Phenylisothiocyanate Derivatization Research Articles

Vliyanie zamorazhivaniya na dinamiku aminokislotnogo sostava belkov telyatiny

The results of study of effect of freezing temperature on the change of amino acid content and the ratio of essential and nonessential amino acids in veal proteins are given. The meat of calves at the age of 3 months, bred in the Leningrad region of the RF, was the object of research. The amino acid composition of veal proteins was analyzed using the Shimadzu 20AD Chromatograph with precolumn phenylisothiocyanate derivatization and spectrophotometric detection at 254 nm by reversedphase gradient highperformance liquid chromatography (HPLC). The Kjeldahl method was used to define the protein content on the basis of the sample mineralization. It was shown that the change of the amount and ratio of essential and nonessential amino acids of veal proteins depended on the freezing temperature as well as on structural features of Rside radicals. Also it was established that the total amount of essential and nonessential amino acids in veal proteins during freezing at -24 and -35 °С temperatures decreased by 13,5 and 8,2%, respectively; the total of essential amino acids by 11 an 7%, nonessential ones by 22 and 11%, respectively. It was proved that significant changes were characteristic of amino acids with polar anionic and cationic radicals. So, the amount of amino acids with polar negatively charged radicals during veal freezing at temperatures -24 and -35 ° С decreased by 22 and 16,5%; with polar positively charged radicals - by 17 and 13,5%, respectively. At the same time, the freezing temperature affects slightly the change in the content of amino acids with nonpolar (hydrophobic) Rgroups and polar nonionogenic radicals. It was shown that the total amount of free amino acids increased independently of the freezing temperature. However, more essential amino acids were accumulated in veal frozen at a temperature of -24 ° С (2,33 times) than at -35 ° С (1,80 times). The amount of free nonessential amino acids changed to a lesser extent

Immunomodulatory Effects of Hydrolyzed Seawater Pearl Tablet (HSPT) on Th1/Th2 Functionality in a Mice Model of Chronic Obstructive Pulmonary Disease (COPD) Induced by Cigarette Smoke.

Chronic obstructive pulmonary disease (COPD) is predicted to become the third leading cause of death around the world. The present study is designed to investigate whether hydrolyzed seawater pearl tablet (HSPT) has immunoregulatory effects on the Th1/Th2 functionality in cigarette smoke-induced COPD model mice. The determination of the amino acid composition of HSPT was carried out by high-performance liquid chromatography (HPLC) with precolumn phenylisothiocyanate (PITC) derivatization. COPD model mice were constructed by cigarette smoking (CS) treatment and HSPT was administered. HSPT inhibited the infiltration of inflammation in the airway of the lung, reduced influx of eosinophils (EOSs), lymphocytes (LYMs), neutrophils (NEUs), and macrophages (MACs) in the bronchoalveolar lavage fluid (BALF), decreased the levels of IFN-γ, IL-2, IL-4, and IL-10 in the serum and lung, and decreased the expression of aforementioned cytokines in the spleen and lung in CS-treated mice. Besides, HSPT also had the ability to reduce the amount of CD3+CD4+ T cells and modulate the Th1/Th2 balance. Taken together, this study supports the consensus that CS is a critical factor to induce and aggravate COPD. HSPT could regulate the balance of Th1/Th2 in CS-induced COPD model mice, indicating its effects on inhibiting the development of COPD.

A novel method for detecting amino acids derivatized with phenyl isothiocyanate by high-performance liquid chromatography–electrospray ionization mass spectrometry

High-performance liquid chromatography–electrospray ionization mass spectrometry (HPLC–ESI-MS) is an important technique for amino acid analysis. As a common derivatization reagent, phenyl isothiocyanate (PITC) is widely used in this field. However, researchers have long faced the critical problem of coupling efficient separation with simultaneous, sensitive MS detection. In this work, a novel HPLC–ESI-MS method based on PITC derivatization was established for detecting amino acids. Complete separation of 15 amino acid derivatives was achieved with an Agilent Eclipse plus C18 column maintained at 30°C in 29min. The mobile phase was 0.05% formic acid aqueous solution (A) and 70:30 (v/v) acetonitrile–water (B). The spectral data of PITC-amino acids are indicative of losses of 17Da (NH3), 18Da (H2O) and/or 46Da (CO+H2O) from the parent protonated molecules. Assessment of this method revealed that response curves were linear within the range of 630–20,156μmolL−1. LOD and LOQ ranged within 111–816 and 311–2721pmolμL−1, respectively. The method was validated with precision values of 0.924–6.578% (intraday, n=6) and 3.327–8.962% (interday, n=6 in 3d). Therefore, the proposed method is a simple, robust alternative to current methods of detecting amino acids.

Isolation and characterization of a C12‐lipopeptide produced byBacillus subtilisHSO 121

A new lipopeptide with C12 fatty acid has been isolated from the cell broth of Bacillus subtilis HSO121 by chromatographic methods, which is believed to be the homologue of lipopeptides. The fatty acid portion was methylated and analyzed by GC/MS, ESI Q-TOF MS and 1H-NMR. The peptide portion, of which the amino acid composition was obtained by HPLC combined with a phenyl isothiocyanate (PITC) derivatization methods, was analyzed by ESI Q-TOF MS. Comparing the obtained results with surfactin C13 showed that the new lipopeptide has a peptide moiety similar to that of surfactin and the difference exists in the fatty acid portion, which is an iso-C12 beta-hydroxy fatty acid. The critical micelle concentration (CMC) of this new homologue is estimated to be 6.27 x 10(-5) mol/l in 10 mmol/l phosphate buffer solution (PBS, pH 8.0) at 30 degrees C, and the surface tension at CMC (gamma CMC) achieved is as little as 27.71 mN/m. The hemolytic activities of the C12-lipopeptide on 2% human erythrocytes showed a HC50 of 26.5 micromol/l.

Characterization of amino acids in silk sericin protein from Gonometa rufobrunnae by MEKC with phenyl isothiocyanate derivatization

An MEKC method was developed for the separation and characterization of phenyl-isothiocyanate (PITC)-labeled amino acids derived from Gonometa rufobrunnae silkworm after microdialysis sample cleanup. The influence of the buffer and SDS concentration on the resolution of the amino acids was investigated. A buffer system consisting of 25 mM phosphate, 10 mM borate buffer at pH 9.00, and 70 mM SDS showed the best results, with 13 PITC-amino acid derivatives being resolved out of 15 possible amino acids that were under study. Microdialysis sampling demonstrated its efficiency as a sample cleanup technique. Sericin protein from G. rufobrunnae was found to be characterized by at least 11 positively identified amino acids. These included His, Tyr, Ser, Ala, Phe, Lys, Gly, Arg, Cys, Glu, and Asp. Leu/Met and Val/Thr were coeluting pairs and hence could not be positively confirmed.

Synthesis and Characterization of a Collagen Model δ-O-Phosphohydroxylysine-Containing Peptide

The existence of δ-O-phosphohydroxylysine (HylP) residues in collagen has been reported. However, such phosphorylated residues have not been isolated nor has their location within the protein sequence been identified. To develop the analytical chemistry necessary for the identification of HylP in proteins, a model HylP-containing peptide, Phe-dl-HylP-Gly-Gln-Pro-Ala-Ile-Gly-Phe (I), was synthesized. The peptide was assembled using 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase synthesis; Nα-Fmoc-dl-hydroxy-dl-Lys(Nε-tert-butyloxycarbonyl)-OH was used to incorporate Hyl, and global phosphitylation/oxidation was used to introduce the phosphate group. The pKa2 of the phosphate group, as determined using 31P NMR, was 5.6. Phosphoamino acid analysis of I was performed using either dabsyl chloride or phenylisothiocyanate derivatization followed by microbore reversed-phase HPLC separation of Nα,ε-didabsyl-HylP or Nα,ε-diphenylthiocarbamyl-HylP. HylP was found to be relatively resistant to acid hydrolysis, allowing for its quantitation. Solid-phase Edman degradation of I was used for positive identification of Nα-phenylthiohydantoin-Nε-phenylthiocarbamyl-HylP. The masses of the phenylthiohydantoin and dabsyl derivatives of HylP were confirmed by electrospray ionization triple-quadrupole (ESI-MS). Peptide I was positively identified as a phosphopeptide by ESI-MS/precursor-ion scanning. Low-energy ESI-MS/MS confirmed the position of HylP within the sequence of I. Phosphorylation of Hyl led to complete resistance of I to lysine-specific endopeptidases.

Compositional analysis of small peptides using capillary electrophoresis and Ru(bpy) 33+-based chemiluminescence detection

Ru(bpy) 3 3+-based chemiluminescence detection of underivatized amino acids was coupled to capillary electrophoresis and evaluated as a separation and detection methodology for the compositional analysis of peptides and proteins. Two tripeptides were studied: glycine–phenylalanine–alanine, and valine–proline–leucine, and the method was demonstrated to be quantitative and reproducible at the 10-μg and 5-μg peptide levels, with a limit of detection (LOD) of 2.5 pmol for gly–phe–ala and 80 fmol for val–pro–leu (S/N=2). Ru(bpy) 3 3+-based chemiluminescence detection coupled to CE offers a sensitive and time-efficient method for the determination of the amino acid composition of peptides or proteins without derivatization. The detection limits for most amino acids were demonstrated to be at, or below the level achievable using ninhydrin or phenyl isothiocyanate derivatization. Amino acids were identified by their migration time through the capillary and their relative luminescence. The relative luminescent response obtained upon reaction of Ru(bpy) 3 3+ with amino acids was dependent upon the amino acid R-group at the α-carbon, and the relative response may be useful in broadly classifying a number of amino acids.

Improved HPLC determination of acidic opines by phenylisothiocyanate derivatization and its application to marine animals.

We present here a reliable and sensitive method for the determination of acidic opines such as meso-alanopine, beta-alanopine, tauropine and strombine in biological samples. Interfering primary amino acids were eliminated by reaction with o-phthalaldehyde, and the derivatized compounds were passed through Sep-Pak Plus PS-1 cartridges. The acidic opines were recovered by flushing the cartridges with water, then determined by high performance liquid chromatography after a second derivatization with phenylisothiocyanate. All 4 acidic opines were detected within 30 min. This method ensured good separation and guaranteed almost full recovery of all acidic opines. This method was applied to analyze opines in marine animals and to test whether opines are metabolized in the livers of the rat and fish.

Protein Crosslinking by the Maillard Reaction: Dicarbonyl-Derived Imidazolium Crosslinks in Aging and Diabetes

α-Dicarbonyl compounds that arise from various metabolic pathways react with proteins to form a variety of adducts in a reaction known as the Maillard reaction. These adducts are collectively known as advanced glycation end products or AGEs. Methylglyoxal (MG) and glyoxal (GXL) are two such dicarbonyls. They react with proteins to produce lysine–lysine imidazolium crosslinking AGEs. The imidazolium crosslinks derived from MG (MOLD-methylglyoxal-lysine dimer) and GXL (GOLD-glyoxal-lysine dimer) are present in human tissue proteins. In this study, we report an HPLC method for the simultaneous quantification of GOLD and MOLD in biological specimens. The method consists of reverse-phase HPLC of acid-hydrolyzed proteins, collection of eluate-containing imidazoliums, phenylisothiocyanate derivatization, followed by a second reverse-phase HPLC. This method was linear for both the imidazolium compounds in the range of 0.5–300 pmol. The levels of GOLD and MOLD in aging lenses (20 to 80 years) were trace–8.4 pmol and 15–93 pmol per milligram of protein, respectively. Cataractous lenses showed significantly higher levels of both GOLD and MOLD (mean ± SD, 14.5 ± 1.8 and 141 ± 18.4 pmol per milligram of protein, P < 0.05). Brunescent lenses had the highest levels of imidazolium crosslinks (GOLD, 18.36 ± 2.5; and MOLD, 179.2 ± 32.3 pmol per milligram of protein, P < 0.05). The GOLD and MOLD levels were higher in diabetic plasma proteins when compared to that of normal (GOLD, 17.5 ± 6.34 pmol per milligram of protein vs 43.5 ± 15.96 pmol per milligram of protein; and MOLD, 172.5 ± 32.53 pmol per milligram of protein vs 273 ± 62.67 pmol per milligram of protein, P < 0.05). GOLD and MOLD are significant in terms of tissue damage in aging and diabetes because they represent protein crosslinking by compounds that are major precursors of AGEs. Our method can be used for quantification of imidazolium crosslinks in tissue proteins to assess α-dicarbonyl-mediated protein damage in vivo.

Column chromatography and verification of phytosiderophores by phenylisothiocyanate derivatization and UV detection

A high-performance liquid chromatographic method was developed for the separation of phytosiderophores and for verification of the Fe 3+-complexing ability of chromatographic separates. The chromatographic procedure involves separation on a C 18 reversed-phase column by linear gradient elution with sodium acetate (pH 6.4) and methanol with UV detection, following pre-column derivatization of the phytosiderophores with phenylisothiocyanate (PITC). A modification of the procedure involving pretreatment of the sample with Fe 3+, which blocks the derivatization by PITC, was used to confirm whether a given chromatographic peak was attributable to an Fe 3+-complexing compound. The practical detection limit was approximately 100 pmol of phytosiderophore.

Analysis of amino acids in nectar from pitchers of sarracenia purpurea (Sarraceniaceae)

Sarracenia purpurea L. (northern pitcher plant) is an insectivorous plant with extrafloral nectar that attracts insects to a water-filled pitfall trap. We identified and quantified the amino acids in extrafloral nectar produced by pitchers of S. purpurea. Nectar samples were collected from 32 pitchers using a wick-sampling technique. Samples were analyzed for amino acids with reverse-phase high-performance liquid chromatography with phenylisothiocyanate derivatization. Detectable amounts of amino acids were found in each of the 32 nectar samples tested. Mean number of amino acids in a nectar sample was 9 (SD = 2.2). No amino acid was detected in all 32 samples. Mean amount of amino acids in a nectar sample (i.e., amount per wick) was 351.4 ng (SD = 113.2). Nine amino acids occurred in 20 of the 32 samples (aspartic acid, cysteine, glutamic acid, glycine, histidine, hydroxyproline, methionine, serine, valine) averaging 263.4 ng (SD = 94.9), and accounting for ~75% of the total amino acid content. Nectar production may constitute a significant cost of carnivory since the nectar contains amino acids. However, some insects prefer nectar with amino acids and presence of amino acids may increase visitation and capture of insect prey.

Determination of non-protein amino acids and toxins in Lathyrus by high-performance liquid chromatography with precolumn phenyl isothiocyanate derivatization

A simple procedure for the precolumn derivatization of toxic and non-toxic non-protein amino acids occurring in the legume crop Lathyrus sativus and other Lathyrus species with phenyl isothiocyanate and an HPLC method for the separation of the derivatives in the nano- and picomole range are reported. The results are compared with those of conventional ion-exchange chromatography for the separation of non-protein amino acids with postcolumn ninhydrin reaction. The relative standard deviations for the two methods are compared.

Protein determination in polychromed stone sculptures, stuccoes and gesso grounds

Abstract Various procedures were used to extract and determine proteinaceous material from artificial specimens and samples taken from works of art. Polychromed stone sculptures (twelfth-thirteenth century), the gesso ground from a wooden statue (fifteenth century), fresco plasters (sixteenth century) and stuccoes of various ages were examined. The best results were obtained by extracting protein with 1N NaOH at 80°C for 180 minutes and determination by a modified Folin phenol assay. The amino acid composition of proteins was determined by direct hydrolysis of the samples and desalting on a sulphonic resin. High performance liquid chromatography (HPLC) using phenyl isothiocyanate (PITC) derivatization permitted the identification of some of the proteins used in the works of art and demonstrated the presence of amino acids deriving from microorganism activity and environmental contamination. The extraction method used here and direct hydrolysis appeared to be complementary: the colorimetric procedure permi...

Semi-automated chromatographic procedure for the isolation of acetylated N-terminal fragments from protein digests

Several published procedures have been combined to develop a general strategy for the specific identification and isolation of the acetylated-N-terminal fragment from all other proteolytic fragments. This ruse can be divided into four steps: (i) succinylation of the substrate to block lysine NH 2 groups; (ii) enzymatic digestion of the modified protein; (iii) automated phenylisothiocyanate derivatization of the protease derived fragments to block newly generated “free” N-termini; and (iv) reversed-phase high-performance liquid chromatography with on-line photodiode array spectroscopy. The individual phenylthiocarbamyl-peptide species exhibit an increased reversed phase retention time and a greater UV (210–297 nm) profile compared to the corresponding control (-phenylisothiocyanate) digest. The N-terminal acetylated fragment shows neither a retention time shift nor an augmented UV profile. To validate each process step, synthetic peptides and acetylated-N-terminal proteins of known sequence were used as test samples. The desired fragment was isolated from three proteins and positively identified by electrospray mass spectrometry and amino acid composition, Proteins with other N-terminal blocking groups should be amenable to this procedure.

DNA sequence and transcriptional analysis of the tblA gene required for tabtoxin biosynthesis by Pseudomonas syringae

The tblA gene of Pseudomonas syringae is required for tabtoxin biosynthesis and is under the control of a regulatory gene, lemA. We have determined the nucleotide sequence of the tblA gene and identified the 5' end of the tblA gene transcript. The sequence of the tblA gene was identified to that of the recently reported open reading frame 1 gene of the tabA region of the BR2 chromosome. The open reading frame of the tblA gene potentially encodes a protein of 231 amino acids. mRNA from the tblA gene was detected at all phases of cells grown in minimal medium. This result is correlated with the constitutive production of tabtoxinine-beta-lactam (the biologically active part of the toxin) by P. syringae BR2R in minimal medium, as quantitated by a phenylisothiocyanate derivatization method.

Effects of acoustic overstimulation and perilymph perfusion--with Joro spider toxin on the concentrations of amino acids in the perilymph

Glutamate (Glu) is considered one of the most probable neurotransmitter candidates in primary afferent nerves of the cochlea. In the present study, the amino acid profile in the perilymph was determined in order to investigate the effects of acoustic overstimulation and perilymph perfusion with Joro Spider Toxin (JSTX), a specific antagonist of the quisqualate sensitive Glu receptor, on the stimulus-induced release of Glu. In the first series of experiments (control group; n = 10), samples of the scala tympani perilymph (PST) were collected from albino guinea pigs at 60 and 75 min after initiating cochlear perfusion with an artificial perilymph. In the second series of experiments (acoustic overstimulation group; n = 7), the PST samples were collected before (at 60 min after the initiation of perilymph perfusion) and after (at 75 min after the initiation of perilymph perfusion) exposure to a pure tone (2000 Hz, 110 dBSPL for 15 min). In the third series of experiments (JSTX group; n = 8), the PST samples were collected as described above under condition of cochlear perfusion with an artificial perilymph containing of 3.54 x 10(-8) M and 3.54 x 10(-6) M of JSTX. The concentrations of Glu, aspartate, glycine, alanine and leucine were measured by high-performance liquid chromatography (HPLC) using phenylisothiocyanate derivatization. There was no significant effect of acoustic overstimulation or perilymph perfusion with JSTX on the levels of amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Precolumn phenylisothiocyanate derivatization and liquid chromatography of free amino acids in biological samples

A method for the analysis of free amino acids using precolumn phenylisothiocyanate derivatization and liquid chromatography was tested on a range of sample matrices: bovine plasma, dry cat food, dry infant milk formula and potatoes. The chromatographic resolution of the phenylthiocarbamyl derivatives of free amino acids was satisfactory, with few unidentified compounds present. Standard deviations for triplicate analyses were generally less than 10% of their means. 5-Sulfosalicylic acid (SSA) and a small pore membrane ultrafilter were tested for the deproteinization of bovine plasma. Recoveries of standards were highest when 1% and 4% SSA were used for deproteinization (98% average recovery), and lower (91%) when the ultrafilter was used. The 4% SSA treatment gave higher average free amino acid contents and protein removal than the other deproteinization techniques. These higher values were not due to protein hydrolysis by SSA. Interaction of free amino acids with plasma protein remaining after deproteinization may account for the lower free amino acid contents measured after 1% SSA and ultrafilter treatment. Deproteinization of plasma with 4% (w/v) (final concentration after mixing with the sample) SSA is recommended.

Identification of a tyrosine residue in rat guanidinoacetate methyltransferase that is photolabeled with S-adenosyl-L-methionine.

Exposure of rat guanidinoacetate methyltransferase to ultraviolet light in the presence of S-adenosyl-L-[methyl-3H]methionine ([methyl-3H]AdoMet) results in covalent linking of radioactivity to the enzyme protein. The incorporation of radioactivity shows no lag and is linear with respect to time up to 1 h. The photolabeling is saturable with [methyl-3H]AdoMet, and the binding constant of the enzyme for AdoMet determined in this experiment is similar to that obtained by equilibrium dialysis. Low concentrations of competitive inhibitors S-adenosyl-L-homocysteine and sinefungin effectively prevent the photoinduced labeling by AdoMet. Although guanidinoacetate methyltransferase is irreversibly inactivated upon ultraviolet irradiation in the absence of AdoMet, the enzyme inactivated by 1-h exposure to ultraviolet irradiation has been shown to bind AdoMet with an affinity identical to that of the native enzyme. These results indicate that photolabeling occurs at the active site. Following proteolysis of the [methyl-3H]-AdoMet-labeled enzyme with chymotrypsin, a radioactive peptide is isolated having a sequence Asp-Thr-X-Pro-Leu-Ser-Glu-Glu-Thr-Trp. The peptide corresponds to residues 134-143, with X being modified Tyr-136. The same peptide is photolabeled when [carboxy-14C]AdoMet is used. High-performance liquid chromatography of this peptide after acid hydrolysis and phenyl isothiocyanate derivatization suggests that the entire molecule of AdoMet is attached to Tyr-136.

Determination of cysteine plus half-cystine in protein and peptide hydrolysates: Use of dithiodiglycolic acid and phenylisothiocyanate derivatization

Cysteine and half-cystine in proteins and peptides can be determined by acid hydrolysis in the presence of dithiodiglycolic acid, derivatization with phenyl isothiocyanate, and HPLC separation of the phenylthiocarbamyl amino acids. The determination requires no additional sample handling, oxidation, reduction, alkylation, or desalting. Samples containing cystine are treated in the same manner as those containing cysteine. Derivatization of cysteine or half-cystine by dithiodiglycolic acid has no effect on any other amino acids. Recovery of the PTC-cysteine derivative is greater than 90% over the linear range of 5 pmol to 1 nmol. Precision of the cysteine plus half-cystine determination is equivalent to that observed for all other amino acids. Structure work on the derivative indicates that it is a mixed disulfide containing cysteine and thioglycolic acid.

High-performance liquid chromatographic determination of desmosine and isodesmosine after phenylisothiocyanate derivatization

A new sensitive and selective high-performance liquid chromatographic method for the analysis of desmosine and isodesmosine in human and rat tissues is described. This method requires a purification step with column chromatography, followed by precolumn derivatization with phenylisothiocyanate. The reaction products are then separated by isocratic chromatography on a C 18 column and quantitated by ultraviolet detection at 254 nm. The recovery of standards of both compounds added to tissue samples and analysed by this method is usually greater than 90%, and the absolute detection limit is 0.5 ng for both compounds. The method is sensitive enough to measure both substances in tissue fragments of 30 mg of wet mass, which means that it can be used to study elastin in small human biopsies.