Abstract: Apparatus and methods for processing liquids are provided. The apparatus include open-ended, hollow reactor compartments which are detachably connected to receptor ends of tube systems. In preferred embodiments, a portion of the tube system is fixed to a rotor arm which can be rotated in a circle and moved up and down. Another portion of the tube system is coupled to a metering syringe which serves for drawing in or ejecting fluids into or from the reactor compartment and/or the tube system. A plurality of process stations such as bottles containing reagents or solvents can be arranged on a circular tray such that they can be accessed by the reactor compartment or by the receptor end if the reactor compartment has been detached from the tube system. The invention can be used, for example, for automatic protein sequencing, whereby the proteins to be sequenced are held in place by a matrix in the reactor compartment.

Abstract: Activated carboxylic acid modified polyethylene membranes useful in the C-terminal sequencing of a peptide are disclosed. The membranes comprise activated carboxylic acid modified polyethylene having a linker coupled to an amide linkage to the surface carboxyl radicals of the membrane. Activation is accomplished by reaction of the carboxylic acid modified polyethylene membrane with DCC, CDI, EDC, BOP, DICD or 2-fluoro-1-methyl pyridine.

Abstract: A method of forming a thiohydantoin from an N-protected amino acid. The method employs a uronium or phosphonium compound to activate the terminal carboxyl group of the amino acid and a thiocyanate reagent to cyclize the activated amino acid to the thio-hydantoin. The thiohydantoin can be cleaved from its N-protecting group, for use in C-terminal peptide sequencing. Particularly preferred uronium compounds include salts of 2-chlorouronium. Preferred thiocyanate reagents include trimethylsilyl isothiocyanate and crown ether adducts of metallothiocyanates, such as the 18-crown-6 adduct of KSCN.

Abstract: A detection system for determining the quantity of amino acid in a sample stream is provided based on the reaction of a buffer at a pH level from 10 to 11, with a reagent Ru(bpy).sub.3.sup.3+, which is generated electrochemically on site. The detection system is further characterized by immediate luminescence upon reaction of the buffer in the pH range containing amino acid, with the reagent Ru(bpy).sub.3.sup.3+. The detection system is capable of not only immediate detection of the quantity of amino acid in a sample stream, but even in very low concentrations.

Abstract: A method for the generation of phenylthiocarbamyl (PTC) amino acids from phenylthiohydantoin (PTH) or anilinothiozolinone (ATZ) amino acids. The method involves a base-catalyzed ring opening of the PTH or ATZ in the presence of a reducing agent. The method affords an alternative to the established aqueous acid conversion reaction of the Edman degradation in which ATZ and PTC amino acids are converted to the PTH amino acid. A further method is described for the generation of reactive ATZ amino acids from PTH amino acids. These methods facilitate the analysis of protein at low molar amounts by allowing the synthesis of amino acid derivatives which can be analyzed in quantities which are much lower than those required for conventional PTH amino acid analysis.

Abstract: A computer system and method are disclosed for determining a protein's tertiary structure from a primary sequence of amino acid residues. The system uses a dynamic Monte Carlo method with Metropolis sampling criterion, and a selected (2,1,0) lattice model, to simulate protein folding during the transition of the protein from an unfolded (denatured) state to its native, folded state. The system generates, for display, a folding trajectory representing successive three-dimensional images of the protein at a level of two Angstrom resolution as it folds to its native conformation. The system permits interaction between all proximate pairs of sidechains of the protein and provides faster processing through the use of the lattice.

Abstract: A method for monitoring the reaction between an activated carboxylic acid derivative and an amine, such as the formation of a peptide bond in peptide synthesis, whereby an inert anionic dye component and an inert cationic component are included in the reaction system, in small relative amounts compared to the carboxylic acid derivative. The distribution of the dye component to the cationic component, when separated from the amine component, is monitored and at a given maximum value indicates a substantially quantitative reaction.

Abstract: A method for analysis of peptides and proteins. A peptide or protein is exposed to a coupling reagent and a buffer. The coupling reagent derivatizes the terminal amino acid residue of the peptide or protein. A cleaving-reagent is then passed across the peptide or protein in the form of a spray to cleave the derivatized terminal amino acid residue from the peptide or protein. The apparatus includes a suitable support on which a peptide or protein can be disposed. The support is disposed in a reaction chamber. Pressurized sources of a suitable coupling reagent and a buffer are connected to the reaction chamber to allow exposure of the peptide or protein on the support to the coupling reagent and the buffer. A valve is also connected to the reaction chamber. The valve includes a sliding member which causes a gas and a liquid cleaving reagent to combine, whereby a spray is formed which is conducted from the valve to the reaction chamber.

Abstract: Reagents and method for the C-terminal sequencing of proteins and peptides are disclosed. The reagents include sodium trimethylsilanolate and trimethyl N-oxide. Derivatized, activated polyethylene supports for peptide samples subjected to C-terminal sequencing are described.

Abstract: A protein microsequencing method for use in conjunction with the thiobenzoylation degradation of polypeptides and proteins is disclosed. The process involves reaction of the N-terminal amino acid of a polypeptide with an excess of a thiobenzoylating reagent. The derivatized polypeptide is subjected to cleavage by acid which forms a 4-substituted 2-phenyl-5(4H) thiazolone. The thiazolone is acylated to form a 5-acyloxy-2-phenylthiazole and subjected to detection by both gas chromatography and chemical ionization mass spectroscopy.

Abstract: An automated apparatus for use in cleaving, deprotecting and purifying synthesized polypeptides immobilized on solid phase particles. The apparatus includes cleavage and extraction vessels where peptide cleavage from solid-phase particles, and extraction of scavenger reagents occurs, respectively, and structure for automated transfer of (i) reaction solutions into and out of the cleavage vessel, (ii) peptide solution from the cleavage vessel to the extraction vessel, and (iii) extraction solvent into the extraction vessel, in dispersed form. Excess extraction solvent is removed from a side arm in the extraction vessel.

Abstract: The present invention provides methods and reagents for sequencing amino acids. One embodiment of the method for determining the terminal amino acid of a substantialy pure polypeptide comprises the steps of (a) attaching the polypeptide to a solid support, (b) reacting the polypeptide with a compound described below, under conditions and for a time sufficient for coupling to occur between the terminal amino acid of the polypeptide and the compound, thereby yielding a polypeptide with a derivatized terminal amino acid, (c) washing the solid support to remove unbound material, (d) cleaving the derivatized terminal amino acid from the polypeptide with a cleaving agent, (e) ionizing the cleaved derivatized terminal amino acid, and (f) determining the molecular weight of the derivatized terminal amino acid, such that the terminal amino acid is determined.Within one embodiment, the compound is p-isothiocyanato phenethyl trimethylammonium and counterion salts thereof.

Abstract: A method for analyzing amino acids in biological liquids wherein a buffer liquid including unknown components to be analyzed is introduced to a separation column for separating amino acids. After reaction of the buffer liquid in a reactor, the amino acids are detected by a photometer. The flow rate of the buffer liquid is maintained at a predetermined value until the asparagine, glutamine acid and glutamine are separated from the separation column and the flow rate of the buffer liquid is varied stepwise or in linear gradient after separation of the above three components from the separation column.

Abstract: The present invention is directed to a method for amino acid sequence analysis comprising either the steps of labeling the sample with a fluorescent reagent and quenching the excess fluorescent reagent remaining after said labeling with an ammonium salt, or the steps of degrading amino acid from the amino terminus of peptides or proteins using a fluorescent Edman reagent and quenching the excess fluorescent Edman reagent remaining after said degrading with an ammonium salt. The method of the present invention makes it possible to eliminate the interference of identification by the chromatographic peak of fluorescent reagent by quenching the excess fluorescent reagent in the sample.

Abstract: A method for the C-terminal sequencing of a peptide in which an alkyl or aryl tin isothiocyanate coupling reagent is used to form a thiohydantoin derivative. Free halogen producing activating agents, such as 2-halo-1-methyl pyridinium salts, may be used as an activating agent for the coupling reagent.

Abstract: Sequential degradation of peptides for sequencing purposes by successive cleavage of amino acids from one end of the peptide chain is performed in an adsorption column with flows of the degradation reagents and wash liquids passing through the column in both directions. Migration of the peptide in one direction is thereby compensated by a subsequent migration in the other, and loss of peptide from the column over repeated cleavages is avoided. In preferred embodiments, the column contains two serially arranged adsorbent zones with differing adsorption characteristics, and the directions of flow of the various system components through the column are selected with a view toward a difference in peptide partitioning between the stationary and mobile phases in one zone vs. the other. The result is that any migration of the peptide occurring at any stage of a given cycle of the procedure occurs at a greater rate toward the zone interface than away from it.

Abstract: The DNA sequencing apparatus contains the following components. A first reaction vessel contains a reaction chamber into and out of which can be transferred reagents, reactants and reaction products. A device for separating individual oligonucleotides and polynucleotides on the basis of length, such as an electrophoretic unit, receives reaction products from the first reaction vessel. A second reaction vessel is designed for oxidizing pentose sugars. It comprises a reaction chamber having a device for transferring reactants and reagents into the reaction chamber and gaseous by-products of a reaction out of the reaction chamber. Separated oligonucleotides and polynucleotides can be selectably transferred from the separating device alternatively into the first or the second reaction vessel. The device also includes a second transfer device for transferring the gaseous by-products out of the second reaction vessel and a collection chamber for collecting the gaseous by-products.

Abstract: A method of detection of amino acids comprising the application, to the material being tested, of a reagent comprising the following basic formula: ##STR1## where X, Y and/or Z may be nitrogen C--H, C-alkyl or C-aryl and wherein A, B, C and/or D may be an alkyl or aryl substituent either alone or in combination.

Abstract: Disclosed is a method for enhancing the cleavage of an acyl thiohydantoin bond, for example, for use in C-terminal peptide sequencing. An acyl thiohydantoin is alkylated to form an adduct on the thiohydantoin, and the adduct-containing thiohydantoin is cleaved at its acyl bond by reaction with a cleaving agent under substantially anhydrous, acidic conditions. In C-terminal amino acid sequencing, the cleaved product is analyzed to identify the C-terminal amino acid.

Abstract: A carboxy terminal protein sequencing process is disclosed which utilizes phosphoroisothiocyanatidate for the derivatization step.

Abstract: An apparatus for the stepwise performance of chemical reactions characterized by a cup-shaped reactor vessel spinning around its longitudinal axis, and a pipette for applying samples, reagents, and solvents onto the inner wall of the vessel. The pipette is rotatable around an axis, and can be positioned at a location from which it can be lowered into reservoirs for drawing in or for ejecting liquid. The pipette can furthermore be positioned at a location from which it can be lowered into the vessel for applying sample, reagents or solvents onto its inner wall or it can be positioned at a location from which it can be lowered to remove reaction products and excess products from the bottom of the vessel. The apparatus can be used for automatically sequencing proteins and/or peptides.

Abstract: A chemical processing system is disclosed for the automated dissolution, dispersing and reaction of chemicals, especially for synthesizing proteins. The system includes a plurality of storage cartridges containing a first chemical in fluid communication with a reservoir containing a second chemical to be reacted with the first chemical. Each cartridge includes a pump (e.g., a plunger) which operates by changing the internal volume of the cartridge. The pump permits bi-directional flow of the first chemical into and out of the reservoir and cartridge to promote mixing and reacting with the second chemical in the reservoir to produce a third chemical.

Abstract: A reverse-phase HPLC method is disclosed which allows the separation of DPU and PTH-Trp and, therefore, the correct assignment of tryptophan residues in an amino acid sequence. The method is based on a modification of the conventional HPLC gradient commonly used to elute and separate all PTH amino acids of interest in automated and manual sequencing. In one embodiment, using automated instruments manufactured by Applied Biosystems, gradient modification is achieved by changing the manufacturer-supplied gradient program which controls the operation of the HPLC pumps in the instrument. Using the methods of the present invention, the correct and reliable assignment of tryptophan residues is possible and was reproducible over time, even with small sample sizes.

Abstract: A compact apparatus having a gravity assisted, substantially linear, vertical fluid flow path through vertically aligned elements for the N-terminal and C-terminal sequencing of proteins and peptides is disclosed.

Abstract: Peptides or proteins are sequenced by stepwise degradation while immobilized on a glass support derivatized with a silica-binding substance bearing a free acid group, especially a sulfonic acid group. The support is preferably derivatized with 2-(4-chlorosulfonyl phenyl) ethyl trimethoxysilane.Peptide sequencing performance is improved if the support is also derivatized with a monomeric silica-binding substance bearing a free quaternary ammonium group, such as N-trimethyoxysilyl propyl -N,N,N-trimethyl ammonium chloride.

Abstract: On-column enrichment is provided for a sample having both polar and nonpolar analytes by mixing the sample with surfactant in a polar solvent. The surfactant causes the nonpolar analytes to be incorporated in micelles that dissolve in the polar solvent. The concentration of surfactant is maintained at a level sufficient to maintain the micelles as the mixture is introduced onto the head of a chromatographic column. Once sample introduction is completed, the mixture is diluted so that the surfactant concentration drops below that required to maintain the micelles, which thus break up. The analytes are then separated using reverse-phase gradient elution.

Abstract: A monitoring method for application in solid phase peptide synthesis. In one aspect, the synthesis starts with an amino acid residue protected by an N-alpha-amino protecting group and involves the steps: (a) removing the N-alpha-amino protecting group to obtain an N-alpha-amino group, (b) adding an amino acid residue protected by an N-alpha-amino protecting group, via a peptide bond, to the N-alpha-amino group obtained in step (a) by use of a reactive protected amio acid derivative, and (c) repeating steps (a) and (b). The reaction system includes 3-hydroxy-1,2,3-benzotriazin-4(3H)-one or a derivative thereof; and the color of the reaction system or of a component thereof is monitored during the synthesis.

Abstract: This invention provides a method for purifying and isolating a carboxyl-terminal peptide. A peptide bond between a lysine residue in a polypeptide and the adjacent amino acid residue is specifically cleaved on its carboxyl-terminal side. The resulting peptide mixture is then reacted with a solid carrier possessing on its surface functional groups capable of reacting and covalently bonding with free amino groups. Subsequently the peptide bond between the aminoterminal amino acid residue and the adjacent amino acid residue of each peptide is cleaned by acid treatment, and the peptides thus released from the solid carrier are isolated.

Abstract: A method is provided for sequencing peptides and proteins in which the coupling and cleavage reagents are supplied through a valve block assembly with internal delivery channels and a common outlet channel. The outlet channel communicates with a reaction chamber in which the sample is placed. Access of reactants to the delivery channels is controlled by diaphragm valves, preferably equipped with relief grooves to accommodate deformation of the diaphragm. Reservoirs feeding the delivery channels have an inert atmosphere controlled by pressurizing and venting channels in the valve block. Exposure of plastic tubing to oxygen is kept to a minimum. Reactants are thereby supplied without significant cross-contamination or oxidative decomposition.

Abstract: A computer-implemented method for designing at least one anti-peptide sequence having affinity for target peptide or a fragment thereof suitable for synthesizing peptides and micromolecules, assaying for a target peptides, purifying target peptides, and/or preventing proteolyis of a polypeptide includes identification of the members of the amino acid sequence of the target peptide and their anti-sense or hydropathically complementary amino acids and determining the moving average hydropathy for the target and anti-sense members. The resulting lowest hydropathy identifies the anti-sense amino acid sequence for the target peptide. The members of the target peptide amino acid sequence are obtained along with their member-specific hydropathic values with the hydropathic values summed as a moving average. Anti-sense or complementary amino acid members are identified from the moving average information to generate an array of anti-sense amino acid sequences.

Abstract: Methodology is described for the carboxyl-terminal sequencing of proteins and peptides using novel coupling reagents.

Abstract: Polypeptides are sequenced from the carboxyl terminus by treating the peptide with an iodoxybenzene, followed by removal of the excess reagent and reaction with 2-aminothiophenol at an elevated temperature. The resulting benzthiazolidine is then analyzed to determine the C-terminal amino acid which has been cleaved. A kit which contains the iodoxybenzene and 2-aminothiophenol reagents is separate containers is also disclosed.

Abstract: A method and apparatus for sequencing polypeptides, including a continuous flow reactor which may include a sample bearing membrane strip.

Abstract: Reagents useful for the C-terminal sequencing of proteins and peptides are disclosed. The reagents include sodium trimethylsilanolate and trimethyl N-oxide. The reagents are used as cleaving reagents in a method for the sequential C-terminal degradation of peptides and proteins.

Abstract: A method of forming an amino acid thiohydantoin from an N-protected amino acid or the C-terminal amino acid of an N-protected peptide. The amino acid is activated by reaction with a ketenimine, and the activated ester is converted to the thiohydantoin by reaction with silyl or pyridine isothiocyanate. The ketenimine is generated by treating an N-substituted isoxazolium compound, such as Woodwards Reagent K with a base, preferably in the presence of the amino acid. Also disclosed is a solid phase support having a derivatized N-substituted isoxazolium or ketenimine group for use in the method.

Abstract: A method for detecting amino acid derivatives in which 2-anilino-5-thiazolinone derivatives of amino acids are reacted with an amino compound of fluorescein or rhodamine derivative amino compounds to form phenylthiocarbamyl amino acids derivatives. The phenylithiocarbamyl amino acids derivatives are detected with a high level of sensitivity by a fluorescence spectrophotometer.

Abstract: A method of C-terminal peptide sequencing. The peptide is reacted with a mixed anhydride of isothiocyanic acid and a carboxylic, carbonic, or sulfonic acid, under basic conditions, to produce a C-terminal peptidyl thiohydantoin. The C-terminal amino acid can be identified by cleaving the thiohydantoin from the peptide and identifying the free amino acid thiohydantoin.

Abstract: A method of C-terminal peptide sequencing. The peptide is reacted with an activated support derivatized with a mixed anhydride of isothiocyanic acid and carboxylic or carbonic acid, under basic conditions. The peptidyl thiohydantoin which forms is separated from the solid support and further reacted with a cleaving agent carried on a second solid support, to release a free C-terminal amino acyl thiohydantoin from the peptide. The free thiohydantoin is analyzed to determine the C-terminal peptide residue. The residual peptide can be recycled through the supports for successive C-terminal residue determinations.

Abstract: A device for the determination of amino acid sequence of a polypeptide comprises two new features offering great advantages in the cost and efficiency of operation of amino acid sequencers. The sequencer is provided with the capability for the bidirectional flow of sequencing reagents and contains a sample chamber having a bicompositional adsorbent for the polypeptide.

Abstract: An automated chemical conversion unit for use in a peptide/protein sequencing process. The improved conversion methodology decreases the time necessary for the conversion reaction chemistry of the specific derivative of the sequenced amino acid to a more stable form. The conversion reaction is designed to be performed directly in a fraction collector tube which operates in one embodiment in conjunction with a thermoelectric temperature element to allow utilization of different temperature levels for different amino acid derivatives.

Abstract: A method of detecting and identifying amino acid derivatives produced by sequential cleavage of N-terminal residues from a polypeptide chain. The polypeptide is first reacted with an isothiocyanate to produce an N-terminal-thiocarbamyl polypeptide. This derivative is then cleaved to form a cyclic ATZ derivative, which is subsequently combined with a detectable alcohol in anhydrous acid to primarily produce a stable thiocarbamyl amino acid ester. This compound may be readily detected and identified.

Abstract: Deblocking of amino terminal N-acetyl serine and N-acetyl threonine residue in proteins and peptides to allow sequencing by the Edman degradation technique is carried out by reacting blocked protein or peptide with anhydrous trifluoracetic acid for 1 to 15 minutes at 30.degree. to 60.degree. C., removing the remaining trifluoroacetic acid and maintaining reacting mixture from which trifluoroacetic acid has been removed at 30.degree. to 100.degree. C. for 60 minutes to 5 days.

Abstract: Peptides or proteins are sequenced by stepwise degradation while immobilized on a glass support derivatized with a silica-binding substance bearing a free acid group, especially a sulfonic acid group. The support is preferably derivatized with 2-(4-chlorosulfonyl phenyl) ethyl trimethoxysilane.Peptide sequencing performance is improved if the support is also derivatized with a monomeric silica-binding substance bearing a free quaternary ammonium group, such as N-trimethyoxysilyl propyl -N,N,N-trimethyl ammonium chloride.

Abstract: A solid support chemistry system which, with a dinitrofluorobenzene based assay, can be used to define titers of ligands and chemistry distal to the support. Cleavable diamine cystamines or 1,6-diamino-3,4-dihydroxyhexane are bonded to the solid support.

Abstract: There is disclosed herein an automated sample preparation system for chemical assay of samples of materials. The sample preparation system includes a sample preparation chamber which includes a removable cup for taking to the location of solid or very viscous samples. The cup may be attached in sealing relationship to a cap through which extends various utilities such as a mixer/grinder to grind solid samples and mix non-homogeneous samples, a fill pipe to pump in liquid samples, an effluent pipe in the sump of the cup to allow pump of samples and solvents and a nozzle to allow liquids to be sprayed against the walls. A sample metering valve associated with the sample preparation chamber allows a known volume of sample to be isolated so that solvent may be pumped in to dilute the sample to a user defined concentration.

Abstract: A preferred embodiment of the invention is a method for constructing a polypeptide chain having a substantially predetermined conformation. Preferably a known stable well-mapped polypeptide structure is used as a starting point, and additional peptide units are incrementally added on while maintaining favorable enthalpic and entropic contributions to stability. Preferably a library of oligopeptide blocks is used to provide candidates for the additional peptide units. Preferably the library includes numerous precomputed parameters for each of the blocks, e.g. parameters for estimating energetic effects of varying the conformation parameters.

Abstract: Methodology involving novel coupling reagents for the carboxyl-terminal amino acid sequence analysis of peptides and proteins is described.

Abstract: Proteins, such as enzymes having enhanced stability are designed through the use of a computer method. The method identifies amino acid residues of a protein which may be replaced with a cysteine residue in order to promote the formation of a protein-stabilizing disulfide bond. The computer-designed, stabilized proteins can be produced using recombinant DNA technology.

Abstract: Primary amines, secondary amines and peptides are fluorometrically assayed using a fluorogenic derivatization reagent of the formula: ##STR1## where Z is an electron withdrawing group or a fused benzene ring, Y and Y', which are identical or different, are nitrogen or carbon and X is a halogen or other leaving group. The above fluorogenic reagent reacts with the primary amine, secondary amine or peptide to form a cyclic fluorescent adduct.

Abstract: Pool or spa water can be monitored for dimethyl hydantoin content, as a residue of disinfection with halogenated hydantoin, by determining the quantity of base required to adjust the pH to 9.3. The pH may be judged by a suitable indicator for example thymol phthalein or thymol blue. The method can be applied either titrimetrically, or by comparison colorimetrically with standard solutions and may be modified for use in relation to other halogenated hydantoins. The method may be applied to the testing of pools for compliance with an advisory dimethyl hydantoin content limit and may be embodied in a kit for easy poolside use.