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PubMedDate not supplied

A survey of potential problems and quality control in peptide synthesis by the fluorenylmethoxycarbonyl procedure.

The routine production of peptides by manual or automated solid-phase synthesis protocols has gained widespread usage among a variety of biological scientists as new synthetic procedures have been introduced over the past several years. We report here a detailed analysis of Fmoc synthesis procedures to identify problematic reactions and to evaluate analytical procedures for monitoring the quality of peptides during and after synthesis. The results of these studies demonstrate double additions of particular amino acids during single coupling cycles, frequent incomplete deblocking of peptides by standard piperidine reactions, and the failure of the Kaiser ninhydrin test to detect free amino groups of certain amino acids at the N-terminus of synthetic peptides. These results suggest the need for more careful monitoring of Fmoc synthesis reactions than previously recognized or recommended in standard protocols. We demonstrate the utility of plasma desorption mass spectrometry (PDMS) in analyzing peptide products and recommend a minimum sequence of analytical quality control including HPLC and PDMS.

Amino Acid Sequence↗
PubMedDate not supplied

Chemical modification and complex formation studies with jack bean proteinase inhibitor.

Pretreatment of the purified jack bean inhibitor with enterokinase activated human pancreatic preparation for 1 hr decreased its inhibitory capacity against crystalline bovine alpha-chymotrypsin by 30% but did not affect its trypsin inhibitory activity. Preincubation of the inhibitor with bovine chymotrypsin for 60 min resulted in partial loss of the inhibitory potency. Complex formation studies by gel chromatography on Sephadex G-100 indicated that the trypsin-inhibitor and chymotrypsin-inhibitor complexes dissociated to release inactivated inhibitor and active proteinases. Gel chromatography of the inhibitor in presence of 1.5 M ammonium sulphate indicated that the inhibitor showed a tendency to aggregate without loss of biological activity. However, in 4.2 M salt medium after 3 hr, antichymotryptic activity was lost completely without any effect on antitryptic activity. Treatment with methylamine, a nucleophile, caused a greater loss of antichymotryptic activity. Trinitrobenzene sulphonate and ethylacetamidate, the amino group modifiers, affected only the antichymotryptic activity. Treatment with ninhydrin, a specific arginine modifier, at pH 9.0 abolished the antitryptic activity whereas only 50% of the antichymotryptic activity was lost. Diethylpyrocarbonate, a histidine reagent, also decreased only the antitryptic activity. Modification of tryptophan and cysteine residues of the inhibitor had no effect on its inhibitory potency. Treatment with mercaptoethanol and sodium borohydride caused nearly 50% loss of antitryptic and antichymotryptic activities. Chloramine-T, a reagent that modifies methionine residues, inactivated the inhibitor.

Binding Sites↗
PubMed1976

Structural similarities between the Ca2+-dependent regulatory proteins of 3':5'-cyclic nucleotide phosphodiesterase and actomyosin ATPase.

Results of studies of the Ca2+-dependent protein modulator of 3':5'-cyclic nucleotide phosphodiesterase isolated from bovine brain are presented which show its structural similarity to the Ca2+-binding subunit of muscle troponin. Both proteins have blocked NH2 termini, similar and characteristic ultraviolet absorption spectra, similar Ca2+-binding properties, very similar amino acid compositions, and co-migrate on sodium dodecyl sulfate-polyacrylamide gels. The primary structures of selected tryptic peptides isolated from bovine brain modulator protein are similar or identical with regions of the primary sequences of rabbit skeletal muscle and bovine cardiac muscle troponin C. Bovine brain modulator protein contains and unidentified ninhydrin-positive basic compound not found in muscle troponin C. An improved procedure is presented which yields 40 to 70 mg of modulator protein per kg of bovine brain.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1991

[Antibiotic activity of marine gram-negative bacteria].

An antimicrobial substance was isolated from the culture fluid of Vibrio fischeri belonging to ++gram-negative sea bacteria. The substance was shown to be highly active against ++gram-positive cocci and less active against some other ++gram-positive bacteria and fungi. The active substance was defined as a low molecular weight compound which appeared to be thermostable and had positive ninhydrin reaction. The antibiotic formed when the culture was grown in the media containing sea water and protein hydrolysates or yeast extract during the exponential phase. The maximum antibiotic activity was observed during the stationary phase of culture development.

Anti-Bacterial Agents↗
PubMed1991

Accumulation of 3-hydroxy-L-kynurenine sulfate and ethanolamine in urine of the rat injected with 1-aminoproline.

By intraperitoneal injection of 1-amino-L-proline, a vitamin B6 antagonist, two unidentified ninhydrin-positive compounds were excreted abnormally in rat urine. One of them was definitely identified as 3-hydroxy-L-kynurenine sulfate by chromatographic and spectroscopic analyses. The other was proved to be ethanolamine. This is the first report on the abnormal excretion of ethanolamine in urine of the vitamin B6-deficient rats.

Animals↗
PubMed1991

Liquid chromatographic analysis of amino acids in physiological fluids: recent advances.

Amino acid analysers have been used for the quantitative investigation of amino acids in physiological fluids for over 30 years. Advances in technology have resulted in a steady decrease in analysis time and detection limits of the cation-exchange chromatography, followed by ninhydrin detection, used by the original instruments. The introduction of pre-column derivatisation and alternative HPLC techniques offers new opportunities for advancement. The analytical characteristics, principles, merits and limitations of both techniques are discussed, and their suitability for clinical purposes assessed. Practical considerations are emphasised and the influence of choice of chemistry and instrument design on the resolution and quality of quantitative data is highlighted.

Amino Acids↗
PubMed1991

[Methodological errors in amino acid assay in biological fluids].

Numerous methodological errors have been observed in the measurement of free-amino acids in the biological fluids. Some of them are related to the presence or the formation of amino acids or ninhydrin-positive compounds in tubes or anticoagulants used for the blood sampling. Hemolysis or blood clotting modify the concentration of some amino acids in plasma. A special error observed with the capillary blood is related to the amino acids present on area of the skin. Bacterial contamination of urines or buffers used for the analysis can also modify the results of the amino acids assays.

Amino Acids↗
PubMed1990

[The formation of the normal intestinal microbiocenosis in newborn infants and under antibiotic therapy].

The dynamic study of the microecological state of the intestine in 41 newborns up to the age of 3 months has been made by biochemical and bacteriological investigation methods. The results of the biochemical rapid analysis have been found to be in complete agreement with those of the bacteriological analysis and, besides, to provide essential additional data. The biochemical rapid analysis permits the evaluation of the state resistance to the colonization of the intestine in children, starting from the age of 6 days. The presence of beta-alanine and ninhydrin-positive unidentified Nos. 7, 8a-1, 8d, 9, 10, 11 and 21 is indicative of a greater decrease in the level of the colonization of the digestive tract.

Anti-Bacterial Agents↗
PubMed1990

[Several methods for determining total titers of free alpha-amino acids in body fluids].

Comparisons were made among different variants of ninhydrin reaction to determine overall titres of free alpha amino acids in body fluids. Included were para-benzoquinone, 2,4,6-trinitrobenzene sulphonic acid, and 2,4-dinitro-1-fluorobenzene (DNFB). The best reproducibility of results was recorded from DNFB which can be used, as well, for analysis of milk or rumen juice.

Amino Acids↗
PubMed1990

Characterization of nonexchangeable radioactivity in L1210 cells incubated with [14C]thiotepa: labeling of phosphatidylethanolamine.

N,N',N''-Triethylenethiophosphoramide ([14C]thiotepa) accumulation by L1210 cells is a biphasic process. A very rapid initial phase is followed by a much slower second phase that reflects accumulation of radioactivity in a form that is not lost or exchanged when cells are resuspended and incubated in drug-free medium for up to 8 h. In this study we attempted to characterize this nonexchangeable radioactivity. Nuclei (10(7)) isolated from L1210 cells and incubated with [14C]thiotepa did not accumulate 14C during incubations of up to 5 h. Similarly, nuclei isolated from 10(7) L1210 cells that had been shown to accumulate nonexchangeable 14C after incubation with [14C]thiotepa did not show an increase in nuclear-associated 14C. Eighty to 85% of nonexchangeable 14C in L1210 cells incubated with [14C]thiotepa was soluble in ethanol or chloroform:methanol (2:1, v/v), and although most of this cell-associated nonexchangeable 14C was precipitated by trichloroacetic acid, subsequent treatment of that precipitate with methanol solubilized most of the 14C so that only 15 to 20% remained with the final precipitate. When chloroform:methanol-soluble nonexchangeable 14C was analyzed with thin-layer chromatography systems suitable for thiotepa or simple lipids, all radioactivity remained at the origin. In contrast, when analyzed with one- and two-dimensional thin-layer chromatographic systems suitable for complex lipids, all chloroform:methanol-soluble radioactivity was associated with a single lipid spot. This lipid cochromatographed with phosphatidylethanolamine, reacted with ninhydrin but not with 4-(p-nitrobenzyl)pyridine or the Dragendorff choline reagent, and was digested by phospholipases C and D, all of which lead to its identification as phosphatidylethanolamine. This extensive labeling of phosphatidylethanolamine in L1210 cells incubated with [14C]thiotepa can be explained by liberation of [14C]aziridine from [14C]thiotepa, hydrolysis of the [14C]aziridine to [14C]ethanolamine, and incorporation of that radiolabeled material into phosphatidylethanolamine via the normal cellular synthetic pathways for that lipid. This information implies that thiotepa serves, at least in part, as a prodrug for aziridine and has implications as to the mechanism of thiotepa-induced cytotoxicity in that aziridine is a monofunctional alkylating agent incapable of producing interstrand, intrastrand, or protein-DNA cross-links.

Animals↗
PubMed1990

[Isolation and primary characteristics of the common species-specific outer membrane protein of Rickettsia prowazekii].

Common species-specific protein is isolated for the first time in the chromatographically pure state from the outer membrane of Rickettsia prowazekii, and its amino acid composition is determined. As revealed by chromatofocusing technique, the protein possesses pI 4.18 +/- 0.03. Three basic ninhydrin-positive compounds, differing from usual amino acids, were discovered in the protein hydrolyzate. Data suggesting the subunit structure of the isolated protein are presented.

Amino Acids↗
PubMedDate not supplied

Coupling efficiencies of amino acids in the solid phase synthesis of peptides.

The "classical" Merrifield method was used to synthesize over 500 peptides using Boc-benzyl strategy. The peptides were prepared either manually or on a Beckman 990B synthesizer or an Applied Biosystems 430A synthesizer. Each coupling of Boc amino acid to the growing peptide on the resin was monitored with the ninhydrin reaction. Couplings were considered "incomplete" if there was 99% or less coupling and "high incomplete" if there was 98% or less coupling. The efficiency of coupling was evaluated in regard to the specific amino acids involved in the coupling reaction and to the length of the peptide at the time of the coupling. The most difficult carboxyl-reacting amino acids were histidine, threonine, arginine, valine, isoleucine and glutamine; the most difficult amine reacting residues were glutamine, leucine, alanine, arginine and isoleucine. The number of "incomplete" and "high incomplete" couplings and the total number of monitored couplings of each of the 20 carboxyl-reacting amino acids when reacting with each of the 20 amine-reacting residues were tabulated. Coupling efficiencies decreased with the length of the peptide. The conclusion of this study is that, with the chemistries and methods used in this group of peptides, no amino acid coupling can be predicted to be complete with a single coupling reaction. The study points to the need for on-line determination of coupling efficiency during the synthesis in which a recoupling step is initiated when the first coupling is incomplete.

Amino Acids↗
PubMedDate not supplied

[The diagnosis of nerve trauma late].

On the basis of examination of 110 patients with sequelae of incised damages to 146 nerves, the author compared the clinical data and the results of electroneuromyography, thermography, polarography, and the ninhydrin test to appraise the anatomical condition of the nerve trunks. Electroneuromyography is a sensitive method in determining the indications for operation, but is insufficiently informative for making the differential diagnosis of intratruncal neurinomas. Complex clinico-instrumental study of the pulsed (electroneuromyography) and the trophic function proper of the nerves (evaluation of microcirculation, thermotopography, and perspiration of the autonomous zones of innervation) allows their preoperative condition to be evaluated more fully.

Electrophysiology↗
PubMed1990

[Syntheses and preliminary bioassays of inhibin segments].

Six segments within the subunit beta-A of the follicular inhibin have been synthesized by an improved solid phase procedure on p-methyl-benzhydrylamine (1% divinylbenzene) resin. The courses of the syntheses were monitored by quantitative ninhydrin assays and amino acid analyses. Following the cleavage by HF-p-cresol (9:1 V/V), the peptides were extracted with anhydrous trifluoroacetic acid containing 1% dithiothreitol and precipitated with dry ethyl ether. The purification was achieved uniquely by reverse phase HPLC and final products were characterized by several TLC systems, analytical HPLC and amino acid analyses. Pituitary cell culture bioassays were performed to ascertain their biological activities. However, among these synthetic peptides, the small peptides Ib-beta A (37-39)NH2(I), Ib-beta A(34-39)NH2(II), and Ib-beta A(30-39)NH2(III) showed no significant suppression on the LHRH-induced FSH secretion; the large peptides Ib-beta A(23-39)NH2(IV), Ib-beta A(16-39)NH2(V), and Ib-beta A(14-39)NH2(VI) lack adequate solubility in neutral media, and other methods are to be sought to test their bioactivities.

Animals↗
PubMed1990

Establishment of an external quality-assessment scheme for amino acid analyses: results from assays of samples distributed during two years.

Ten different samples of lyophilized plasma and two of liquid urine were distributed during two years to 26 laboratories performing quantitative amino acid analyses in a scheme designed to provide external quality assessment. After each distribution, statistical summaries and performance scores based on delta standard deviations and percentage biases from the all-laboratory trimmed means were returned to participants, who also received annual performance summaries based on their accumulated results. Coefficients of variation calculated from returns across all the samples ranged from 13% for glycine to 65% for methionine. Automated ion-exchange amino acid analyzers with ninhydrin detection appeared to perform better than other methods, although there was no clearly superior method and no model of analyzer clearly outperformed the others. These exercises demonstrate that there is room for improvement in the performance of quantitative amino acid analyses and that individual expertise may be more important in maintaining good performance than the choice of method or analyzer.

Amino Acids↗
PubMed1990

3-Methylhistidine determined in plasma by "high-performance" lipid chromatography.

In this fully automated method for determination of 3-methylhistidine (3MH) in plasma we use precolumn derivatization with o-phthaldialdehyde and subsequent separation by HPLC. Total analysis time is 36 min, and peak areas measured vary linearly with the amount of analyte injected, over the range of 0 to 20 pmol of 3MH (R2 = 0.995), with a coefficient of variation (CV) of 1.6%. The method is reliable, accurate, inexpensive, and at least 1000-fold more sensitive than conventional ion-exchange chromatography with ninhydrin. Because of its sensitivity, the method can be used to estimate venous-arterial differences. In four human volunteers the plasma 3MH concentration varied between 4.97 and 6.08 mumol/L, and the difference between "arterialized" and femoral venous blood for 3MH varied between 0.09 and 0.47 mumol/L.

Arteries↗
PubMedDate not supplied

Amino acid analysis of feeds in The Netherlands: four-year proficiency study.

To improve accuracy and precision of amino acid analysis in 12 Dutch feed laboratories, a proficiency study was organized twice annually over a 4-year period. The method used included reflux acid hydrolysis for 22 h followed by evaporation, separation on a cation-exchange resin in an amino acid analyzer, and photometric detection after post-column derivatization with ninhydrin. For the determination of sulfur-containing amino acids, samples were oxidized prior to hydrolysis. For the determination of tryptophan, samples underwent an alkaline hydrolysis excluding oxygen, were separated by liquid chromatography on a Hypersil ODS analytical column, and were assayed by UV or fluorescence detection. The average relative standard deviations within (CVr) and between (CVR) laboratories were 3 and 7%, respectively. For some mixed feed samples, the results from the proficiency study were compared with those obtained by the International Analytical Group. For those samples, the relative standard deviation of the difference between IAG and Dutch groups was only 1.9%. For samples that were analyzed twice during this 4-year period, relative standard deviation of between-series differences was only 2.1%.

Amino Acids↗
PubMed1990

[Study of some official pharmaceutical preparations in the Sixth Edition of Formule Normales by thin-layer chromatography].

Pharmaceutical preparations--containing aminophenazonum, noraminophenazonum sodium mesylicum, acidum acetylsalicylicum and aethylmorphinium chloratum--official in the VI. Edition of Formulae Normales have been investigated. Simple method for studying decomposition products--which can be carried out in pharmacies and can be followed the quality of products with during their usability--has been developed. The method proving and precluding the presence of decomposition products, respectively by means of preparing dilution sets has been capable of semiquantitative determination of decomposition products. DC Alufolien Kieselgel 60 F254 (Merck) layer was used. Temperature of laboratory was 22-26 degrees C and its relative humidity was 30-40%. Developing solvents were: acetone-ether-water (90:15:12) for aminophenazum; benzene-acetone-90% alcohol-wated (35: 33: 26: 11) for noraminophenazonum sodium mesylicum; methenol-glacial acetic acid-ether-benzene (1: 18: 60: 120) for acidum acetylsalicylicum; dichloromethane-methanol-concentrated ammonia (85: 15: 2) for aethylmorphinium chloratum. Detections were carried out under UV light and by using colour reagents (alcoholic ninhydrine solution for aminophenazonum, dimethylamino-benzaldehyde solution for noraminophenazonum sodium mesylicum). Rf-values of intact and decomposed compounds have been given in figures. Interfering effects of other components of preparations were studied too. These components had different Rf-values or did not react with the colour producing reagents in the applied systems.

Chromatography, Thin Layer↗