from that of typical mesenchymal mitogens. We offer the hypothesis that mast cells produce growth factors for fibroblasts, possibly including a novel growth factor, and that this may contribute to pathologic fibrosis.

Peptide synthesis by stabilized trypsin: Industrial kinetic studies under extreme experimental conditions

Studies of the kinetics of peptide synthesis catalysed by trypsin derivatives have been carried out. We have studied equilibrium (ECS) and kinetically (KCS) controlled synthesis using benzoyl arginine or benzoyl arginine ethyl ester as acyl donors and various amino acid derivatives as nucleophiles. In both cases, kinetics studies were carried out under conditions of industrial interest: (a) by using very active and very stabilized enzyme derivatives as catalysts, (b) under the extreme experimental conditions necessary for improving the performance of these synthetic processes (high concentrations of apolar organic co-solvents in ECS or high concentrations of ammonium sulfate in KCS), and (c) by using high concentrations of substrates. The hydrophobic adsorption of nucleophile substrates on the active center of the enzyme seems to be the key kinetic step in both of these synthetic processes, as synthetic activity in both ECS and KCS greatly increases with pH and with nucleophile concentration. In ECS, the presence of high concentrations of apolar organic solvents (necessary for achieving important synthetic yields) greatly inhibits the adsorption of nucleophiles, and hence the best performance of these processes would be obtained using a moderate excess of the nucleophile substrate in order to quantitatively convert the acyl donor substrate into synthetic product at the highest reaction rates. In KCS, we found that the use of a ‘salting out’ agent ( e.g., ammonium sulfate) greatly improves the hydrophobic adsorption of nucleophiles. In addition, the presence of ammonium sulfate, perhaps as a ‘water-ordering agent’, promotes a dramatic reduction in the rate of hydrolysis of the acyl-enzyme complex when the active center of each trypsin molecule is partially blocked by the adsorbed nucleophile at saturating concentrations. Thus synthetic/hydrolytic ratios are dramatically improved by the presence of this salt, and consequently synthetic yields approach 100%. By using these two different synthetic strategies we have been able to quantitatively convert arginyl derivatives into dipeptides under extreme conditions in which our stabilized trypsin derivatives are very active and very stable. We have calculated catalyst productivities of 2 and 300 tons of dipeptide per year per liter of our trypsin catalysts by using ECS or KCS synthetic strategies, respectively.

Stabilization of substilisin E in organic solvents by site‐directed mutagenesis

Subtilisin E was rationally engineered to improve its stability in polar organic solvents such as dimethylformamide (DMF). A charged surface residue, Asp248, was substituted by three amino acids of increasing hydrophobicity, Asn, Ala, and Leu; all three variants were stabilized with respect to wild type in 80% DMF. This stabilization was only observed in the presence of high concentrations of the organic solvent: no stability enhancements were observed in 40% DMF. In contrast, the mutation Asn218 --> Ser alters internal hydrogen bonding interactions and stabilizes subtilisin E in both 40% and 80% DMF. This study provides additional evidence that substitution of surface-charged residues is a generally useful mechanism for stabilizing enzymes in organic media and that the stabilizing effects of such substitutions are unique to highly altered solvent environments. The effects of the single amino acid substitutions on free energies of stabilization are additive in the Asp248 --> Asn + Asn218 --> Ser combination variant, yielding an enzyme that is 3.4 times more stable than wild type in 80% DMF.

Examination of dimethacrylate inhomogeneity by solvent swelling techniques

AbstractDimethacrylate monomers polymerize to form highly crosslinked networks with an inhomogeneous distribution of regions of low and high crosslink density. The systems are probed in this work by observing the effects of solvents (nitrobenzene and water) on dynamic mechanical measurements, diffusion kinetics and fracture toughness experiments. Dynamic mechanical analysis showed that the addition of nitrobenzene is found to have little effect on the position of the glass transition or secondary relaxations for solvent levels of under 3.5 mass %. It is proposed that solvents initially diffuse into low crosslink density regions, impinging little on the methacrylate mainchains which reside largely in regions of high crosslink density. Only at higher solvent concentrations do these chains become plasticized. Confirmation of heterogeneity is provided by diffusion analysis of isothermal solvent uptake. This is also interpreted interpreted in terms of a two‐stage diffusion process due to differential rates of sorption which occur in different microregions. Weighing of the water uptake of samples suspended in water indicated that some 30% of water absorbed at equilibrium exists within void regions. Addition of water causes the fracture mechanism to change from a ‘stick‐slip’ mechanism characteristic of brittle polymers to stable crack growth with a lower fracture toughness. This is also attributed to differential swelling of the material causing internal stresses which aid material failure.

Solvent and Formaldehyde Exposure in Parquet and Carpet Work

Abstract Hazardous, solvent-containing products are still widely used in carpet and parquet work. In this study, exposure of Finnish parquet and carpet workers to solvents was monitored. In addition, formaldehyde concentrations were measured during varnishing work. Three carpet and three parquet firms participated in the study. The measurements were carried out at 13 construction sites during 16 working days. In parquet work, the highest solvent concentrations were measured during puttying and undercoat varnishing. Mean additive concentrations of solvents were 6.5 to 7.3 times higher than the limit value, compared to the American Conference of Governmental Industrial Hygienists Threshold Limit Values 1988–89. For coating parquetry with a urea-formaldehyde-based varnish, the additive concentration of solvents was 3.3 times that recommended, and the mean formaldehyde concentration was 4.3 ppm. Dispersion adhesives used for installing parquetry and carpets did not emit solvent vapors in considerable amounts. However, using contact and synthetic resin adhesives in rooms with poor ventilation resulted in an additive concentration of solvents that was 3–5 times the amount recommended. Exposure of carpet and parquet fitters to solvents can best be reduced By changing from solvent-based products to water-based ones. High solvent concentrations can also be reduced by introducing more effective ventilation and improving upon the use of respirators.

Solvent selection strategies for extractive biocatalysis.

This report follows the development of systematic solvent screening strategies for the identification of superior pure solvents and introduces techniques for the identification of effective coextractants. Specifically, methods to predict the biocompatibility and extractant capability of solvents are discussed. Biocompatibility is predicted by using heuristic data or the correlations between bioactivity and the logarithm of the partition coefficient of the solvent or the concentration of solvent in the cell membrane. A computer program, known as the extractant screening program or ESP, has been developed to effectively predict the behavior of virtually any product in any solvent/aqueous system. It is demonstrated that a biocompatible yet poor solvent can be mixed with a toxic solvent that has better extractant properties to yield a mixture with improved solvent characteristics that is still biocompatible. The fact that solvents do not mix in an ideal manner is exploited by using ESP to identify solvent mixtures that are still biocompatible at relatively high concentrations of toxic solvent.

Two-stage continuous fermentation of Clostridium acetobutylicum: effects of pH and dilution rate

The kinetics of a two-stage continuous fermentation of Clostridium acetobutylicum have been studied. The pH and the dilution rate have been shown to be two essential factors for process optimization. An increase in pH or dilution rate in the first stage decreased solvent production in the second fermentor. To achieve optimal solvent production, the pH had to be maintained at 4.5 in the first stage and between 4.5 and 5.0 in the second stage. Dilution rates of 0.08 h−1 and 0.04 h−1,respectively, in the first and second fermentors allowed a high solvent concentration. When the pH was maintained at 4.5 in each stage and when the dilution rates were 0.08 h−1 and 0.04 h−1 in the first and second fermentors respectively, 21 g/l solvent concentration was achieved. A conversion yield of 0.36 g solvents/g glucose consumed was obtained with total consumption of glucose. Biomass was only produced in the first stage together with 40% of the solvents, indicating that solvent production had to be induced in the first fermentor.

Experimental modeling of solvent‐casting thin polymer films

AbstractAn apparatus was designed and assembled to study the solvent removal from solution‐cast thin polymer films. The computer interfacing of a thermogravimetric analyzer, spectrophotometer, electronic flowmeters, and control valves for the apparatus enabled the preprogramming of the carrier gas velocity, carrier gas solvent content, and temperature profiles to simulate the environment experienced in large parallel flow industrial driers. The apparatus has also been designed and operated to enable the visual observation of the drying film with an optical microscope. Initial experimental studies conducted with the apparatus involved the effect of temperature on solvent removal. The results indicate that high dryer gas temperatures can apparently cause skinning of the film surface resulting in slower solvent removal rates. The skin formation can be suppressed by higher solvent concentration in the carrier gas. The visual observations revealed the formation of standing waves in the film surface during drying at high gas velocities (&gt;2OO cm/min). The wave formation at least partially overcomes the effect of skinning by increasing the surface area of the film, and may be the manifestation of flow instabilities involving circulation within the film.

Influence of Solvent Composition (Water—Dioxane Mixtures) on the Formation Degree of Intramolecular Aromatic-Ring Stacks in Binary Cu(L-Phenylalaninate)2, Cu(L-Tryptophanate)2, and Related Complexes

The stability constants of the binary Cu(AA)+ and Cu(AA)2 complexes, where AA⁻ = L-phenyl-alaninate (Phe⁻) or L-tryptophanate (Trp⁻), have been determined by potentiometric pH titrations in water, and in 30, 50, 70 and 80% (v/v) dioxane—water mixtures (I = 0.1 M, NaNO3; 25 °C); the corresponding data for the complexes with L-alaninate (Ala⁻), L-valinate (Val⁻), L-norvalinate (Nva⁻), and L-leucinate (Leu⁻) are taken from our recent work (G. Liang, R. Tribolet, and H. Sigel, inorg. Chim. Acta 155, 273 (1989)). The overall stability of Cu(AA)+ and Cu(AA), is governed for all amino acetates (AA⁻) by the polarity of the solvent, while the extent of the intramolecular stack formation between the aromatic side chains in Cu(AA)2 is influenced by the hydrophobic solvation properties of the organic solvent molecules (i.e., the ethylene units of dioxane). Based on the stability difference Δ log K*AA = log KCu(AA) Cu(AA) 2-log KCu(AA) Cuit is shown that Cu(Phe)2 and Cu(Trp)2 are more stable than Cu(Ala)2, and this increased stability is used for evaluating the extent of the stack formation (= closed form) in Cu(Phe)2 and Cu(Trp)2: the percentages of the closed forms vary between about 25 and 80% (based on Cu(AA),2/tot,), and those for Cu(Val)2, Cu(Leu)2 and Cu(Nva)2 between about 10 and 30%. The formation degree of the intramolecular side-chain adduct in Cu(AA)2 decreases (in most solvents), as one might expect, within the series: Cu(Trp)2 &amp;gt; Cu(Phe)2 &amp;gt; Cu(Val)2≳ Cu(Leu)2≳ Cu(Nva)2. The corresponding observations are made with M(AA)2 complexes of Co2+, Ni2+, and Zn2+. The influence of the organic solvent on the intramolecular hydrophobic and stacking adducts differs somewhat: (i) Stack formation in Cu(Phe)2 and Cu(Trp)2 is slightly inhibited by the presence of dioxane, but even in 50% (v/v) dioxane—water the formation degree of the aromatic-ring stacks is still more than 50%. (ii) Addition of some dioxane to an aqueous solution containing Cu(Val)2, Cu(Leu)2or Cu(Nva)2 favors the formation of the aliphatic side-chain adducts; the largest formation degree being reached close to a content of 70% dioxane. Both observations contrast with the general experience made at unbridged hydrophobic or stacking adducts: these are considerably destabilized already by the addition of relatively small amounts of an organic solvent to an aqueous solution. Such a destabilization of the closed Cu(AA)2 species occurs only at high concentrations of the organic solvent (usually more than 70%). It should be added that the organic solvent most probably influences the structure of the intramolecular ligand-ligand adducts giving rise to a whole series of “closed” species; a resolution is presently not possible and therefore the whole stability increase is attributed to a (single) so-called “closed” species to allow a quantification of the effect. The relevance of amino acid side-chain interactions regarding cooperativity, selectivity, evolutionary aspects, and low polarity regions, as in active-site cavities of proteins, are shortly indicated.

A trace-enrichment technique for the loading of gel-permeation high-performance liquid chromatography columns

Gel-permeation high-performance liquid chromatography (GP-HPLC) columns provide rapid high-resolution separations but are frequently limited to analytical tasks because the injection volumes must be small. The reduction of volume required for the loading of solutes can often be impractical and lead to poor recoveries. We have developed a trace-enrichment technique to circumvent this problem. By placing a Waters Guard Pak within the loop of a Valco injector and connecting a pump to the injection port it is possible to concentrate proteins and peptides onto the guard column from relatively large volumes. Enrichment onto a reversed-phase guard column insert is achieved by loading solutes in an aqueous solution or one of low organic solvent concentration. Provided that the GP-HPLC is mean-while equilibrated with a solvent system of sufficiently high organic solvent concentration (i.e., 40% acetonitrile containing 0.1% trifluoroacetic acid) it is possible to elute material that has been loaded in this manner by simply placing the injection loop in line with the column. The solvent strength abruptly increases and the peptide or protein sample is loaded onto the column in a very small volume. We have applied this loading principle to both analytical and semipreparative problems. The amino-terminal fragment of pro-opiomelanocortin (POMC) has been extracted from a single human fetal pituitary (18 weeks gestation) and characterized in terms of its molecular weight. This study indicated that no proteolytic processing of the amino-terminal fragment of POMC takes place at this stage in development. In a larger scale application the amino-terminal fragment of POMC was purified from bovine anterior pituitaries. The trace-enrichment GP-HPLC loading technique was used as an initial fractionation procedure. The isolation of the polypeptide by subsequent reversed-phase HPLC was greatly facilitated by the initial removal of high- and low-molecular-weight contaminants.

ChemInform Abstract: Influence of Dioxane on the Extent of Intramolecular Hydrophobic Ligand‐Ligand Interactions in the Binary Cu2+ 1:2 Complexes of L‐Leucinate, L‐Valinate and L‐Norvalinate

Abstract The stability constants of the binary Cu(AA)+ and Cu(AA)2 complexes, where AA− = L-alaninate (Ala−), L-leucinate (Leu−), L-valinate (Val−) or L-norvalinate, have been determined by potentiometric pH titrations in water, and in 30, 50, 70 and 80% (v/v) dioxane-water mixtures (I = 0.1 M, NaNO3; 25 °C). The overall stability of Cu(AA)+ and Cu(AA)2 is governed for all four amino acetates (AA−) by the polarity of the solvent, while the extent of the intramolecular hydrophobic ligand-ligand interaction between the aliphatic side-chains in Cu(Val)2, Cu(Leu)2 and Cu(Nva)2 is obviously influenced by the hydrophobic solvation properties of the organic solvent molecules. Based on the stability difference Δ log KAA* = log KCu(AA)2Cu(AA) - log KCu(AA)Cu it is shown that Cu(Val)2, Cu(Leu)2 and Cu(Nva)2 are more stable than Cu(Ala)2, and this increased stability is taken as evidence for hydrophobic side- chain interactions in Cu(Val)2, Cu(Leu)2 and Cu(Nva)2; such interactions are not possible in Cu(Ala)2 due to the small size of the methyl side-chain. By using the stability data of the Cu2+/Ala− system as a basis for the evaluation, the extent of the hydrophobic ligand-ligand interaction (= closed form) in the other three Cu(AA)2 complexes is calculated: the percentages of the closed forms vary between about 10 and 30% (based on Cu(AA)2/tot). The formation degree of the closed species is influenced by the solvent: addition of some dioxane to an aqueous solution favors their formation, contrary to the experience with simple unbridged hydrophobic adducts which are destabilized. Such a destabilization of the closed Cu(AA)2 species occurs only at high concentrations of the organic solvent (usually more than 70%). The general relevance of the present results, especially with regard to biological systems, is indicated.

Influence of dioxane on the extent of intramolecular hydrophobic ligand-ligand interactions in the binary Cu 2+ 1:2 complexes of L-leucinate, L-valinate and L-norvalinate

The stability constants of the binary Cu(AA) + and Cu(AA) 2 complexes, where AA − = L-alaninate (Ala −), L-leucinate (Leu −), L-valinate (Val −) or L-norvalinate, have been determined by potentiometric pH titrations in water, and in 30, 50, 70 and 80% ( v/ v) dioxane-water mixtures ( I = 0.1 M, NaNO 3; 25 °C). The overall stability of Cu(AA) + and Cu(AA) 2 is governed for all four amino acetates (AA −) by the polarity of the solvent, while the extent of the intramolecular hydrophobic ligand-ligand interaction between the aliphatic side-chains in Cu(Val) 2, Cu(Leu) 2 and Cu(Nva) 2 is obviously influenced by the hydrophobic solvation properties of the organic solvent molecules. Based on the stability difference Δ log K AA * = log K Cu(AA) 2 Cu(AA) - log K Cu(AA) Cu it is shown that Cu(Val) 2, Cu(Leu) 2 and Cu(Nva) 2 are more stable than Cu(Ala) 2, and this increased stability is taken as evidence for hydrophobic side- chain interactions in Cu(Val) 2, Cu(Leu) 2 and Cu(Nva) 2; such interactions are not possible in Cu(Ala) 2 due to the small size of the methyl side-chain. By using the stability data of the Cu 2+/Ala − system as a basis for the evaluation, the extent of the hydrophobic ligand-ligand interaction (= closed form) in the other three Cu(AA) 2 complexes is calculated: the percentages of the closed forms vary between about 10 and 30% (based on Cu(AA) 2/tot). The formation degree of the closed species is influenced by the solvent: addition of some dioxane to an aqueous solution favors their formation, contrary to the experience with simple unbridged hydrophobic adducts which are destabilized. Such a destabilization of the closed Cu(AA) 2 species occurs only at high concentrations of the organic solvent (usually more than 70%). The general relevance of the present results, especially with regard to biological systems, is indicated.

Health care of workers engaged in waste water treatment. 1) The exposure conditions to organic solvents in workers engaged in waste water treatment

In the investigation of the exposure conditions to organic solvents of workers in 35 waste water treatment facilities, it was found that these workers treated various kinds of solvents. Almost all of the facilities treated chloroform, acetone, n-hexane, methanol and carbon tetrachloride. The average exposure concentration of the workers was, however, low. Exposure concentration to organic solvents (chloroform, xylene and carbon tetrachloride) was evaluated in one of the facilities. The average concentration of the respective solvents in the workers did not exceed the threshold limit value of Japan, but the concentration calculated on the basis of combined exposure exceeded the threshold limit value. On the other hand, high concentrations of organic solvents were detected in the room where the solvents were introduced but not in the working rooms, indicating that the workers engaged in waste water treatment were mainly exposed to organic solvents in the former room where they sorted and mixed the solvents and placed them in the incinerator. In conclusion, improvement in working conditions and health care for workers engaged in waste water treatment facilities should be made in order to prevent workers from becoming intoxicated by organic solvents.

Peptide mapping and internal sequencing of proteins electroblotted from two-dimensional gels onto polyvinylidene difluoride membranes : A chromatographic procedure for separating proteins from detergents

Direct sequence analysis of proteins electroblotted from two-dimensional polyacrylamide gels onto immobilizing matrices provides an efficient technique for obtaining N-terminal sequence data for proteins not amenable to purification by reversed-phase high-performance liquid chromatography (RP-HPLC). We present in this paper a procedure for obtaining peptide fragments from electroblotted proteins for internal amino acid sequence analysis. First, Coomassie Blue-stained proteins are extracted from polydivinylidene difluoride membranes, using a detergent mixture of sodium dodecylsulfate and Triton X-100. Proteins are then separated from the detergent mixture by a chromatographic procedure which relies on the ability of proteins to interact with certain reversed-phase sorbents at high organic solvent concentrations. Under these conditions, detergents and Coomassie Blue are not retained and pass through the column. Proteins are recovered by simultaneously: (i) introducing trifluoroacetic acid into the mobile phase and (ii) decreasing the organic solvent concentration. After proteolytic fragmentation, peptides are purified by microbore-column (1–2 mm I.D.) RP-HPLC for microsequence analysis.

Experimental study of systm peaks and elution profiles for large concentration bands in the case of a binary eluent containing a strongly sorbed additive

Band profiles of high-concentrations samples of single compounds eluted from a silica column by a binary mobile phase were recorded under various sets of experimental conditions. The mobile phase was a dilute solution 0.02–2%, v/v) of lights alkanols in dichloromethane or n-hexane. When the concentration of the strong solvent increases at a constant sample size, or the sample size increases at a constant strong solvent concentration, the elution profile, which is first of the type associated with a Langmuir adsorption isotherm with a sharp front and a slanted tail, changes the direction of its asymmetry and becomes similar in shape to the profiles associated with an anti-Langmuir isotherm, with a slanted front and a sharp tail. The shape of the profiles in the transition range is unusual and appears similar to the composite of two or three different bands. Finally, at large sample sizes and high strong solvent concentrations, the profile develops a hump on its front, which eventually may turn into a second peak. These experimental profiles are in excellent qualitative agreement with those predicted by theory and described in the accompanying paper. Their variations with the experimental parameters follow the trends predicted in that study. The competition between the molecules of the strong solvent (or modifier) and those of the samples is entirely responsible for these profiles.