Immunoenzyme assay of human cartilaginous proteoglycans and their antibodies

An ELISA has been developed to measure human cartilaginous proteoglycans (PG) and their auto-antibodies. The assay is described step by step: successively the nature of the microtiter wells, the concentration of the PG to be coated, the optimal dilution of the antiserum, the length of various incubations and their respective temperature, are described. Standard curve obtained with purified PG is linear in a logit-log representation. The sensitivity of the assay is 2 ng/tube. Finally, biological fluids-sérum and synovial fluid-show a good parallelism with PG.     

Separation of acylcarnitines from biological samples using high-performance liquid chromatography

A reverse-phase high-performance liquid chromatography technique to separate carnitine and acylcarnitines from a biological matrix is described. The method utilizes a step gradient to provide baseline resolution of acylcarnitines (individually or by class) for subsequent quantification using a sensitive radioenzymatic assay. The method requires minimal sample preparation and prevents any contamination among groups of acylcarnitines. This technique has been applied to liver tissues of rats obtained under a variety of conditions. These studies demonstrate the validity and utility of the HPLC method while confirming the applicability of the perchloric acid fractionation of acylcarnitines by functional class. The present HPLC method permits resolution of long-chain acylcarnitines in the presence of large excess concentrations of carnitine and short-chain acylcarnitines (coelution of unesterified carnitine with long-chain acylcarnitines less than or equal to 0.05%). Thus, the method will be of use in the study of acylcarnitines in biological systems over a broad spectrum of metabolic conditions.     

Single step protocol to purify recombinant proteins with low endotoxin contents

Endotoxin is an unwanted by product of recombinant proteins purified from Escherichia coli. The inherent toxicity of endotoxins makes their removal an important step for the proteins' application in several biological assays and for safe parenteral administration. The method described in this paper is a one-step protocol which is effective at removing tightly bound endotoxin from over-expressed tagged proteins in E. coli. We combined affinity chromatography with a non-ionic detergent washing step, to remove most of the endotoxin contaminants from the end product. An endotoxin reduction of less than 4 to 0.2 EU mg(-1) was achieved with protein recovery close to a yield 100%. As this new protocol requires only one step to simultaneously purify tagged proteins and eliminate endotoxins, it represents a substantial advantage in time, effort, and expense.     

A simple extraction procedure for prostaglandins in human seminal plasma with specific detection by selected ion monitoring

The extraction of prostaglandins (PGs) from biological samples and in particular from human seminal plasma becomes in practice a rather complex process, usually requiring a significant degree of sample manipulation and clean up procedures. Our work in this field has led to a very simple method based on a direct ultrafiltration of samples of human seminal plasma and extraction of the PGs in the ultrafiltrate into ethylacetate. The type of ultrafiltration membranes used for this purpose retain all substances of molecular weight over 1000, effectively removing proteins and other heavy interfering material. Recoveries of PGs in the first step are of the order of 81,5 to 86,8% as verified with tritiated PGs while the second step is virtually quantitative (>99%). These extractions are both quantitatively and qualitatively reproducible judging from the recovery values obtained from replicate determinations of the same samples as well as from the pattern reproducibility of the corresponding gas chromatographic and selected ion and profiles. The latter are identical to the profiles obtained from samples processed by a different extraction method of proven efficacy which in principle would validate the procedure and results herein described.     

Development of a microporous membrane liquid–liquid extractor for organophosphate esters in human blood plasma: identification of triphenyl phosphate and octyl diphenyl phosphate in donor plasma

An extractor has been developed for microporous membrane liquid–liquid extraction (MMLLE) of lipophilic xenobiotics at trace levels in biological fluids. This new construction allows the sample phase to be stirred, while the organic phase is pumped. The extractor was evaluated using human blood plasma with added organophosphate esters. The size exclusion properties of the membrane reduced lipid co-extraction by 94% compared to ordinary liquid–liquid extraction. In combination with a solid-phase extraction (SPE) step, the method was shown to remove plasma lipids efficiently and thus allow gas chromatographic separation of the compounds. The clean-up method described, including the SPE step, showed a high level of reproducibility, and recoveries of between 72 and 83% were obtained for five of the organophosphate esters after a 200-min extraction period. Using this technique, triphenyl phosphate and an isomer of octyl diphenyl phosphate were detected in human plasma obtained from blood donors. The concentration of triphenyl phosphate ranged between 0.13 and 0.15 μg/g plasma.     

Microwave-mediated analysis for sugar, fatty acid, and sphingoid compositions of glycosphingolipids

For chemical characterization of glycosphingolipids, it is necessary to determine the chemical compositions of three constituents, i.e., sugars, fatty acids, and sphingoids. A new rapid analytical method is described using a one-pot reaction in a household microwave oven, producing sugars, fatty acids, and especially sphingoids free of by-products, from a single aliquot of a biological sample. Glycosphingolipids were hydrolyzed by microwave exposure with 0.1 M NaOH/CH(3)OH for 2 min followed by 1 M HCl/CH(3)OH for 45 s. The alkaline methanolysis step produced intermediate lysoglycosphingolipids virtually free of by-products such as the O-methyl ethers usually seen. The fatty acid methyl esters were extracted with n-hexane, and other reaction products were dried, taken up in aqueous alkaline methanol, and shaken with chloroform. Sphingoids partitioned into the organic phase under these conditions, whereas the sugar portion that partitioned into the aqueous phase was re-N-acetylated and remethanolyzed for 30 s by microwave exposure. Analysis of the profiles of glycosphingolipid constituents obtained using the microwave oven method showed that they were quantitatively and qualitatively comparable to those obtained by time-consuming conventional methods, which require reaction for several hours. Analysis of the three constituents, including analysis by gas chromatography, may be obtained within 1 day using the method described here.     

Iron determination by carbon rod atomizer (author's transl)

A method is described for direct analysis of iron in proteins. The organic and inorganic matrices can be removed during the ashing step. The results obtained with simple aqueous standards were confirmed for all determinations by the standard addition method. The interpretation of the results by means of regression analysis showed, in dependence on the biological material (Haemoglobin, suspension of Microsomes, Cytochrome P 450), relative standard deviations between 4 and 20%.     

Kinetics of the interaction of dihydroalprenolol with beta-adrenergic receptors in rat cerebral cortex

Time and temperature dependence of the binding of 3H-dihydroalprenolol (3H-DHA) to beta-adrenergic receptors in rat cerebral cortex is described. The kinetic data obtained suggest that 3H-DHA binding proceeds through a two-step reaction scheme consisting of a bimolecular association step followed by an unimolecular internal conversion of the radioligand receptor complex (isomerisation). Equilibrium thermodynamic analysis provided evidence that the over-all binding process is associated with a small decrease in enthalpy and a substantial increase in entropy. Within the framework of the two-step binding kinetics, the evaluation of the temperature dependence by the van't Hoff analysis resulted in values for thermodynamic parameters for the single equilibrium steps. The data suggest that the association step can be considered as a bimolecular hydrophobic interaction which is mainly entropy-driven due to the release of structural water, while the isomerisation step is accompanied by a large negative change in both enthalpy and entropy. The large negative change in the activation entropy for the forward reaction of the isomerisation step, obtained from evaluation of Arrhenius plots, indicates an internal conversion to a highly ordered receptor-ligand complex, while the low activation energy points to a small threshold energy for reaching this structure. Thus, these result support a previous assumption that the hydrophobic center of an adrenergic antagonist interacts with the receptor by entering a pocket (Cherksey et al. 1981).     

The analysis of low levels of γ-glutamyltransferase activity by high-performance liquid chromatography with electrochemical detection

A method for the analysis of low levels of the enzyme γ-glutamyltransferase in biological samples by high-performance liquid chromatography with electrochemical detection is described. A γ-glutamyl moiety from glutathione is transferred by the enzyme to glycylglycine to produce a tripeptide which is assayed directly after a purification step using octadecylsilica. Confirmation of the method is by use of the inhibitor AT-125. The method is used to measure the level of enzyme activity in rodent tissues and in cultured cells.     

Visualization of proteases within a complex sample following their selective retention on immobilized bacitracin, a peptide antibiotic.

A method for the visualization of individual proteases within a complex biological sample is described. In a single chromatographic step, proteases can be separated from other biomolecules by selective binding to immobilized bacitracin, a peptide antibiotic. Following desorption, proteases may be separated by SDS-polyacrylamide gel electrophoresis. The application of this method is presented in the visualization of proteases secreted by the fungus Aspergillus niger.     

Calmodulin-free skeletal-muscle troponin C prepared in the absence of urea.

A method is described for the rapid preparation of electrophoretically pure troponin C from rabbit skeletal-muscle myofibrils that avoids the use of urea. The three-step procedure includes extraction od the myofibrils with EDTA-containing buffers, one-step elution from DEAE-Sephadex and Sephadex G-100 chromatography in the presence of EDTA. The procedure gives yields comparable with those of currently used methods that involve dissociation of the troponin complex with urea. Except for the thiol-group reactivity, troponin C produced by our method is physicochemically and functionally indistinguishable from that obtained by the classical procedure. Purified troponin C always contains traces of calmodulin. However, this contamination can be decreased to less than 0.02% by means of a second Sephadex G-100 chromatography step in the presence of EDTA.     

Determination of boronophenylalanine in biological samples using precolumn o-phthalaldehyde derivatization and reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method for the detection of boronophenylalanine is described. Determination was obtained by precolumn reaction of o-phthalaldehyde with a mixture of standard amino acids containing boronophenylalanine and separating the corresponding o-phthalaldehyde derivatives, using a Kromasil C-18, 250 x 4.6 mm, 5-microm particle size column, a step gradient with two buffers, a flow rate of 1.2 ml/min, a column temperature of 23 degrees C, and fluorimetric detection (excitation and emission wavelengths of 330 and 430 nm, respectively). The use of such a method for assaying boronophenylalanine in biological samples was tested in neutralized perchloric acid blood and cerebral tissue extracts of rats treated with intracarotid administration of 300 mg/kg of body weight boronophenylalanine. Results of these experiments showed that the present HPLC method represents a valid alternative to currently available analytical techniques for assaying boronophenylalanine based on boron determination in terms of reproducibility, recovery, or sensitivity. Therefore, it is suggested that the present method may routinely be used in all preclinical and clinical studies in which quantification of circulating and tissue concentrations of boronophenylalanine is critical for the application of boron neutron capture therapy.     

卷枝毛霉Δ6-脂肪酸脱氢酶基因的克隆及在酿酒酵母中的高效表达

为获得产高γ 亚麻酸的酿酒酵母工程菌株,应用RT PCR技术,从卷枝毛霉中扩增出△6 脂肪酸脱氢酶 基因,亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体pYES2.0,在大肠杆菌中筛选到含有目的基因的重组质粒 pYES412,用醋酸锂方法转化到酿酒酵母缺陷型菌株INVScI中,在SC ura合成培养基中筛选到转化酵母,在合适 的培养基及培养条件下,加入外源底物亚油酸,经半乳糖诱导后,收集菌体,通过气相色谱对转化酵母进行脂肪酸 色谱分析,结果表明:γ 亚麻酸占总脂肪的50.07%。迄今为止,这是国内外△6 脂肪酸脱氢酶基因在酿酒酵母表 达量最高的报道。     

The enrichment of plasmid DNAs, in bacterial cell lysates, using an alkaline-pH procedure that does not permanently denature them

The method described permits the enrichment of large (greater than or equal to 54 kb), small (less than or equal to 2.1 kb), or intermediate sizes of plasmid DNAs. It is a modification of the plasmid enrichment method described by Currier and Nester (Anal. Biochem., 76, 431-441, 1976) in that it defers the alkali denaturation step until the pH and temperature can be controlled. This prevents permanent alkali denaturation of some plasmids. In general, total cellular nucleic acids are precipitated with either ethanol or isopropanol after lysates, in 3% w/v NaCl, are extracted with a phenol-chloroform mixture. The nucleic acids are then treated at an alkaline-pH (12.3-12.4), in a buffer, at 0 degree C, for a minimum time of 15 min. Denatured DNA, in 3% w/v NaCl, is removed with phenol. The RNA- containing, plasmid enriched fraction, is once again precipitated with either ethanol or isopropanol, dried in a vacuum, and redissolved. Samples are then digested with various restriction enzymes and/or examined directly on agarose gels after treatment with RNAase A.     

Kinetics of protein release from yeast using enzymatic lysis for selective product recovery

The kinetics of release of four intracellular enzymes from different yeast cell locations using the Differential Product Release (DPR) method has been investigated. The method uses a combination of physical, chemical and biological agents such as lytic enzymes, an osmotic support and a spheroplast stabilizer. Using the DPR technique a wall enzyme, invertase, was released with a very high specific activity in the first step from a breadmaking strain ofS. cerevisiae. Maximum release could be obtained in this step when the incubation time was extended from 60 min to 100 min. Two cytosol enzymes, α-D-glucosidase and alcohol dehydrogenase were released in the second step. Fumarase was released in the third step almost instantaneously after disruption of the mitochondria which reduces considerably, by ca. 1 hour, the total incubation time of DPR. This paper investigates the kinetics of enzyme release during the 3 steps of DPR.     

The purification of cholinesterase from horse serum

A relatively simple method is described by which cholinesterase was purified about 19000-fold starting from horse serum. Typically 20 litres of serum were processed to yield 15-18mg of electrophoretically pure cholinesterase in the form of an active salt-free dry powder. The method included two stages: fractionation with (NH(4))(2)SO(4) and ion-exchange chromatography. The (NH(4))(2)SO(4) stage included, in principle, the acid (pH3) step of the Strelitz (1944) procedure. The step took advantage of the stabilizing effect that 33%-satd. (NH(4))(2)SO(4) has on cholinesterase activity at pH3 and it is recognized that in the absence of (NH(4))(2)SO(4) the enzyme is rapidly destroyed at pH3. Cholinesterase was significantly more stable to pH3.0 at 2 degrees C than at 24 degrees C, and the acid step was done at both temperatures. The specific activities of the final products obtained by way of acid steps were the same at either temperature, thus indicating that the step has not harmed the enzyme active sites. The product from the first two stages was purified over 18000-fold and was 85-90% cholinesterase. The remaining impurities were removed by preparative gel electrophoresis. The product was about 40% more active and contained 40% more active sites per unit weight than electrophoretically pure cholinesterase prepared from partially purified commercial starting material. Although the number of active sites per molecule was not determined with certainty, a value of at least 3 and possibly 4 was indicated. The partial specific volumes were determined with a precision density meter, on the ultracentrifuge and from the amino acid and carbohydrate composition. The values by these independent methods were 0.688, 0.71 and 0.712ml/g, respectively. The amino acid and carbohydrate composition was determined. The cholinesterase contained 17.4% carbohydrate including 3.2% N-acetylneuraminic acid.     

A modified colorimetric method for the determination of pyrrolidone carboxylyl peptidase activity in bacteria

The direct spectrophotometric estimation of ferrocyanide (1) suggested the possibility of measuring small quantities of reducing sugars. However, as previously described (1), the method is not applicable to the measurement of reducing sugars because of the high temperature required for the required oxidation step.Due to the relative differences in molar extinction coefficients at the wavelengths concerned (1,5), the measurement of ferrocyanide rather than ferricyanide (5) provides a large increase in sensitivity.A comparison of such a method with a modified folin-Wu method (2–4) for estimation of blood glucose should indicate the possibility of its application to biological fluids containing cells and proteins.     

Novel cyclization chemistry especially suited for biologically derived, unprotected peptides.

A novel method is described for the cyclization of peptides--or segments of polypeptides--which requires a free N-terminal alpha-amino group and a distal amino acid residue containing a nucleophilic side chain. The reaction is conducted in two steps, both in the aqueous phase. The first step involves acylation of the N-terminal alpha-amino group with iodoacetic anhydride at pH 6. This acylation reaction has greater than 90% specificity for peptide alpha-amino groups and gives no alkylation of Arg, His, Lys or Met by the iodoacetate side product (R. Wetzel et al., Bioconjugate Chem., 1, 114-122, 1990). In the second step, the acylation reaction mixture or the isolated iodoacetyl-peptide is incubated at room temperature to give the cyclic peptide formed by reaction of the nucleophilic side chain with the iodoacetyl moiety. The pH dependence of the cyclization reaction by Met, Lys, Arg or His is consistent with the pKa of the nucleophilic side chain. Thus, peptides containing Met plus other nucleophilic amino acids should preferentially cyclize via Met at low pH. In this paper, preparation of cyclic peptides containing 3-6 amino acids is described; the full range of ring sizes and sequences which can undergo this cyclization has not been further explored. Preliminary results suggest that this method is also fairly general with respect to the amino acid sequence being cyclized. The reaction appears to be particularly suited for cyclization via Lys and Met side chains. All of the cyclized products are sufficiently stable for many biological applications.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature differentially affects encounter and docking thermodynamics of antibody--antigen association

Using BIACORE SPR, we have examined the mechanism of temperature effects on the binding kinetics of two closely related antibody Fabs (H10 and H26) which recognize coincident epitopes on hen egg-white lysozyme (HEL), and whose association and dissociation kinetics are best described by the two-step conformational change model which we interpret as molecular encounter and docking. Time-course series data obtained at a series of six temperatures (6, 10, 15, 25, 30 and 37 degrees C) showed that temperature differentially affects the rate constants of the encounter and docking steps. Docking is more temperature-sensitive than the encounter step, and energetically less favorable at higher temperatures. At elevated temperatures, the time required for docking is longer and the apparent increase in off-rate reflects the greater proportion of the molecules failing to dock and remaining in the less stable encounter state. As a consequence, distribution of free energy change between the encounter and docking steps is altered. At physiological temperature (37 degrees C) the docking step of the H26 complex is energetically unfavorable and most complexes essentially do not dock. There is a significant decrease in total free energy change of the H26 complex at higher temperatures. Elevated temperature changes the rate-limiting step of H26--HEL association from the encounter to the docking step, but not that of H10--HEL. Our results indicate that the mechanism by which elevated temperature reduces the affinities of antigen--antibody complexes is to decrease the net docking rate, and/or stability of the docked complex; at higher temperatures, a smaller proportion of the complexes actually anneal to a more stable docked state. This mechanism may have broad applicability to other receptor--ligand complexes.     

A mechanical dissociation method for preparation of muscle cell cultures

A cell preparation method by which large numbers of embryonic chick skeletal muscle cells may be obtained is described. The procedure requires fewer manipulations and much less time than standard trypsinization. By the criteria used, both methods are comparable with respect to percent viable cells and survival of plated cells. However, in addition to the ease of preparation, the mechanical dissociation method offers the significant advantage that the cell suspension is greatly enriched for myoblasts without the necessity of an additional preplating step.