Phenylthiohydantoin Amino Acids Research Articles

The use of mixed surfactants in micellar electrokinetic chromatography.

The use of mixed surfactants for the separation of nine phenylthiohydantoin amino acids (PTH amino acids) by micellar electrokinetic chromatography (MEKC) was examined. The potential of a mixed surfactant system, consisting of cationic and anionic surfactants, for enhancing the selectivity of MEKC separations was explored. Both sodium dodecyl sulfate (SDS) and cetyltrime-thylammonium bromide (CTAB) in the electrophoretic medium were found to improve the separation of the PTH-amino acids compared to the use of CTAB or SDS alone. Complete separation of the PTH amino acids was achieved using a buffer containing 2.5 mM CTAB and 40 mM SDS. The analysis was performed without the need to reverse the polarity of the electrodes, typically required if oppositely charged micelles are used. The migration behavior of the nine PTH amino acids using the mixed surfactant system was compared with MEKC in presence of SDS alone.

Micellar capillary electrophoresis separation and thermo-optical absorbance detection of products from manual peptide sequencing.

Micellar capillary electrophoresis is optimized for separation of phenylthiohydantoin (PTH) amino acids produced in manual Edman degradation reaction for protein sequencing. There are also two major side-products produced by the Edman degradation reaction: diphenylthiourea and dimethylphenylthiourea. We report the complete separation of 19 PTH amino acids plus the two major side-reaction products in 10 min. Capillary electrophoresis is used to identify the five residues generated by manual Edman degradation sequencing of a pentapeptide.

Micellar electrokinetic chromatography with electroosmotic step gradient elution.

The peak capacity, defined as the maximum number of resolvable peaks, is dependent on the range of the migration-time window in micellar electrokinetic chromatography (MEKC). By applying various radial electric potential gradients across the capillary wall, the direct control of the zeta potential and the electroosmotic flow in MEKC results in a significant increase in the migration-time window and a great enhancement of the separation resolution. The enhancement in the separation resolution is obtained at a large expense in analysis time. To optimize both the separation efficiency and the resolution in MEKC, the dynamic control of electroosmosis and the migration-time window during the separations is demonstrated with the use of neutral and charged phenylthiohydantoin-amino acids (PTH-amino acids) as a model system. A simple mathematic model which is capable of predicting migration times of analytes in electroosmotic step gradient elution MEKC is presented.

Identification of Cys-150 in the active site of phosphomannose isomerase from Candida albicans.

Candida albicans phosphomannose isomerase (PMI) (EC 5.3.1.8) has been recently cloned and overexpressed in Escherichia coli. The enzyme can be irreversibly inactivated by iodoacetate in 50 mM borate buffer, pH 9.0, in a time-dependent manner at a rate of 4.2 +/- 0.03 min-1 M-1. This inhibition can be prevented by the substrate mannose 6-phosphate with a Ks of 0.22 +/- 0.05 mM, slightly lower than its Km value. However, metals such as zinc and cadmium, which are reversible, competitive inhibitors for PMI, do not protect the enzyme against modification. The protein has been labeled by using [2-14C]iodoacetate, in the presence or absence of substrate, and the protein is fully inactivated when 1.0 thiol group is modified per molecule of enzyme. Tryptic maps of the modified protein have been produced. The protected peptide has been identified and sequenced, and the phenylthiohydantoin amino acids have been collected. The modified amino acid is Cys-150. This cysteine residue is conserved in mammalian and yeast phosphomannose isomerases, but not in bacterial species where it is replaced with asparagine. We therefore purified PMI from E. coli and showed that this enzyme is not sensitive to inactivation by iodoacetate. The iodoacetate is presumably inhibiting PMI by sterically blocking the mannose 6-phosphate binding site. Multiple sequence alignment procedures were used to try to identify potential ligands of the zinc atom that is essential for enzyme activity and thus to delineate the active site region.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of Cysteine Residues at the 10-pmol Level by Carboxamidomethylation of Protein Bound to Sequencer Membrane Supports

A procedure is described for the in situ carboxamidomethylation of cystine/cysteine residues in protein samples of as little as 10 pmol, prior to automated protein sequence analysis. Previous in situ methods for the modification of cysteines are limited to proteins available in quantities greater than 100 pmol due to contaminants which interfere with HPLC identification of phenylthiohydantoin amino acids, and cannot be performed on polyvinylidenedifluoride (PVDF)-bound samples. In our procedure, protein samples, immobilized on either PVDF- or polybrene-treated glass filters, are reduced with tributylphosphine followed by alkylation with iodoacetamide prior to automated sequence analysis. Carboxamidomethylcysteine is formed in high yield with no discernable side reactions in standard proteins (insulin, human transferrin, lysozyme) or experimental samples. Both initial and repetitive yields of carboxamidomethylated proteins were either comparable to or better than nonalkylated proteins. No apparent increase in background nor any sequence preview due to partial amino-terminal alkylation was observed. The carboxamidomethylation procedure described here successfully overcomes the limitations of available methods for reduction and alkylation of less than 100 pmol of protein directly on sequencer membrane supports.

Direct control of electroosmosis and retention window in micellar electrokinetic capillary chromatography

The ζ potential and the electroosmotic florw in micellar electrokinetic capillary chromatography (MEKC) can be directly controlled by using a radial electric potential gradient across the capillary wall. In this study, the separations of neutral phenylthiohydantoin amino acids as the model system in MEKC with direct control of electroosmosis and retention window are presented. Significant enhancement in the separation resolution of phenylthiohydantoin amino acids under the direct control of electroosmosis is demonstrated

Optimization of phenylthiohydantoinamino acid separation by micellar electrokinetic capillary chromatography

Optimization of phenylthiohydantion (PTH)-amino acid separation by micellar electrokinetic capillary chromatography was achieved by the use of a weighted variable-size simplex algorithm. The optimization procedure concerned the pH of the aqueous buffer, the sodium dodecyl sulphate concentration and the percentage of organic solvent; the organic solvent used was either methanol or acetonitrile. In both instances the optimization procedure led to very similar final experimental conditions and migration order and times of the PTH-amino acids, showing that the organic solvent probably provides a better polydispersity of the micellar phase. The elution pattern observed in the two instances suggests that ionic interactions and polar repartition play a role in the separation mechanism, but other types of interaction cannot be excluded.

Reversed-Phase High Performance Liquid Chromatographic Separation of Small Molecules on Nonporous C18 Silica Gel Column

Abstract Reversed-phase high-performance liquid chromatographic (RP HPLC) separation of small molecules on a nonporous octadecylsily (C18) spherical silica gel was compared with the separation or. conventional porous packings. Various alkylbenzenes were separated on three different types, nonporous, macroporous (pore diameter; 30 nm), and microporous (pore diameter; 10 nm), C18 bonded silica columns under both isocratic and gradient elution. conditions. Under the isocratic conditions, column efficiency of nonporous silica was inferior to that of others. In case of the gradient elution, the nonporous silica column gave the most. favorable separation in a short time. Fast separation of srall molecules on the nonporous column could be performed by using a conventional HPLC system with a steep gradient condition. because the void volume of the nonporous packing was extremety smaller than the porous ones. Various series compounds such as fatty acid derivatives, phenylthiohydantoin (PTH) amino acids, and vitamins were well separated within 1 or 2 min on the C18 nonporous silica column under steep gradient conditions.

N-Isopropyliodoacetamide in the Reduction and Alkylation of Proteins: Use in Microsequence Analysis

A new reagent, N-isopropyliodoacetamide (NIPIA), for alkylation of sulfhydryl groups on proteins for microdigestion and microsequencing is described. The utility of this reagent in both of these procedures has been demonstrated. NIPIA was shown to be especially useful in microsequence analysis, where it yields high sensitivity in detection of Cys residues. This is because the phenylthiohydantoin (PTH) derivative of NIPIA-alkylated cysteine [PTH-Cys(NIPCAM)] appears as a sharp peak in a standard reverse-phase HPLC analysis of PTH amino acids, and elutes between PTH-Tyr and PTH-Pro where no other peaks are present. Thus the use of NIPIA circumvents various problems associated with HPLC analysis of PTH-Cys when other commonly used agents are employed for sulfhydryl alkylation, such as coeluting peaks or low signal levels. Procedures for the synthesis of NIPIA and other analogs, as well as PTH-Cys(NIPCAM), are also presented, and HPLC retention times for their corresponding PTH-Cys derivatives are compared.

Isolation of transglutaminase-reactive sequences from complex biological systems: a prominent lysine donor sequence in bovine lens.

The transglutaminase (protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13)-catalyzed cross-linking of proteins in biological systems can often be inhibited by inclusion of small primary amines or glutamine-containing peptides, which act as site-specific blockers of the relevant acceptor (i.e., glutamine) and donor (i.e., lysine) functionalities of the natural substrates. Compounds such as dansylcadaverine and dansyl-epsilon-aminocaproyl-Gln-Gln-Ile-Val are particularly useful in sorting out acceptor-donor relationships among lens crystallins. Apart from its fluorescent properties, the dansyl hapten offered special advantages as a "handle" for the rapid isolation of transglutaminase targets even in the complex system of lens cortical homogenate. The dansylated peptide was incorporated into bovine lens proteins under the influence of the Ca(2+)-activated intrinsic transglutaminase and, after digestion by endoproteinase Glu-C, the tracer-containing fragments were isolated by affinity chromatography on an anti-dansyl antibody column. The major fluorescent peak was isolated by HPLC and sequenced by Edman degradation, which yielded phenylthiohydantoin amino acid derivatives for the first 10 cycles, EKPAVTAAPK, and none for the next 2. The sequence, corresponding to residues 165-174 of alpha B-crystallin, unambiguously identifies the known carboxyl-terminal domain, EK-PAVTAAPKK, as the prominent lysine-donating fragment in bovine lens.

Sub-femtomole determination of phenylthiohydantoin-amino acids: capillary electrophoresis and thermooptical detection

Twenty phenylthiohydantoin-amino acide (PTH-AA) are separated by micellar capillary electrophoresis in 13.5 min. A low-energy krypton fluoride laser ise used in a thermooptical absorbance detector to produce typical detection limits (3σ) of 9×10 -7 M PTH-glycine injected in a 0.6-nL volume, corresponding to 0.5 fmol (100 fg) of PTH-glycine injected onto the capillary. Detection limits are roughly 3 orders of magnitude superior conventional liquid chromatographic determinations

The development of high-performance liquid chromatographic analysis of allyl and allyloxycarbonyl side-chain-protected phenylthiohydantoin amino acids

Ten phenylthiohydantoin (PTH) amino acids possessing allyl (Al) or allyloxycarbonyl (Aloc) side-chain-protecting groups have been characterized by high-performance liquid chromatography for use in Edman degradation sequence analysis. Optimized separation of side-chain-protected and -unprotected PTH amino acids was achieved on a C-18 reversed-phase column with a two-step gradient spanning 32 min. Five of the side-chain-protected amino acids [Cys(Al), Cys(Aloc), Lys(Aloc), Thr(Aloc), Tyr(Al)] were completely stable to the conditions of PTH derivatization, four [Asp(OAl), Arg(Aloc) 2, Glu(OAl), Ser(Aloc)] were partially deprotected during PTH derivatization, and one [His(Aloc)] was completely deprotected during PTH derivatization. All allyl-based derivatives were well resolved from their side-chain-unprotected counterparts. Studies on the stability to piperidine treatment showed Asp(OAl), Cys(Al), Glu(OAl), Lys(Aloc), Thr(Aloc), and Tyr(Al), and possibly Arg(Aloc) 2 and Ser(Aloc), to be suitable for peptide synthesis by 9-fluorenylmethoxycarbonyl (Fmoc)-based chemistry. Edman degradation of AI and Aloc side-chain-protected Conus geographus Lys 9-α-conotoxin GI synthesized on 4-methylbenzhydrylamine-copoly(styrene-1%-DVB)-resin demonstrated the usefulness of these derivatives for solid-phase preview sequence analysis.

Sequence Analysis of Cysteine and Cystine Containing Peptides and Proteins by a Gas-Phase Sequencer with Isocratic Separation of PTH-Amino Acids

Abstract A facile method for routine sequence analysis of cysteine and cystine containing peptides or proteins is described. An automated gas-phase protein sequencer, Shimadzu PSQ-1 System, consisting of an Edman-reaction unit and an isocratic separation mode for the phenylthiohydantoin amino acid analysis with equivalent sensitivity and resolution to that of the current gradient mode, allows easy determination of the intact second half cystine, of which phenylthiohydantoin derivatives appear as a dithiothreitol adduct of dehydroalanine, cysteine and S- pyridylethylated cysteine. The optimal sequencing protocol for in situ pyridylethylation was established. This protocol gives an N-terminal amino acid sequence with half cystines, which are automatically converted to the S-pyridylcthyl derivative in the sequencer. Sequence analyses both intact and S-pyridyiethylated cysteine can identify a disulfide form if only one disulfide linkage exists in the sample. Sequence analysis of synthetic peptides containing ...

Sites of glycation of human and horse liver alcohol dehydrogenase in vivo.

Sites of in vivo glycation of human and horse liver alcohol dehydrogenase were identified by cleavage of the borotritide-treated enzyme with trypsin, followed by gas-phase sequencing of the resulting tritium-labeled glycated peptides. A blank sequencing result, i.e. failure to detect an amino acid phenylthiohydantoin after completion of an Edman degradation cycle, was ascribed to an N-(1-deoxyhexitolyl)lysyl residue, which represented a glycation site on the original enzyme subunit. In human liver alcohol dehydrogenase the sites affected were the epsilon-amino groups of lysines 10, 39, 231, 248, and 325, which were glycated to the relative extents of 10, 5, 75, 5, and 5%, respectively. The site specificity of in vivo glycation of the horse enzyme is similar; 70-75% of it had occurred at lysine 231. A computer image of the crystal structure of horse liver alcohol dehydrogenase was examined. As a result, it was proposed that the high rate of glycation at lysine 231 is due to acid-base catalysis of the Amadori rearrangement by the imidazole group of histidine 348. This hypothesis was supported by showing that imidazole groups were close to sites of glycation in several other proteins.

Primary structure of bovine conglutinin, a member of the C-type animal lectin family.

We report the complete amino acid sequence of bovine conglutinin obtained by structural characterization of peptides derived from the protein by various chemical and enzymatic fragmentation methods. The protein consists of 351 amino acid residues including 55 apparent Gly-X-Y repeats with two interruptions. This 171-residue-long collagenous domain separates a short noncollagenous NH2-terminal region of 25 residues from the 155-residue-long globular COOH terminus revealing the structural relation of conglutinin with mannose-binding proteins, pulmonary surfactant-associated proteins, and a complement component C1q. Eight hydroxylysine residues were found in the collagenous domain. All of these hydroxylysine residues which occupy a Y position in a Gly-X-Y triplet are possible glycosylation sites since no phenylthiohydantoin amino acid was identified in automated Edman degradation cycles corresponding to these sites. The noncollagenous COOH domain of conglutinin, on the other hand, contains a carbohydrate recognition domain which shares substantial sequence homology with C-type animal lectins. Conglutinin has the greatest sequence similarity with mannose-binding proteins and pulmonary surfactant-associated proteins.

Intracellular Immature Subunits of Human Chorionic Gonadotropin in First Trimester Placental Cells: Purification and Characterization<xref ref-type="fn" rid="fn1"><sup>1</sup></xref>

As we previously reported [Sakakibara et al. (1986) Biochem. Biophys. Res. Commun. 137, 443-452; and Tominaga et al. (1989) J. Biochem. 105, 992-997], subunits of human chorionic gonadotropin (hCG) containing immature N-linked sugar chains (immature subunits), i.e., the 21 kDa form of alpha-subunit and the 23 and 19 kDa forms of beta-subunit, are present predominantly in first trimester placental cells. The molecular mass of intracellular hCG consisting of these subunits, based on gel filtration, was approximately 200 kDa, suggesting homo- or hetero-oligomerization of intracellular hCG. In the present study, we purified the 21 kDa form of alpha-subunit as well as the 23 and 19 kDa forms of beta-subunit from fresh normal first trimester placental tissues by gel filtration and reverse-phase high-performance liquid chromatography. Purified subunits were hydrolyzed (with a decrease in their molecular weighs) by endoglycosidase H and alpha-mannosidase but not by sialidase or sialidase followed by O-glycanase, indicating that those forms have presumably only high-mannose-type N-linked sugar chains but not O-linked sugar chains of the type present in mature beta-subunit. Fifteen cycles of Edman degradation of the purified forms of the subunits were performed. Only one phenylthiohydantoin amino acid, which was the same amino acid as in the urinary beta-subunit, was detected at each step for the mixture of 23 and 19 kDa forms of beta-subunit, indicating that the protein backbones of both forms are identical to each other as well as to the urinary beta-subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Microsequence analysis of peptides and proteins: IX. An improved, compact, automated instrument

We describe the construction of an improved, compact protein sequencer with a vertical flow path and continuous flow reactor (CFR). Unique features include a hexagonal valve for six fluid inputs to the CFR, which connects vertically to a transfer valve that allows sample, reagent, and solvent input to a conversion flask (CF). The simplified CF contains only two inputs at the top, one for sample, reagent, and solvent input, and the other a vent. The CF drains from the bottom, connecting to a switching valve which allows either delivery to waste or to an on-line HPLC for the analysis of phenylthiohydantoin amino acid derivatives. Approximately 90% of the sample is analyzed by use of a sonic flow detector. The overall vertical flow path of the sequencer is about 16 cm. The size of the instrument (25 w × 38 h × 44 d cm) is smaller than that of commercially available sequencers or HPLC systems. The performance of the instrument includes reduced background peaks and high-sensitivity sequence analysis at the 5–10 pmol level. The simplified sequencer is more economical and portable than conventional sequencers.

ATTOMOLE DETERMINATION OF PHENYL THIOHYDANTOIN DERIVATIVES OF AMINO ACIDS BY CAPILLARY ZONE ELECTROPHORESIS SEPARATION AND ULTRAVIOLET-LASER EXCITED THERMO-OPTICAL ABSORBANCE DETECTION

Capillary zone electrophoresis provides a rapid method for the analysis of minute amounts of amino acids. A laser-based absorbance technique is used to determine phenylthiohydantoin amino acids with detection limits of 10 -15 mol; separation of 20 amino acids, based on micelle modified zone electrophoresis, requires 13 minutes

Protein D of Haemophilus influenzae. A novel bacterial surface protein with affinity for human IgD.

Protein D, a novel surface protein of the bacterial species Haemophilus influenzae with affinity for human IgD, was isolated after solubilization with sonication and Sarcosyl-extraction by a single SDS-PAGE step. From 1 ml of packed bacteria was prepared 0.25 mg of purified protein D. The apparent m.w. of protein D was estimated to 42,000 by SDS-PAGE and gel chromatography. Edman degradation cycles of protein D produced no amino acid phenylthiohydantoin derivatives and the amino-terminal end of the single protein D polypeptide chain is thus probably blocked. Protein D differs from all previously described outer membrane proteins (protein 1 to 6) of H. influenzae. Thus, protein D did not react with antibodies against protein 1 or protein 2 and the latter proteins did not bind IgD. Protein D was found to exhibit unique Ig-binding properties. Thus, in dot blots protein D bound four different human IgD myeloma proteins but not IgG, IgM, IgA, IgE, or some additional proteins. On the IgD molecule, constant parts of the H chains both in the Fab and Fc fragments appear responsible for the interaction with protein D. This novel Ig-binding reagent promises to be of theoretical and practical interest in immunologic and microbiologic research.

A method for the unambiguous identification of tryptophan in automated protein sequence analysis

Some widely used standard protocols for the separation of phenylthiohydantoin amino acid derivatives by reverse-phase gradient HPLC do not provide separation of the phenylthiohydantoin derivative of tryptophan (PTH-Trp) from diphenylurea (DPU), a by-product generated during Edman degradation of proteins in variable amounts. Furthermore, PTH-Trp is usually recovered in low yield under typical experimental conditions used with automated sequencing equipment. These factors may compromise the unambiguous assignment of tryptophan residues in automated protein sequence analysis, especially when sequencing is performed at high sensitivity. We devised a reverse-phase HPLC method which allows the separation of DPU and PTH-Trp and therefore the correct assignment of PTH-Trp. The method is based on a modification of the HPLC gradient used to elute and separate all PTH amino acids of interest. With Applied Biosystems Model 477A protein sequencers with on-line PTH amino acid identification, the correct assignment of tryptophan was consistent and reproducible even when sequencing at very high sensitivity (5 pmol).