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Here, we aimed to use graphene oxide to improve the selectivity and sensitivity of Tyr determination via the reaction with 1-nitroso-2-naphthol as a selective reagent of Tyr. The reaction between Tyr and 1-nitroso-2-naphthol in absence and presence of GO was studied spectrophotometrically. Different parameters such as concentrations, temperature, incubation time were optimized. The obtained data showed that the maximum absorbance was achieved by using 2 mL of 0.03% 1-nitroso-2-naphthol at temperature 60 °C for 10 min. On the basis of calibration curve of various concentrations of Tyr in the presence of 20 μg mL -1 GO, the limit of detection was 6.4 × 10 -6  M (1.15 μg mL -1 ), where in absence of GO was 1.1 × 10 -5  M (19.9 μg mL -1 ). The selectivity of Tyr in presence of other amino acids and phenols was studied with and without GO. The data obtained revealed that the selectivity of Tyr in presence of GO with respect to some amino acids and phenols was improved. The proposed method has been applied for the determination of Tyr in urine and serum samples. Therefore, GO is a powerful catalytic surface for the sensitive and selective determination of Try in biological fluids., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Protein tyrosine sulfation (Tyr-O-SO3) is a common post-translational modification (PTM), which is important for protein function. Absolute quantitation of Tyr-O-SO3 in recombinant therapeutic proteins has been challenging. We report here an MRM method used for absolute quantitation of Tyr-O-SO3 in the hydrolysate of a recombinant Fc-fusion protein. Quantitation is achieved by monitoring the sum of two transitions: the loss of carboxylic acid from tyrosine sulfate (major transition) and sulfate group from tyrosine sulfate sodium salt. The method exhibits a good sensitivity with a limit of quantitation of 1.4 ng/mL, linearity over three orders of magnitude, good repeatability, precision and accuracy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We synthesized and characterized a new tagging reagent, (3R,4S)-1-(4-(aminomethyl)phenylsulfonyl)pyrrolidine-3,4-diol (APPD), for the selective fluorogenic derivatization of 3-nitrotyrosine (3-NT) residues in peptides (after reduction to 3-aminotyrosine) and affinity enrichment. The synthetic 3-NT-containing peptide, FSAY(3-NO(2))LER, was employed as a model for method validation. Furthermore, this derivatization protocol was successfully tested for analysis of 3-NT-containing proteins exposed to peroxynitrite in the total protein lysate of cultured C2C12 cells. The quantitation of 3-NT content in samples was achieved through either fluorescence spectrometry or boronate affinity chromatography with detection by specific fluorescence (excitation and emission wavelengths of 360 and 510 nm, respectively); the respective limits of detection were 95 and 68 nM (19 and 13 pmol total amount) of 3-NT. Importantly, the derivatized peptides show a strong retention on a synthetic boronate affinity column, containing sulfonamide-phenylboronic acid, under mild chromatographic conditions, affording a route to separate the derivatized peptides from large amounts (milligrams) of nonderivatized peptides and to enrich them for fluorescent detection and mass spectrometry (MS) identification. Tandem MS analysis identified chemical structures of peptide 3-NT fluorescent derivatives and revealed that the fluorescent derivatives undergo efficient backbone fragmentations, permitting sequence-specific identification of protein nitration at low concentrations of 3-NT in complex protein mixtures., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A fully automated chiral capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) method was developed for enantiomeric quantification of 3,4-dihydroxyphenylalanine (DOPA) and its precursors, phenylalanine (Phe) and tyrosine (Tyr). To avoid MS source contamination, a negatively charged chiral selector, sulfated β-cyclodextrin (sulfated β-CD), that migrated away from the detector was used in combination with the partial filling technique. The six stereoisomers were simultaneously quantified in less than 12 min. Detection limits were 0.48 and 0.51 μM for l- and d-DOPA enantiomers, respectively. Assay reproducibility values (relative standard deviations [RSDs], n=6) were 4.43, 3.15, 4.91, 5.16, 3.96, and 3.25% for l- and d-DOPA, l- and d-Tyr, and l- and d-Phe at 10 μM, respectively. Thanks to the high enantioseparation efficiency, detection of trace d-DOPA in l-/d-DOPA mixtures could be achieved. The assay was employed to study the metabolism of DOPA, a well-known therapeutic drug for treating Parkinson's disease. It was found that l-DOPA was metabolized effectively in PC-12 cells. Approximately 88% of l-DOPA disappeared after incubation at a cell density of 2×10(6)cells/ml for 3 h. However, d-DOPA coexisting with l-DOPA in the incubation solution remained intact. The enantiospecific metabolism of DOPA in this neuronal model was demonstrated., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Measurement of nitrotyrosine levels in biological fluids can serve as a biomarker for oxidative/nitrative damage arising from formation of reactive nitrogen species, including peroxynitrite. Peroxynitrite is formed by the reaction of the superoxide radical (O2.-) with the nitric oxide radical (.NO) that is generated by nitric oxide synthase (NOS). This article describes an immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure 3-nitrotyrosine at very low (picomolar) levels. Incorporation of a pronase digestion step prior to the immunoaffinity LC-MS/MS allowed for measuring not only free amino acid but also protein 3-nitrotyrosine in biological fluids. The use of an in-line antibody column allowed for increased specificity as compared with previously reported assays. The assay is linear over a range of 5 to 500 pg/ml (0.022-2.20 nM, r(2)=0.9987), with the lower detection limit being 5 pg/ml. In addition to its increased sensitivity and specificity, this assay showed great nitrotyrosine recovery from biological fluids when either nitrotyrosine or nitrotyrosine-containing peptides were added exogenously. The utility of this assay for nitrotyrosine as a clinically translatable biomarker was demonstrated by quantifying both free and total nitrotyrosine levels in various biological fluids, including urine, plasma, serum, cerebrospinal fluid (CSF), and synovial fluid (SF) from both preclinical species and human subjects. Thus, whether in an animal model of human disease or in a clinical setting, the quantification of nitrotyrosine levels should provide support for NOS-driven pathology and its blockade following therapeutic intervention.

A simple and sensitive spectrophotometric method to resolve ternary mixtures of tryptophan (Trp), tyrosine (Tyr), and histidine (His) in synthetic and water samples is described. It relies on the different kinetic rates of the analytes in their oxidative reaction with potassium ferricyanide (K(3)Fe(CN)(6)) in alkaline medium. The absorbance data were monitored on the analytical wavelength (420 nm) of K(3)Fe(CN)(6) spectrum. Synthetic mixtures of the three amino acids were analyzed, and the data obtained were processed by principal component-artificial neural network (PC-ANN) models. After reducing the number of spectral data using principal component analysis, an artificial neural network consisting of three layers of nodes was trained by applying a back-propagation learning rule. Tangent and sigmoidal transfer function were used in the hidden and output layers, respectively. The analytical performance of this method was characterized by relative standard error. The method allowed the determination of Trp, Tyr, and His at concentrations between 10 and 55, 10 and 60, and 10 and 40 microg ml(-1), respectively. The results show that the PC-ANN is an efficient method for prediction of the three analytes.

In this paper, we propose an evanescent wave-based infrared (IR) spectroscopic sensing method for the selective and sensitive detection of tyrosine in aqueous solution. In this approach, alpha-cyclodextrin (alpha-CTD) was chemically immobilized onto the surface of an IR-sensing element to attract tyrosine specifically to the surface of the sensing element. Theoretical equations were developed for the quantitative analysis of tyrosine. Based on its IR spectra, the synthesized alpha-CTD phase was stable in water. Optimal detection with this system occurred when the pH of the solution was ca. 10.5. Based on the absorption bands, we confirmed that alpha-CTD was most effective at attracting tyrosine under basic conditions. Using the unique absorption band of tyrosine at 1500 cm(-1), the alpha-CTD phase allowed the detection of tyrosine selectively from among a range of potentially interfering amino acids and other species commonly present in biological samples. For quantitative analysis, this CTD-modified phase was most suitable for sensing tyrosine at concentrations below 100 microM because of limits in the surface adsorption mechanism. The detection times were, in some instances, lower than 5 min. For a detection time of 10 min, the detection limit of tyrosine was ca. 0.4 microM.

3-Nitrotyrosine (3-NT) is formed by the reaction of peroxynitrite with either free or protein-bound tyrosine residues and has been proposed as a biomarker of oxidative stress caused by reactive nitrogen species. This study describes the development of an HPLC electrochemical detection assay for free 3-NT capable of measuring this metabolite at the very low (nanomolar) levels encountered physiologically. We employed a dual-cell coulometric approach in which 3-NT is first reduced at an upstream cell to 3-aminotyrosine, which itself is then oxidized at the downstream cell. The method was shown to be linear over the range of 1-500 nM (r = 0.999), with a detection limit (signal/noise ratio of 3) of 0.5 nM (25 fmol on column). Ten consecutive injections of 2 and 20 nM 3-NT standards produced coefficients of variation of 5.88 and 1.87%, respectively. Validation of the identity of the 3-NT peak was confirmed by coelution with authentic standards and by the in vitro production of 3-NT by incubation of 3-morpholinylsydnoneimine (SIN-1, 100 microM), a molecule releasing nitric oxide and superoxide in solution at a pH of 7.0 or higher with tyrosine (10 microM). Using this method, 3-NT was detected in human liver microdialysate (levels up to 2.6 nM), although levels in rat spinal cord dialysate were below the limit of detection.

Tyrosine nitration is a well-established protein modification that occurs in disease states associated with oxidative stress and increased nitric oxide synthase activity. Nitration of specific tyrosine residues has been reported to affect protein structure and function, suggesting that 3-nitrotyrosine formation may not only be a disease marker but may also be involved in the pathogenesis of some diseases and in normal regulatory processes. It has been, however, difficult to identify sites of nitration. We describe a method that combines specific isolation of nitrated proteins with mass spectrometric determination of the amino acid sequence and the site of nitration of individual proteins. A complex protein mixture, e.g., serum or cell lysate, was enriched for nitrotyrosine-containing proteins by immunoprecipitation with antinitrotyrosine antibodies. The nitrotyrosines were then reduced to aminotyrosines with a strong reducing agent in parallel in-gel and in-solution procedures. Using nitrated human serum albumin as a model, we reduced the disulfide bonds with dithiothreitol and alkylated the free sulfhydryl groups with iodoacetamide. The nitrotyrosines were next reduced to aminotyrosines with sodium dithionite, and-at pH 5.0-cleavable biotin tags were selectively attached to the aminotyrosines and the albumin was then digested with trypsin. The biotinylated tryptic peptides were purified on a streptavidin affinity column and identified by mass spectrometry. We have also purified nitrated human serum albumin from an enriched sample of SJL mouse plasma and confirmed its identity by peptide mass fingerprinting and MASCOT.

3-Nitrotyrosine, a product of tyrosine nitration, is useful as a marker for the generation of reactive nitrogen oxide species with short half-lives such as peroxynitrite. A reverse-phase high-pressure liquid chromatographic method using a dual-mode electrochemical detector in series with a photodiode array detector has been developed to determine the levels of 3-nitrotyrosine in biological samples. The principle of this method involves reduction of 3-nitrotyrosine at an upstream gold amalgam electrode and oxidation of the resulting product(s) at a downstream glassy carbon electrode. 3-Nitrotyrosine is quantified by the amount of the current generated at the downstream electrode, and a femtomole detection level can be achieved. The disappearance of the corresponding peak when the electrochemical detector is used only in the single oxidative mode provides additional evidence for the identity of 3-nitrotyrosine in the sample. Tyrosine from the same sample is determined by its UV absorption at 280 nm, thus eliminating the need for an internal standard. With this method a dose-dependent increase of 3- to 10-fold in the levels of protein 3-nitrotyrosine was observed in the blood plasma, and a 2- to 4-fold increase in the lung cytosols, of rats treated with the lung carcinogen and nitrating agent tetranitromethane., (Copyright 2000 Academic Press.)

A number of methods are commonly employed for the determination of protein in biological samples. Unfortunately, several compounds that are constituents of biological buffers interfere with these methods, limiting their application. Previous studies have demonstrated that tyrosine rapidly undergoes nitration in nitric acid to yield 3-nitrotyrosine, which has a lambdamax of 358 nm. Utilizing this reaction, we have developed a one-step method for the assessment of protein content in biological samples. Common interfering substances, including SDS, urea, glycerol, ammonium sulfate, and beta-mercaptoethanol, do not interfere with this method. Because of its simplicity, this reaction might be useful for estimating protein content in a variety of biological samples., (Copyright 1999 Academic Press.)

Oxidative damage by reactive nitrogen species has been implicated in the pathogenesis of atherosclerosis and other inflammatory diseases. The mechanisms of tissue damage are poorly understood, however, because the toxic intermediates are short-lived. Previous in vitro studies have suggested that 3-nitrotyrosine represents a specific marker of protein oxidation by reactive nitrogen species. The detection of this nitrated aromatic amino acid may thus serve as an indicator of tissue injury by nitrogen species in vivo. Here we describe a highly sensitive and specific analytical method for quantifying free and protein-bound 3-nitrotyrosine. The assay involves acid hydrolysis of proteins, isolation of 3-nitrotyrosine by ion exchange chromatography, and reduction of 3-nitrotyrosine to 3-aminotyrosine with dithionite. The reduced amino acid is then converted to its n-propyl, per-heptafluorobutyryl derivative and quantified by isotope dilution gas chromatography negative-ion chemical ionization mass spectrometry. Attomole levels of 3-nitrotyrosine can be reproducibly measured in this manner. Quantifying 3-nitrotyrosine levels of tissues by stable isotope dilution gas chromatography/mass spectrometry should provide a powerful tool for exploring the impact of reactive nitrogen species on oxidative reactions in vivo.

We describe an improved method for the analysis of pheomelanin in biological samples. The method is based on a chemical degradation of the melanin polymer and HPLC analysis of specific degradation products. Hydriodic hydrolysis provides 4-amino-3-hydroxyphenylalanine (AHP) and 3-amino-l-tyrosine (AT) which are detected with an electrochemical detector. We have examined each step of the analysis and the results are presented in this paper. First the samples are hydrolyzed for 16 h. AT and AHP are then isolated from the hydrolysates by ion-exchange chromatography and then separated and quantitated by HPLC and electrochemical detection. The method shows good reproducibility with a total imprecision below 5.6%. The linearity of the method was shown from 0 to 490 ng AT and 0 to 850 ng AHP per sample, using a melanoma cell suspension (27 mg protein/ml) with up to 24-fold dilutions of the original sample. For cultured "normal" human melanocytes a minimal amount of 0.1 mg protein is sufficient for analysis of pheomelanin in the samples. This method provides the opportunity to study the composition of the formed melanin in cell lines, cultured in different growth media., (Copyright 1997 Academic Press.)

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been implicated in myriad disease etiologies and may represent an obligate pathologic sequelus of inflammation. Unfortunately, few sensitive and specific analytical techniques exist for the routine assay of biomarkers indicative of ROS and RNS elaboration. In this study, high-performance liquid chromatography is used in conjunction with coulometric electrochemical array (HPLC-EC) detection to allow ultrasensitive determination of protein-bound 3-nitrotyrosine and 3, 4-dihydroxyphenylalanine (3-hydroxytyrosine) as specific in situ biomarkers of protein exposure to reactive nitrating and oxidizing species. Tyrosine and derivatives can be analyzed simultaneously with practical detection limits for tyrosine, 3-NT, and 3,4-Dopa being 10, 50, and 2 pmol, respectively, in as little as 20 microL of sample. HPLC-EC array detection allows two-dimensional resolution of chromatograms, greatly facilitating peak detection and confidence assignment. A method of sample preparation wherein tyrosine analogs are enzymatically hydrolyzed from protein without the need for sample extraction, concentration, or derivatization is reported., (Copyright 1997 Academic Press.)

This article describes chromatographic and spectroscopic techniques that have multiple applications in the preparation, isolation, and analysis of dityrosine. A three-step chromatographic procedure facilitates the preparation of 120 mg or more (yield > 26% of theoretical maximum) of dityrosine from the enzyme-catalyzed oxidation of tyrosine. DEAE-cellulose chromatography performed in a boric acid-sodium borate buffer removes most of the contaminating pigments. Two-dimensional pH-dependent chromatography on BioGel P-2 separates dityrosine from tyrosine, residual pigments, salts, etc. Elemental analysis indicates that the purified product is approximately 92% dityrosine by weight. Fast atom bombardment mass spectrometry and two types of reverse-phase high-performance liquid chromatography (HPLC), monitored in fluorescence and absorbance measurements, verify the purity of the dityrosine. The distinctive pH-dependent chromatography of dityrosine on BioGel P-2, with reversible adsorption to the matrix occurring at pH values less than 3, is useful for the isolation of varying quantities of dityrosine and for analysis per se. Affinity chromatography on immobilized phenyl boronate (Matrex Gel PBA-60) is an alternate method for the isolation and determination of dityrosine, which undergoes specific interactions with the boronate moiety and possible hydrophobic association with the phenyl group. Two new reverse-phase HPLC techniques expedite the analysis of picomole quantities of dityrosine. One employs isocratic elution (92% H2O, 8% acetonitrile, and 0.1% trifluoroacetic acid) of an ODS II Spherisorb column, with both fluorometric and spectrophotometric detection. The other procedure may be performed in conjunction with total amino acid analysis. A rapid gradient program, developed with a Phenomenex Ultracarb 20 column, clearly separates dabsylated dityrosine and tyrosine from other dabsylated amino acids. It is especially useful when dityrosine is a trace component.

An improved method for the quantitative derivatization of amino acids with fluorenylmethyl chloroformate (FMOC-Cl) is described. Amino acids are derivatized in borate buffer at pH 11.4 for 40 min at ambient temperature. All amino acids resulted in stable derivatives. In particular, improved derivatization was obtained with the troublesome amino acids His and Tyr: exclusively monosubstituted His and disubstituted Tyr were formed, eluting as free peaks in the chromatogram. These derivatives show a higher fluorescence response than their disubstituted and monosubstituted counterparts, respectively, resulting from other protocols. Under the new conditions, considerable less of the hydrolysis product of FMOC-Cl is seen in the chromatograms. Baseline noise was substantially reduced at a higher emission wavelength (630 nm instead of 313 or 340 nm). With simple precautions, extensive adsorption of the disubstituted derivatives (Lys, Hyl, and Tyr) on plastic or glass surfaces could be prevented. Calibration curves were linear over a 10 to 300 molar ratio of FMOC-Cl to total amino acid. The detection limits are in the femtomole range and the derivatives are stable for more than 48 h, thus permitting automated analysis of multiple samples.

We have developed a new method for the quantification of phosphoserine, phosphothreonine, and phosphotyrosine as dabsyl derivatives in acid-hydrolyzed extracts of 32P-labeled A431 cells. In the first step the phosphamino acids are concentrated using a disposable anion-exchange column. Subsequently, the phosphoamino acid fraction is treated with dabsyl reagent (28.8 mM) for 10 min at 70 degrees C. After cleanup with a second anion-exchange column followed by separation on a disposable C18 column, the covalently modified phosphoamino acids are separated on silica TLC sheets using a one-dimensional solvent system. The major advantages of this method are the complete separation of dabsylated P-Ser, P-Thr, and P-Tyr on silica aluminum sheets in a very reproducible way without the interference of 32P contaminants originating from hydrolyzed cell extracts. Very clean chromatograms are obtained, enabling the fast and unambiguous quantification of the phosphoamino acids by simply cutting out the relevant spots from the aluminum sheets. A high sensitivity is achieved by the removal of the amino acids before derivatization of the sample. This allows the use of relatively low amounts of [32P]orthophosphate to load up the cells. Most important, the method allows the simultaneous analysis of dozens of samples within 1 day, making it a very convenient technique for routine analysis of the phosphorylation state of cultured cells. Consequently the method is well suited to implementation in large screenings for inhibitors of protein kinases, e.g., PTK inhibitors, in whole-cell studies.

A method for quantitation of tryptophan and tyrosine residues in proteins by fourth-derivative ultraviolet spectroscopy is described. The direct quantitation of tryptophan is based on measurement of a tryptophan-specific trough at 292 nm in the fourth derivative of a protein's ultraviolet absorption spectrum. A peak overlapping the tryptophan and tyrosine signatures at A282 is used to quantify tyrosine content. The procedure is accomplished by adding back known quantities of tyrosine to the sample and subtracting the contribution of tryptophan to the A282 peak to obtain an internal calibration curve. This curve is linear, with the ordinate axis intercept relating the quantity (residues/mole) of tyrosine present in the protein. This nondestructive and facile method was used to successfully predict the tryptophan and tyrosine content of a variety of well-characterized proteins. The utility of this method was further demonstrated by resolving the number of tryptophan and tyrosine residues in proteins oxidized by N-bromosuccinimide.

We describe new colorimetric methods for the direct determination of total solid-supported sulfydryl, aldehydo, hydrazido, and N-hydroxysuccinimido carboxylate groups as well as of immobilized cysteine, tyrosine, and thyroxin using only the commercially available bicinchoninic acid/copper protein assay reagent. The method is based on the ability of these groups to reduce Cu2+ to Cu+, which forms a chelate complex with bicinchoninic acid absorbing at 562 nm. Each assay requires only one incubation step of the solids with the reagent for 1 h at 60 degrees C. The quantitation of the different groups is finally carried out through standard curves of appropriate substances. Using the assays developed we determined the amount of the above-mentioned functional groups and ligands onto several commercially available solid supports. The values obtained were in agreement with those provided by relative literature methods and/or by the manufacturers. The assays were found to be accurate, precise (interassay CV less than 3%), and very sensitive, allowing the determination of nmol quantities of functional groups per assay tube.

Two continuous assays for protein tyrosine phosphatases (PTPases) have been developed using phosphotyrosine containing peptide substrates. These assays are based on the marked differences in the spectra of the peptide before and after the removal of the phosphate group. The increase in the absorbance at 282 nm or the fluorescence at 305 nm of the peptide upon the action of PTPase can be followed continuously and the resulting progress curve (time course) can be analyzed directly using the integrated form of the Michaelis-Menten equation. The procedure is convenient and efficient, since both kcat and Km values can be obtained in a single run. The difference absorption coefficient (delta epsilon) at 282 nm is relatively insensitive to the pH of the reaction media. These techniques were applied to two homogeneous recombinant PTPases employing six phosphotyrosine-containing peptides. Km and kcat values obtained from the progress curve analysis were similar to those determined by the traditional initial rate inorganic phosphate assay. The peptides corresponding to autophosphorylation sites in Neu, p56lck, and p60src proteins show distinct behavior with the Yersinia PTPase, Yop51*, and the mammalian PTPase (PTP1U323). In both cases, the kcat values were relatively constant for all the peptides tested whereas the Km values were very sensitive to the amino acid sequence surrounding the tyrosine residue, especially in the case of Yop51*. Thus, both Yop51* and PTP1U323 show differential recognition of the phosphotyrosyl residues in the context of distinct primary structure of peptide substrates.

A continuous activity assay for protein tyrosine phosphatases (PTPs), employing phosphotyrosine (P-Tyr) as a substrate, has been developed and applied to measure the activities of two purified enzymes, namely, the full length T-cell protein tyrosine phosphatase (TC PTP) and its truncated form (TC delta C11 PTP). The reaction was followed by changes in ultraviolet absorption and fluorescence resulting from the dephosphorylation of P-Tyr. Both enzymes obey Michaelis-Menten kinetics, with Km = 304 microM, Vmax = 62,000 units/mg for TC PTP and Km = 194 microM, Vmax = 73,000 units/mg for TC delta C11 PTP. The D- and L-forms of P-Tyr are equally effective as substrates. The optimum pH for both enzymes is 4.75. The known effectors of PTPs have the predicted effects on catalytic activity.

We have employed near-uv second-derivative spectra of DNA, N-acetyl-L-tryptophanamide, N-acetyl-L-tyrosinamide, N-acetyl-L-phenylalanine ethyl ester, and phenol in a matrix least-squares multicomponent analysis algorithm to detect the presence of tryptophan, tyrosine, phenylalanine, and/or phenol in DNA preparations. With this method, each of these compounds can be detected in a DNA sample (absorbance, 0.1) at absorbance levels of less than 0.002. In practice, the presence of proteins can be detected at absorbance levels of less than 0.003. Using second-derivative spectra of proteins, contents of mixtures of proteins and DNA can be determined with less than 1% error. Mixtures of DNA and RNA can also be quantitatively analyzed with an error of approximately 2%. This technique can be easily implemented with computer-controlled spectrophotometers equipped with standard spectral analysis software. With prerecorded standard spectra, the time of analysis does not exceed a few seconds.

A method for the determination of the sites of tyrosine phosphorylation in proteins and peptides at the low picomole level for "cold" phosphopeptides and at the subpicomole level for 32P-labeled phosphopeptides is presented. The procedure is based on solid-phase sequence analysis of phosphopeptides immobilized on carrier discs and the "on-line" detection by reverse-phase high-performance liquid chromatography of the phenylthiohydantoin derivative of phosphotyrosine. The procedure is sensitive and automated and allows the identification of phosphotyrosine derivatives in the same operation as the detection of the derivatives of the other common amino acids. Essentially quantitative extraction of the phosphotyrosine derivatives from the sequencer makes this method ideally suited for the quantitative assessment of protein-tyrosine kinase and protein phosphatase activities and for the determination of their respective recognition sequences.

A micromethod for nondestructive amino acid analysis was developed. Peptides were digested with amino-peptidase M followed by prolidase. The whole procedure was performed in a microvial for the autosampler of the analyzer. The resulting method has a working range from 10 to 600 pmol, at least, of sample taken into work. The usefulness of the method was demonstrated by its ability to determine the sulfation of tyrosine preceding proline in chicken gastrin.

A sensitive, automated, and nonisotopic assay for protein-tyrosine kinases and phosphatases has been developed. The assay uses commercially available antiphosphotyrosine monoclonal antibodies and the recently developed particle concentration immunofluorescence immunoassay technology. The assay is specific for phosphotyrosine residues, can be performed faster, and is at least 100-fold more sensitive than the current standard filter type radioassay. Myelin basic protein and a synthetic peptide corresponding to the autophosphorylation site of p56lck performed equally well in the detection of p56lck kinase activity. Myelin basic protein phosphorylated on tyrosine residues by p56lck was successfully used as substrate in the detection of phosphatase activity and vanadate or molybdate were shown to inhibit the phosphatase activity. The assay is particularly useful for the rapid detection of enzyme activities in column fractions from biochemical procedures steps and also for screening of large numbers of potential inhibitors or activators of protein-tyrosine kinases and phosphatases.

A reverse-phase, high-performance liquid chromatographic system (HPLC) is described that makes possible optimal resolution and quantitation of picomole levels of phosphoamino acids, both with or without the presence of a large excess of nonphosphorylated amino acids. The assay involves precolumn derivatization of an amino acid mixture with phenyl isothiocyanate (PITC) at room temperature, followed by separation of phosphoamino acids from other amino acids by HPLC. The liquid chromatography was carried out on a C18 reverse-phase column at pH 7.4 and 30 degrees C using gradient elution with eluent A as 157 mM sodium acetate containing 2% acetonitrile and eluent B as 60% acetonitrile in water. A uv absorption at 254 nm is employed for detection of the PITC-derivatized amino acids eluting from the column. Amino acids are eluted with baseline resolution in the following order: phosphoserine, phosphothreonine, aspartic acid, glutamic acid, and phosphotyrosine followed by other amino acids. The sensitivity is in the picomole range, and the separation time, injection to injection, is 36 min. Phosphoserine, phosphothreonine, and phosphotyrosine are resolved within the first 8 min. This procedure enables determination of as low as 5 pmol of nonradioactive phosphoamino acids in a 100-fold excess of amino acids, as is usually present in most phosphoproteins in the natural state. Phosphoamino acids in polypeptides separated by sodium dodecyl sulfate-polyacrylamide electrophoresis and transferred to polyvinylidene difluoride (PVDF) membrane, or protein samples directly blotted on the membrane, can also be analyzed by this procedure after acid hydrolysis of the proteins bound to the PVDF membrane.

4'-Dimethylaminoazobenzene-4-sulfonyl chloride is a chromophoric reagent commonly used to detect amino acids at picomole levels. This article describes a single-column reverse-phase high-performance liquid chromatography system which allows the resolution and analysis of the dabsyl chloride derivatives of several modified amino acids. Highly derivatized C18 (22 and 31%) columns from Phenomenex are run at pH 8.1 to separate dityrosine, the phosphorylated amino acids (o-phosphoserine, o-phosphothreonine, and o-phosphotyrosine), and the 17 other amino acids normally present in protein hydrolysates. In order to gain additional sensitivity and to verify the presence of dityrosine, the dityrosine and lysine peaks are collected and run at pH 4.1 on the same columns. Our experience indicates that, with the described setup, the lower limit for accurate and reproducible detection is near 1 pmol. This method has been applied to the analysis of dityrosine in uv-irradiated calmodulin and cardiac troponin C and to the detection of phosphorylation sites in several polypeptides.

The microfibrillar proteins of human hair have been studied by reversed-phase high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A chromatographic procedure which isolates the microfibrillar proteins from other hair-matrix proteins and separates them into collectable fractions has been introduced. These fibrous proteins fall into two major subgroups which are resolved into six components. The same procedure has also resulted in the identification and simultaneous separation of a group of proteins rich in glycine and tyrosine never before detected in human hair. Comparative electrophoretic studies of the crude microfibrillar proteins reveal five bands with apparent molecular weights of 47,000, 50,000, 53,000, 57,000, and 62,000. The relationship between the electrophoretic bands and the chromatographic fractions is now under investigation.

A synthetic tetradecapeptide derived from the phosphorylation site of the beta-subunit of phosphorylase kinase (Arg-Thr-Lys-Arg-Ser-Gly-Ser-Val-Tyr-Glu-Pro-Leu-Lys-Ile) is a highly efficient substrate for the cAMP-dependent protein kinase, exhibiting a 36% decrease in the intrinsic tyrosine fluorescence on phosphorylation. The fluorescence changes in continuous assays were monitored to demonstrate the roles of protein kinase effectors (cAMP, the type II regulatory subunit, and the 8000-Da heat-stable inhibitor) in the regulation of the enzyme and to determine Km and Vmax. The phosphorylation reaction requires 1 mol ATP/mol peptide. Amino acid analysis demonstrates the presence of phosphoserine in the phosphorylated peptide. Auxiliary experiments show that tyrosine phosphorylation can also be detected fluorometrically and distinguished from serine or threonine phosphorylation.

A new spectrophotometric method for quantitative determination of tryptophan and tyrosine in peptides and proteins is described. It is based on two specific color reactions, the reaction of tryptophan with formaldehyde and the reaction of tyrosine and tryptophan with hydroxylamine and ceric cations. By combination of these two reactions both tyrosine and tryptophan can be determined simultaneously. Tyrosine and/or tryptophan bound in peptides and/or proteins react independently of the rest of the peptide or protein molecule. The method is simple, accurate, and sensitive. Hydrolysis is not necessary.

Reaction of avian pancreatic polypeptide with an iodine monochloride reagent at both pH 4 and pH 7.5 results in the differential modification of the four tyrosine residues in this peptide hormone. A total of 19 distinct iodinated derivatives were isolated by reverse-phase high-performance liquid chromatography, and their sites of iodination were characterized by both tryptic mapping and leucine aminopeptidase techniques coupled with HPLC. The pH 4 reaction produced 16 derivatives which, overall, represented substantial iodination at each tyrosine residue, whereas the pH 7.5 reaction was more directed, producing only 7 derivatives. Iodination at the C-terminal tyrosineamide 36 predominated at both pH values, and diiodo-Tyr 36 was found in the majority of the pH 7.5 derivatives. The relative of the four tyrosine residues with ICl were as follows: at pH 7.5, Tyr 36 much greater than Tyr 21 much greater than Tyr 27 greater than Tyr 7; at pH 4, Tyr 36 greater than Tyr 27 greater than Tyr 7 greater than Tyr 21.

The hydrolysis of fluorotyrosine phosphate by the calmodulin-activated phosphatase calcineurin has been monitored by 19F nuclear magnetic resonance spectroscopy. Previous work had established that the 19F nuclear magnetic resonance shift of the fluorine nucleus was altered after the phosphorylation of the phenolic hydroxyl group (B. Martin, C.J. Pallen, J.H. Wang, and D.J. Graves (1985) J. Biol. Chem. 260, 14592-14597). The disappearance of substrate and the appearance of product can be measured simultaneously with this approach. Application of the integrated form of the Michaelis-Menten equation yields estimates of the kinetic parameter, KM, close to the values obtained by initial rate kinetics. The velocity term, VM, was also evaluated to be approximately the same value. Calcineurin was determined not to be inactivated over the time period of the reaction. The results demonstrate that 19F nuclear magnetic resonance spectroscopy can be applied to the examination of enzyme-catalyzed reactions.

The characteristic absorption spectra of aromatic amino acids between 240 and 310 nm were used to identify tryptophan, tyrosine, and phenylalanine-containing peptides. In acidic solution, the absorption spectra of these amino acids exhibit minima or maxima at 255, 270, and 286 nm. Based on these characteristics, the content of the aromatic amino acid in peptide can be estimated. For this study, 2 nmol of tryptic peptides from human apolipoprotein A-1 was separated by high-performance liquid chromatography using a reverse-phase column. The peptide fragments were monitored by a photodiode-array spectrophotometer. This new approach offers a rapid, simple, sensitive, and direct identification of peptides containing aromatic amino acids. Those containing Trp, which may be of interest for DNA sequencing and important in sequence analysis of proteins, can be selectively purified using this technique.

A procedure has been developed for the analysis of tyrosine O-sulfate in proteins. Samples are subjected to base hydrolysis with Ba(OH)2, neutralized with sulfuric acid, and the majority of other amino acids removed by chromatography on Dowex AG 50 X 8. The average recovery of tyrosine O-sulfate from these procedures was 43%. Tyrosine O-sulfate was identified by reverse-phase HPLC as the phenylthiocarbamyl derivative following precolumn derivatization with phenyl isothiocyanate. The method has been applied to bovine fibrinogen giving a tyrosine O-sulfate content ranging from 0.59 to 1.23 mol/mol. These procedures were also shown to be suitable for the analysis of the incorporation of [35S]sulfate into tyrosine O-sulfate residues in proteins by intact cells.

A method for determining relative tyrosyl radical scavenging activity of antioxidants which requires only a standard fluorometer and commercially available materials is presented. Ultraviolet irradiation of aqueous tyrosine solutions containing superoxide dismutase and catalase produces fluorescent dityrosine residues via dimerization of photogenerated tyrosyl radicals. Added antioxidants suppress the buildup of fluorescence by scavenging the tyrosyl radicals. A correlation exists between the ability of a substance to suppress dityrosine formation and the substance's one-electron oxidation potential. This method demonstrates that ovothiol A scavenges tyrosyl radicals much more efficiently than glutathione or cysteine, resembling instead the known biological radical scavengers uric acid and ascorbic acid and the alpha-tocopherol analog trolox.

A technique to detect and analyze phosphotyrosine containing peptides after separation of total cellular proteins by two-dimensional gel electrophoresis is described. This is achieved by electroblotting of proteins on nylon membranes followed by alkali treatment. In comparison with direct alkali treatment of the polyacrylamide gel, this procedure is easier to perform; avoids the diffusion of proteins out of the gel during alkali treatment; allows a more precise localization of phosphotyrosine containing peptides on the untreated membrane; and is less time consuming with respect to extraction of proteins for phosphoamino acid analysis.