Analysis of pH-dependent protein interactions with gel filtration medium.

A prepacked Superose 12 HR 10/30 column was used to study the effects of elution ionic strength and pH on the chromatographic behaviour of a strong hydrophobic Clostridium thermocellum endoglucanase (1) and two weak hydrophobic proteins, Clostridium thermocellum endoglucanase C and egg white lysozyme. Ion-exclusion or ion-exchange interactions between weakly hydrophobic proteins and the gel matrix were observed at low ionic strength, depending on whether the pH of the elution buffer was higher or lower than the pI values of the proteins. These interactions were due to the presence of negatively charged groups on the surface of Superose and could be eliminated at any pH by adding electrolyte at a concentration determined by its chemical identity. The optimum results were observed with sodium sulphate at a concentration of 100 mM. The chromatographic behaviour of strong hydrophobic endoglucanase (1) on a Superose column as a function of pH was much more complex because of two interplaying effects, electrostatic and hydrophobic. Ideal size-exclusion chromatography could be achieved only in a narrow range of the conditions: first, the mobile phase must contain a weak salting-out electrolyte such as NaCl, and second, the mobile phase pH must be high enough that hydrophobic interactions between the solute and support are balanced by their electrostatic repulsion. At pH greater than pI, the retardation of endoglucanase (1) gradually increased with decreasing pH as a result of lowering of repulsive electrostatic interactions whether or not the buffer ionic strength was high. At pH less than pI a drastic increase in the capacity factor k' was observed owing to the additivity of hydrophobic and ion-exchange effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of total fructan in foods by enzymatic/spectrophotometric method: collaborative study

An AOAC collaborative study was conducted to evaluate the accuracy and reliability of an enzyme assay kit procedure for measuring oligofructans and fructan polysaccharide (inulins) in mixed materials and food products. The sample is extracted with hot water, and an aliquot is treated with a mixture of sucrase (a specific sucrose-degrading enzyme), alpha-amylase, pullulanase, and maltase to hydrolyze sucrose to glucose and fructose, and starch to glucose. These reducing sugars are then reduced to sugar alcohols by treatment with alkaline borohydride solution. The solution is neutralized, and excess borohydride is removed with dilute acetic acid. The fructan is hydrolyzed to fructose and glucose using a mixture of purified exo- and endo-inulinanases (fructanase mixture). The reducing sugars produced (fructose and glucose) are measured with a spectrophotometer after reaction with para-hydroxybenzoic acid hydrazide. The samples analyzed included pure fructan, chocolate, low-fat spread, milk powder, vitamin tablets, onion powder, Jerusalem artichoke flour, wheat stalks, and a sucrose/cellulose control flour. Repeatability relative standard deviations ranged from 2.3 to 7.3%; reproducibility relative standard deviations ranged from 5.0 to 10.8%.     

Measurement of reduced and total mercaptamine in urine using liquid chromatography with ultraviolet detection

A simple liquid chromatographic method for the determination of reduced and total mercaptamine in human urine is described. The method is based on derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate followed by ion-pairing reversed-phase liquid chromatography separation and ultraviolet-absorbance detection at 355 nm. Total mercaptamine was determined by reductive conversion of its oxidized fraction to the thiol form before the derivatization step. Baseline separation was achieved on an analytical Zorbax SB C(18) (5 microm, 150 x 4.6 mm) column with a mobile phase consisting of pH 2.0 0.05 mol L(-1) trichloroacetic acid buffer (component A) and acetonitrile (component B) pumped at 1.2 mL min(-1). Gradient elution was used: 0-3 min 12% B, 3-9 min 12-30% B, 9-12 min 30-12% B. The response of the detector was linear within the ranges studied, from 0.1 to 50 micromol L(-1) for reduced mercaptamine and from 0.4 to 400 micromol L(-1) for total mercaptamine. The imprecision ranges for reduced and total mercaptamine were within 1.45-11.71 and 0.73-10.61%, respectively. The analytical accuracy for determined compounds was from 98.79 to 109.77%. The lower limits of detection and quantitation were 0.05 and 0.1 micromol L(-1) of urine for reduced mercaptamine, and 0.2 and 0.4 micromol L(-1) of urine for total mercaptamine, respectively. This method can be used for routine clinical monitoring of the title thiol-drug and its reduced and oxidized fractions. Moreover, cysteine and cysteinylglycine can be measured concurrently, if needed.     

Unsteady state flux response: a method to determine the nature of the solute and gel layer in membrane filtration

The unsteady-state permeate flux response to a step change in transmembrane pressure is shown to result in unique flux–pressure profiles for the three types of solutes common in membrane ultrafiltration (UF): (a) solutes which exert an osmotic pressure but do not form a ‘gel’; (b) solutes which do not exert an osmotic pressure but form a ‘gel’ and (c) solutes which exert an osmotic pressure and also form a ‘gel’. It is also shown that for stirred cell UF, changes in the bulk feed solution properties (concentration, volume) are negligible on the time scale needed to attain a stable permeate flux. Unsteady-state permeate flux measurements could therefore be made at short filtration times so that the results would not be masked by changes in bulk properties.     

Spot volume vs. amount of protein loaded onto a gel: a detailed,statistical comparison of two gel electrophoresis systems

The long-term goal of this research program is to clarify the molecular mechanisms that participate in the formation of human pituitary macroadenomas. One approach to that goal is to characterize the differentially expressed proteins that are found by a comparison of the proteomes of control pituitary vs. macroadenoma tissues. In order to accurately perform a comparative proteomics study, based on the combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and PDQuest 2-D analysis software, a reproducible 2-DE separation system with a wide linear dynamic measure range is needed. A typical horizontal system is the Multiphor II system that analyzes one gel at a time, using a precast gradient gel (180 x 245 x 0.5 mm); a typical vertical system is the Dodeca system that analyzes up to 12 gels at a time on a single-concentration gel (190 x 205 x 1.0 mm). We have evaluated (Zhan and Desiderio, Electrophoresis 2003, 24, 1834-1846) the spatial and quantitative reproducibility of the two second-dimensional gel systems to separate a human pituitary proteome; that study showed a higher reproducibility for the Dodeca gel system. This present study investigated the relationship between the spot volume and the amount of protein loaded onto the gel for those two 2-D systems. The results demonstrated that the Dodeca gel system provides a wider linear dynamic range to measure the changes in the protein abundance in pituitary proteome.     

Radiation induced DNA strand breaks measured by a modified method of gel scanning

Gel electrophoresis is an effective method for assaying plasmid DNA fractions, and UV lights with long wavelengths such as 315 nm is used to image the gel. In the present work, the sensitivities of detecting the fluorescence emitted from ethidium bromide (EB) stained DNA bands in the gel illuminated with UV lights of various wavelengths were compared. It was found that, in the range 245 to 320 nm, shorter excitation wavelength had higher detection sensitivity, thus 260 nm was selected for further studies. With this excitation light, as little as 0.7 ng DNA was detected. The fluorescence of DNA-EB bands had a good linear response to DNA quantity in a wide range. In addition, measured via this modified method, the yield of DNA strand breaks and the second-order rate coefficient of the reaction between DNA and √OH radical were consistent with many previous studies.     

Comparison of a novel thin-layer chromatographic-fluorescence detection method with a spectrofluorometric method for the determination of 7-hydroxycoumarin in human urine.

A novel method for the determination of 7-hydroxycoumarin in human urine which combines thin-layer chromatography (TLC) with fluorescence detection (FD) has been devised. The limit of detection (1 ng/ml) enables determination of 7-hydroxycoumarin after both administration of coumarin and environmental exposure to this fragrance material. When compared to a spectrofluorometric method of analysis, the TLC-FD method proved to be more selective for the analysis of 7-hydroxycoumarin in human urine.     

Problems of traceability of total protein and catecholamine determinations in human urine

Some problems arising in the establishment of the traceabili- ty of the certified reference materi- al (CRM) CZ 6007a (total protein, creatinine and stress indicators) are discussed. Bovine serum albumin is recommended as a calibration standard for total protein determi- nation Received: 12 November 1999 Accepted: 16 August 2000     

CP/MAS NMR investigations of silica gel surfaces modified with aminopropylsilane

Summary Aminopropyl chemically bonded phases for high performance liquid chromatography (HPLC) have been prepared using mono- and trifunctional methoxyor ethoxysilanes. Three types of silica gel with different surface characteristics were used as support for the chemically bonded phases (CBPs). Surface characteristics of the packings before and after chemical modification were determined by porosity parameters, elemental analysis and CP/MAS NMR spectroscopy.²⁹Si and¹³C CP/MAS NMR investigations gave informations about different interactions between aminosilyl ligands and/or these ligands and/or water molecules condensed in the pores of the silica gel surface. With decreasing pore diameter of the silica gel the proportion of protonated aminopropyl ligand increases.     

Conducting properties of a gel ionite modified with zirconium hydrophosphate nanoparticles

The organo-inorganic composites based on a strongly acid gel resin including zirconium hydro-phosphate nanoparticles and their aggregates were studied by impedance spectroscopy, electron microscopy, and standard contact porosimetry. The porous structure of the polymer was transformed under the action of the inorganic filler. The nanoparticles in the transport pores provided a three- to fivefold increase in the electric conductivity of the nanocomposites compared with the conductivity of the nonmodified ionite and a decrease in the percolation threshold. The nanocomposite ionites demonstrated stability against the accumulation of organic substances during electrodeionization to extract Na⁺ from low-concentrated solutions.     

Guidance for selecting the measurement conditions in the dye-binding method for determining serum protein: theoretical analysis based on the chemical equilibrium of protein error.

A methodology for selecting the measurement conditions in the dye-binding method for determining serum protein has been studied by a theoretical calculation. This calculation was based on the fact that a protein error occurs because of a reaction between the side chains of a positively charged amino acid residue in a protein molecule and a dissociated dye anion. The calculated characteristics of this method are summarized as follows: (1) Although the reaction between the dye and the protein occurs up to about pH 12, a change in the color shade, called protein error, is observed only in a pH region restricted within narrow limits. (2) Although the apparent absorbance (the absorbance of the test solution measured against a reagent blank) is lower than the true absorbance indicated by the formed dye-protein complex, the apparent absorbance correlates with the true absorbance with a correlation coefficient of 1.0. (3) At a higher dye concentration, the calibration curve is more linear at a higher pH than at a lower pH. Most of these characteristics were similarly observed experimentally in the reactions of BPB, BCG and BCP with human and bovine albumins. It is concluded that in order to ensure the linearity of the calibration curve, the measurement should be performed at a higher dye concentration and sufficiently high pH where the detection sensitivity is satisfied.     

Comparison of the Kjeldahl method and a combustion method for total nitrogen determination in animal feed

The features of the Dumas combustion method (CM) and those of the Kjeldahl method (KM) were compared as they apply to total nitrogen determination in animal feed. Both methods achieved similar repeatability (S.D., 0.11-0.38 from Kjeldahl and 0.15-0.36 from combustion) and similar intra-laboratory reproducibility (S.D., 0.11-0.39 from Kjeldahl and 0.15-0.37 from combustion). R.S.D. is always below 2%. These results show that the CM is suitable for the analysis of protein content in animal feed (5-75% protein content). The CM is recommended owing to its shorter analysis time, its cost and its environmental suitability.     

Comparison of microwave-assisted digestion procedures for total trace element content determination in calcareous soils

The aim of the study was to determine total trace (Cd, Co, Cr, Cu, Mn, Pb and Zn) and major (Al and Fe) element concentrations in calcareous soils using microwave-assisted digestion procedures. The literature showing lack of consensus regarding digestion procedures and unsatisfying recoveries for calcareous materials, four procedures using various acid combinations (HCl, HNO₃, H₂O₂, HF) and volumes were tested using a certified reference material (CRM 141R) and natural calcareous soil samples. Digests were analysed by inductively coupled plasma-atomic emission spectrometry (ICP-AES). Repeatability (R.S.D. <5%) and recoveries (82-116%) showed that the procedures were precise and accurate for most elements. Five calcareous soil samples from a Champagne vineyard plot were, then, subjected to these procedures. In calcareous materials, the presence of HF resulted in Al being severely underestimated (recovery <5%) and Co overestimated (recovery >124%) due to complex formation or spectrochemical interferences, respectively. As digestion was not significantly influenced by the addition of H₂O₂, the procedure corresponding to Aqua regia (HCl-HNO₃) appeared as the best compromise and was selected for further multielemental environmental studies on calcareous materials, even if the absence of HF could lead to incomplete digestion of accessory silicate minerals. Results for a vineyard plot showed that the soils were contaminated (3.65 mg kg⁻¹ Cd, 67 mg kg⁻¹ Cr, 278 mg kg⁻¹ Cu, 143 mg kg⁻¹ Pb and 400 mg kg⁻¹ Zn) as a consequence of urban waste and copper-treatment applications.     

Comparison of two vibrational procedures for the direct determination of mancozeb in agrochemicals

The direct determination of mancozeb in agrochemicals has been made by diamond attenuated total reflectance (ATR) Fourier transform infrared spectroscopy in the middle range (DATR-MIR) and diffuse reflectance infrared Fourier transform spectroscopy in the near range (DR-NIR) methods using in both cases a previous identification of the samples using a dendrographic classification and an appropriate partial least squares (PLS) calibration established from a set of nine external standards and optimized for each type of sample. It was analyzed a heterogeneous population of 11 samples obtained from the Spanish market, containing different co-formulated products, such as fosetyl-Al, copper oxychloride, metalaxyl or cymoxanil. High performance liquid chromatography (HPLC) was used as reference method for validation of both vibrational strategies. The close agreement between values found for both, DATR-MIR and DR-NIR methods, and reference HPLC values indicates the accuracy and reliability of the proposed techniques for the direct determination of mancozeb in commercially available formulations.