Preparative isoelectric focusing of reduced wheat gluten proteins

Proteins extracted from gluten of the bread wheat cultivar Fiorello 2 in the presence of 2-mercaptoethanol or dithiothreitol were separated by isoelectric focusing in a free solution in a pH 3-10 gradient containing 50% v/v 1-propanol or urea. The collected fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% gels (high and medium molecular weight glutenin subunits) and 16% gels (low molecular weight gliadins). The isoelectric focusing pattern of gluten polypeptides in 50% v/v 1-propanol was comparable to that obtained on two-dimensional gel electrophoresis, based on isoelectric focusing and polyacrylamide gel electrophoresis or nonequilibrium pH gradient electrophoresis and polyacrylamide gel electrophoresis. A similar isoelectric focusing pattern was also observed when 3M urea was used as solvent. New gluten polypeptides, similar in mobility to the high molecular weight subunits of glutenin were detected at acidic pH.     

Qualitative and quantitative changes in barley seed protein patterns during the malting process analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with respect to malting quality.

Seeds of two barley cultivars, similar in total protein content and malt extract yield but different in their final attenuation values, were malted. Samples taken at daily intervals during the malting process were extracted sequentially with Tris-HCl buffer, aqueous 2-propanol, aqueous 2-propanol containing 0.5% dithiothreitol, and 4 M urea, containing 0.5% dithiothreitol and 1% Nonidet P-40. The protein composition of these extracts was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and computer densitometry to determine whether differences observed in the rate or extent of protein modification are related to the malting quality character final attenuation. It was found that, common to both cultivars, the albumin and globulin proteins were relatively resistant to proteolysis, whereas the hordeins suffered a dramatic breakdown during malting, with the D hordein being degraded most rapidly, followed by the B and C hordeins. Besides these similarities, differences between both cultivars were observed in the relative rates of D hordein degradation, as this rate was considerably higher in the cultivar with high malting quality. Similar, but much less distinct kinetics were seen with certain B hordeins. Since a possible relationship might exist between the rate of proteolysis of the D hordeins and the character final attenuation, we analyzed a larger number of barley cultivars with different final attenuation values with a simplified technique. For the ten cultivars examined, differences during germination were again seen in the rates of modification of the D hordeins. However, significant correlations between the D hordein breakdown and final attenuation values were not obtained, so that we propose that there exists at best a loose correlation between the relative rate of proteolysis of these proteins and the malting quality character final attenuation.     

An HPLC Method for the Determination of Diltiazem and Desacetyldiltiazem in Human Plasma

Abstract

A high performance liquid chromatographic method is presented for the determination of diltiazem and its metabolite desacetyldiltiazem in human plasma. Diltiazem and desacetyldiltiazem are extracted from plasma basified with 0.5M dibasic sodium phosphate (pH 7.4) using 1% 2-propanol in n-hexane containing diazepam as an internal standard. A reversed phase cyanopropylsilane column was used with a mobile phase of 45% acetonitrile and 55% 0.05M acetate buffer (pH 4.0). The minimum detectable limit was 2ng/ml of plasma. The effect of the pH, molarity, and percent acetonitrile of the mobile phase on the capacity factor was studied. Possible interferences from other drugs administered concurrently are presented.     

Effective Solubilization Procedure for Analysis of Silkworm Hemolymph Proteins by Two-Dimensional Gel Electrophoresis

The outstanding capability of two-dimensional gel electrophoresis in separating all types of proteins basically depends on the efficiency of sample preparation. Sample preparation is one of the most critical steps in two-dimensional gel electrophoresis. Unfortunately, due to severe solubility, resolution of protein on gel is usually hampered, and thus, analysis remains a difficult task. However, technically several problems are generally encountered during protein extraction and isoelectric focusing. In the present investigation, we emphasized on evaluation and comparison of six different protein solubilization methods intended for resolving and analyzing silkworm hemolymph proteins by two-dimensional gel electrophoresis. Our findings revealed that the buffer composition of 8 M urea, 4 % 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 40 mM Tris base, 65 mM dithiothreitol, and 0.2 % ampholyte can effectively solubilize and yields maximum protein spots.     

Inhibition of unwanted proteolysis during sample preparation: evaluation of its efficiency in challenge experiments

Measures to counteract proteolysis during sample preparation are widely used; among them, protein extraction at a basic pH (Tris pH 11.0), sample boiling in sodium dodecyl sulfate (SDS), extraction in denaturing lysis solutions and the use of proteinase inhibitors combined with some of these approaches. Here, we tested their efficiency under stringent conditions using a high proteinase (trypsin and a mixture of pancreatic proteinases) contamination and as substrate, streptokinase, a protein highly sensitive to proteolytic degradation. Total degradation was observed in Tris pH 11.0. There was an efficient inhibition for the pancreatic proteinases after boiling in 1% SDS, 1% dithiothreitol (DTT), while trypsin inhibition was dependent on the enzyme-to-substrate ratio. A panel of 21 lysis solutions with variable concentrations of urea, thiourea and detergents was essayed for the ability to counteract proteolysis. In all solutions containing 7-9 M urea, detergents and proteinase inhibitors but not containing thiourea, there was a strong proteolysis. However, in all samples containing 2 M thiourea, proteolysis was inhibited. Moreover, inhibition was dependent on the thiourea concentration. According to these results, we are prompted to consider that the well-known benefits of incorporating thiourea into the lysis solution are a result of two factors, its efficiency in solubilizing proteins and the inhibition of the proteolysis of sensitive substrates; both contributing to the detection of a higher number of species in two-dimensional electrophoresis (2-DE) gels.     

Investigation of 2-Methoxyethanol as a Modifier in Acetonitrile for the Reversed Phase HPLC of Proteins

Abstract

The results of using 2-methoxyethanol as a mobile phase modifier combined with acetonitrile for the separation of a standard mixture of proteins is described. Selectivity comparisons are made between this system and similar mobile phase systems containing acetonitrile, methanol, n-propanol and iso-propanol.     

Temperature optimization for improved determination of phosphatidylserine species by micro liquid chromatography with electrospray tandem mass spectrometric detection

A sensitive method for determination of disaturated phosphatidylserine species in the presence of their monounsaturated analogs has been developed, using micro liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The hydrophobic nature of the phosphatidylserine species required a combination of low-eluting sample solvents and sub-ambient temperatures in order to focus large sample volumes up to 20 microL. The samples were dissolved in 2-propanol:hexane:water (20:10:4, v/v/v) prior to 1:9 dilution with ammonium formate buffer:2-propanol:tetrahydrofuran (30:55:15, v/v/v) and final 1:4 dilution with ammonium formate buffer (10 mM):2-propanol: tetrahydrofuran (55:37.5:7.5, v/v/v). The analytical column was a 0.5 x 150 mm stainless steel column packed with 5 microm C30 particles, while the mobile phase contained ammonium formate buffer (10 mM): 2-propanol:tetrahydrofuran (30:55:15, v/v/v). A temperature program from 5 degrees C (hold for 3 minutes) to 75 degrees C at 8 K/min provided separation of the disaturated phosphatidylserine species from their monounsaturated analogs, making available a sensitive determination of the isobaric species. The mass limit of detection for dipalmitoyl phosphatidylserine was 100 pg, corresponding to a concentration limit of detection of 5 pg/microL when using an injection volume of 20 microL. This is an improvement by a factor of 20 as compared to previously reported numbers obtained with conventional LC columns. The within-assay precision of dipalmitoyl phosphatidylserine was 11.9% RSD (n = 3), while the retention time precision was 4.1% RSD (n = 6).     

Preparation of active ribosomal proteins by HPLC

Summary The proteins of the large ribosomal subunit fromEscherichia coli have been separated by size-exclusion, ion-exchange and reversed-phase high-performance-liquid chromatography (HPLC) using various buffer systems. The biological activity of the isolated proteins was tested via their ability to assemble into active 50S subunits (total reconstitution). The activity of the reconstituted subunits was measured with poly(U)-dependent poly-(Phe) synthesis. Reversed-phase HPLC techniques yielded active proteins (80–100%) by application of 2-propanol or acetonitrile. Proteins prepared by size-exclusion chromatography employing ammonium acetate as buffer also gave highly active proteins (70%). On the other hand, separation of the proteins on ion-exchange columns, using urea containing buffers, resulted in reduced activity (up to 50%).     

Thermochemical investigations of hydrogen-bonded solutions. Part 11. Expressions for predicting anthracene solubilities in alcohol+alkoxyalcohol mixtures based on mobile order theory

Experimental solubilities are reported for anthracene dissolved in eight binary mixtures containing 2-ethoxyethanol with 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-1-propanol, 1-pentanol, 3-methyl-1-butanol and 1-octanol, and also in binary 1-pentanol+2-methoxyethanol and 2-methyl-1-propanol+2-propoxyethanol solvent systems at 25°C. Results of these measurements, combined with previously reported anthracene solubility data in 22 different alcohol +2-alkoxyethanol (2-methoxyethanol, 2-propoxyethanol and 2-butoxyethanol) solvent mixtures, are used to test the limitations and applications of expressions derived from Mobile Order theory. The first predictive expression assumes only formation of homogeneous self-associated hydrogen-bonded species, whereas the second equation includes additional terms to account for heterogeneous complex formation between the dissolved alcohol and 2-alkoxyethanol solvent molecules. Both equations predict the observed anthracene solubilities to within an average absolute deviation of about 3%.     

Catalytic dechlorination of aromatic chlorides with Pd/C catalyst in alkaline 2-propanol: activity enhancement by the addition of methanol

Summary p- and o-Chloroanisole were catalytically dechlorinated with Pd/C at 30°C in a solution of NaOH in mixtures of 2-propanol and methanol. The dechlorination rate of p-chloroanisole in the mixture containing 1% methanol was 12 times as high as that in 2-propanol alone. The dechlorination rate of o-chloroanisole was also enhanced by the addition of a small amount of methanol.