Sequence analysis of rat hypothalamic corticotropin-releasing factor with the o-phthalaldehyde strategy

Sequence analysis was performed on a 41-residue polypeptide that has been identified as the predominant form of high intrinsic corticotropin-releasing activity of rat hypothalamus. The sequence of residues 1-39 of this corticotropin-releasing factor (CRF) was determined by Edman degradation of a partially purified peptide in a highly sensitive spinning cup sequencer after selective blocking of CRF or its main contaminant with o-phthalaldehyde. This approach was validated by peptide mapping of CRF of a highly purified preparation. Peptide mapping was accomplished with reverse-phase high-pressure liquid chromatography of CRF fragments obtained by digestion with clostripain. The identities of the fragments cleaved from CRF were established by chromatographic comparison with synthetic peptides, amino acid analysis, and Edman degradation. On the basis of these experiments, the primary structure of rat hypothalamic CRF was established to be H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn - Arg-Lys-Leu-Met-Glu-Ile-Ile-NH2. It is expected that the o-phthalaldehyde strategy will facilitate the sequence analysis of partially purified peptides containing proline residues.     

Covalent attachment of peptides to cytochrome C for automated sequence determination.

Because small peptides are lost into the organic solvents used, it is virtually impossible to obtain the complete amino acid sequence of a small peptide using only an automated peptide sequencer of the spinning cup type. To overcome this problem we have extended peptides at the carboxy terminus by attachment to equine cytochrome c by a water soluble carbodiimide, relying on the acetylated N-terminus of the cytochrome to minimize its direct contribution to recovery of PTH-amino acids. The Model Peptide H-Leu-Trp-Met-Arg-phe-Ala-OH was used for most experiments. After reaction of 3H-peptide with cytochrome c, about one-third of the tritium counts migrated with cytochrome c during gel filtration. After attachment, the amino acid sequence of the hexapeptide was readily determined with a single cleavage Quadrol program in a Beckman 890B sequencer, whereas only the N-terminal residue was recovered without attachment. The repetitive yield after attachment was 95-96%, with 21-27+ overlap and an initial yield of 18-20%. Sequence data with other peptides illustrate applications and present limitations of our approach.     

Covalent Attachment of Peptides to Cytochrome C for Automated Sequence Determination

Because small peptides are lost into the organic solvents used, it is virtually impossible to obtain the complete amino acid sequence of a small peptide using only an automated peptide sequencer of the spinning cup type. To overcome this problem we have extended peptides at the carboxy terminus by attachment to equine cytochrome c by a water soluble carbodiimide, relying on the acetylated N-terminus of the cytochrome to minimize its direct contribution to recovery of PTH-amino acids. The Model Peptide H-Leu-Trp-Met-Arg-Phe-Ala-OH was used for most experiments. After reac-tion of³H-peptide with cytochrome c, about one-third of the tritium counts migrated with cytochrome c during gel filtration. After attachment, the amino acid sequence of the hexapeptide was readily determined with a single cleavage Quadrol program in a Beckman 890B sequencer, whereas only the N-terminal residue was recovered without attachment. The repetitive yield after attachment was 95–96%, with 21–27% overlap and an initial yield of 18-20%. Sequence data with other peptides illustrate applications and present limitations of our approach.     

Design and operation of a completely automated Beckman microsequencer

A unique, efficient, and inexpensive system has been designed and built for the automatic conversion of anilinothiazolinone derivatives extracted from a Beckman spinning-cup sequencer with subsequent on-line high-pressure liquid chromatography separation of the phenylthiohydantoin derivatives. The Auto Converter-Auto Sampler system is controlled by a tape programmer or microprocessor and operates by transfer of the sample from the conversion vial into an HPLC injection loop by nitrogen pressure. Incorporation of a minor programming change on the sequencer allows the introduction of nitrogen vapor into the spinning cup during phenylisothiocyanate coupling. These modifications have resulted in a completely automated subnanomole protein sequencer.     

Automated carboxy-terminal sequence analysis of peptides and proteins using diphenyl phosphoroisothiocyanatidate.

Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on the automation of the thiocyanate chemistry using acetic anhydride and trimethylsilylisothiocyanate (TMS-ITC) to derivatize the C-terminal amino acid to a thiohydantoin and sodium trimethylsilanolate for specific hydrolysis of the derivatized C-terminal amino acid (Bailey, J.M., Shenoy, N.R., Ronk, M., & Shively, J.E., 1992, Protein Sci. 1, 68-80). A major limitation of this approach was the need to activate the C-terminus with acetic anhydride. We now describe the use of a new reagent, diphenyl phosphoroisothiocyanatidate (DPP-ITC) and pyridine, which combines the activation and derivatization steps to produce peptidylthiohydantoins. Previous work by Kenner et al. (Kenner, G.W., Khorana, H.G., & Stedman, R.J., 1953, Chem. Soc. J., 673-678) with this reagent demonstrated slow kinetics. Several days were required for complete reaction. We show here that the inclusion of pyridine was found to promote the formation of C-terminal thiohydantoins by DPP-ITC resulting in complete conversion of the C-terminal amino acid to a thiohydantoin in less than 1 h. Reagents such as imidazole, triazine, and tetrazole were also found to promote the reaction with DPP-ITC as effectively as pyridine. General base catalysts, such as triethylamine, do not promote the reaction, but are required to convert the C-terminal carboxylic acid to a salt prior to the reaction with DPP-ITC and pyridine. By introducing the DPP-ITC reagent and pyridine in separate steps in an automated sequencer, we observed improved sequencing yields for amino acids normally found difficult to derivatize with acetic anhydride/TMS-ITC. This was particularly true for aspartic acid, which now can be sequenced in yields comparable to most of the other amino acids. Automated programs are described for the C-terminal sequencing of peptides covalently attached to carboxylic acid-modified polyethylene and proteins (200 pmol to 5 nmol) noncovalently applied to Zitex (porous Teflon). The generality of our automated C-terminal sequencing methodology was examined by sequencing model peptides containing all 20 of the common amino acids. All of the amino acids tested were found to sequence in good yield except for proline, which was found not to be capable of derivatization. In spite of this limitation, the methodology should be a valuable tool for the C-terminal sequence analysis of peptides and proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Primary structure of the major β-chain of rat haemoglobins

The amino acid sequence of the major β-chain, ^IIβ, from rat haemoglobins was established with an automated sequencer. Amino acid heterogeneities were found that appear to result from allelic variation at particular residues. We applied several new or unusual techniques in determining the sequence: (1) reaction of the polypeptide with dansylaziridine for detection of cysteine; (2) blockage of the N-terminal residue and the ε-amino group of lysine residues with 1-fluoro-2-nitro-4-trimethylammoniobenzene iodide and subsequent identification of the modified lysine phenylthiohydantoin by absorbance at 420nm; (3) identification of histidine phenylthiohydantoin by its blue fluorescence under long-wave u.v. light; (4) cleavage of the chain into two or three fragments and subsequent sequencing without purification [a detailed statement giving the major phenylthiohydantoins assigned at each step for each sequence run before their alignment in individual sequences has been deposited as Supplementary Publication SUP 50084 (10 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5]; (5) separation of fragments produced by CNBr cleavage by cation-exchange chromatography; (6) peptide sequencing after attachment of the peptide to cytochrome c. The amino acid sequence was confirmed by amino acid compositions of the complete chain, of CNBr fragments 1 and 3, and of 11 purified tryptic peptides.     

Automated carboxy-terminal sequence analysis of peptides.

Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on solution phase conditions for formation of the peptidylthiohydantoins with trimethylsilylisothiocyanate (TMS-ITC) and for hydrolysis of these peptidylthiohydantoins into an amino acid thiohydantoin derivative and a new shortened peptide capable of continued degradation (Bailey, J. M. & Shively, J. E., 1990, Biochemistry 29, 3145-3156). The current study is a continuation of this work and describes the construction of an instrument for automated C-terminal sequencing, the application of the thiocyanate chemistry to peptides covalently coupled to a novel polyethylene solid support (Shenoy, N. R., Bailey, J. M., & Shively, J. E., 1992, Protein Sci. I, 58-67), the use of sodium trimethylsilanolate as a novel reagent for the specific cleavage of the derivatized C-terminal amino acid, and the development of methodology to sequence through the difficult amino acid, aspartate. Automated programs are described for the C-terminal sequencing of peptides covalently attached to carboxylic acid-modified polyethylene. The chemistry involves activation with acetic anhydride, derivatization with TMS-ITC, and cleavage of the derivatized C-terminal amino acid with sodium trimethylsilanolate. The thiohydantoin amino acid is identified by on-line high performance liquid chromatography using a Phenomenex Ultracarb 5 ODS(30) column and a triethylamine/phosphoric acid buffer system containing pentanesulfonic acid. The generality of our automated C-terminal sequencing methodology was examined by sequencing model peptides containing all 20 of the common amino acids. All of the amino acids were found to sequence in high yield (90% or greater) except for asparagine and aspartate, which could be only partially removed, and proline, which was found not be capable of derivatization. In spite of these current limitations, the methodology should be a valuable new tool for the C-terminal sequence analysis of peptides.     

Structural basis for the species selectivity of a fibrin-specific monoclonal antibody

The structural determinant underlying the species specificity of a monoclonal anti-fibrin antibody (59D8) is the leucyl residue at position 5 in beta-chains of human fibrin. Anti-fibrin antibody 59D8 which had been elicited by immunization with human beta(1-7) peptide, Gly-His-Arg-Pro-Leu-Asp-Lys, binds to human and canine fibrins but not to bovine, ovine, or porcine fibrins. A comparison of the available amino acid sequence data suggested that the ability of anti-fibrin antibody 59D8 to discriminate among various fibrin beta-chains might be due to the amino acid at position 5. This was confirmed by competitive inhibition studies using synthetic fibrin-like peptides and determination of the amino acid sequences of the N-termini of ovine and porcine fibrin beta-chains. Edman degradation employing o-phthalaldehyde blocking permitted use of fibrin monomer rather than its separated constituent polypeptide chains. The same sequencing strategy was used to obtain partial sequence data for the alpha-chains of bovine, ovine, and porcine fibrin.     

High-performance liquid chromatography of side-chain-protected amino acid phenylthiohydantoins

Eighteen side-chain-protected amino acids, routinely employed in solid-phase peptide synthesis, were derivatized to their phenylthiohydantoins (PTH) by one cycle of the Edman degradation. All of these side-chain-protected PTH amino acids elute, with almost-baseline resolution, in less than 18 min by high-performance liquid chromatography, utilizing a biphasic gradient of acetonitrile in 0.01 n sodium acetate, pH 4.5, or a linear gradient of 0 to 100% acetonitrile with the exception of the coelution of a O-benzyl-threonine and carbobenzoxy-lysine phenylthiohydantoin amino acids. The derivatized amino acids were subjected to reverse-phase chromatography on a Zorbax ODS column and monitored at 254 nm. None of the PTH amino acids coelute with side-chain-protected PTH amino acid counterparts, although PTH-tosyl-histidine undergoes deprotection to PTH-histidine in the Edman degradation. A protected decapeptide attached to a chloromethylated polystyrene resin was degraded on a solid-phase sequencer in 16 h. The PTH amino acids resulting from the automated Edman degradation on the decapeptide were fully resolved and quantified in less than 3 h demonstrating that automated high-performance liquid chromatography can keep pace with both the automated sequencer and synthesizer which requires minimally 2–3 h for attachment of each residue to the growing peptide chain.     

The amino acid sequence of the alpha chain of the major haemoglobin of the rat (Rattus norvegicus).

1. A partial amino acid sequence of the alpha chain from the rat (Wistar, Rattus norvegicus) major haemoglobin is reported. The soluble tryptic peptides prepared from aminoethylated alpha-globin were separated by peptide 'mapping'. Sequencing of the tryptic peptides was carried out by the dansyl-Edman method and by the overlapping of smaller peptide fragments derived from secondary enzymic digestion. The insoluble 'core' peptides were further digested with chymotrypsin, thermolysin and pepsin to give smaller soluble peptides for sequencing. The tryptic peptides were ordered on the basis of their homology with the corresponding peptides of human alpha chain. 2. The proposed sequence is compared with that obtained by using an automated sequencer [Garrick et al. (1975) Biochem. J. 149, 245-258]. The differences in sequence resulting from the two methods are discussed. 3. It is suggested that the externally situated cysteine (residue 13) is responsible for the observed inhibition of crystallization of rat haemoglobin at alkaline pH. 4. Detailed evidence for the sequence has been deposited as Supplementary Publication SUP 50047 (9 pages) at the British Library (Linding Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from which copies can be obtained on the terms given in Biochem. J. (1975) 145, 5.     

Amino acid sequence of plastocyanin from Solanum crispum using automatic methods

The complete amino acid sequence of plastocyanin of Solanum crispum L. was determined on 6 micromole protein using automatic solid-phase and spinning cup sequencer methods. Overlap between residues Lys 54 and Ile 55 was however not demonstrated. The protein consists of 99 residues.     

Primary structure of hemoglobin from monitor lizard (Varanus exanthematicus albigularis--Squamata)

The primary structure of the major hemoglobin component from the Monitor Lizard Varanus exanthematicus albigularis is presented. The polypeptide subunits were separated by reversed-phase high-performance liquid chromatography on Nucleosil C-4 column. The amino-acid sequence was established by automatic Edman degradation of the native polypeptide and its tryptic and hydrolytic cleavage products in a spinning cup sequencer. The structural data are discussed with reference to other reptiles.     

Complete amino acid sequence of amelogenin in developing bovine enamel

Pure amelogenin protein in developing bovine incisor enamel was isolated and its primary structure was investigated by sequencing the peptides obtained after clostripain and chymotrypsin digestions and CNBr degradation with an automated Edman sequencer. The enamel protein was found to be composed of 170 amino acid residues with one phosphate having a molecular weight of 19,350 and its complete amino acid sequence was elucidated. This protein has no sequence homology with any other tissue or secretory protein of known structure.     

Sensitization of Edman amino acid derivatives using the fluorescent reagent, 4-aminofluorescein

The ability to analyze amino acid derivatives at the femtomole level is one of the most interesting challenges in the field of protein microsequencing. 2-Anilino-5-thiazolinone amino acids, obtained by Edman degradation, were quantitatively derivatized with fluorescent primary amines. The most fluorescent reagent tested was 4-aminofluorescein. The amino acid derivatives sensitized with this reagent were separated using reversed-phase high-performance liquid chromatography and identified at the 100 attomole level. Incorporation of this method into the operation of a conventional automated sequencer is also described.     

Primary structure and synthesis of a blocked myotropic neuropeptide isolated from the cockroach, Leucophaea maderae

A peptide which stimulates the contractile activity of the cockroach hindgut was isolated from head extracts of the cockroach, Leucophaea maderae. The inability of aminopeptidase M to degrade the peptide and the presence of glutamic acid in the hydrolysate suggested N-terminal blocking by pyroglutamic acid. The N-terminal pGlu was removed enzymatically and the unblocked fragment was sequenced with an automated gas-phase peptide sequencer. The structure determined (pGlu-Thr-Ser-Phe-Thr-Pro-Arg-Leu-NH2) was synthesized and shown to be both chemically and biologically identical with the natural product.     

An apparatus for continuous analysis of protease-catalyzed acyl transfer reactions

An apparatus that allows continuous analysis of protease-catalyzed acyl transfer reactions is described. Hydrolysis reaction is assayed using automatic titration. A continuous determination of amino group concentration by reaction with o-phthalaldehyde gives the rate of peptide bond formation. The apparatus allows the determination of the partition constant for the nucleophile at various nucleophile concentrations from one run.     

Development of an automated procedure for fluorescent DNA sequencing

We describe here the development of a procedure for complete automation of the dideoxynucleotide DNA sequencing chemistry using fluorescent dye-labeled oligonucleotide primers. This procedure combines rapid preparation of template DNA using a modification of the polymerase chain reaction, automation of the DNA sequencing reactions using a robotic laboratory workstation, and subsequent analysis of the fluorescent-labeled reaction products on a commercial automated fluorescent sequencer. Using this procedure, we were able to produce sufficient quantities of template DNA directly from bacterial colonies or bacteriophage plaques, perform the DNA sequencing reactions on these templates, and load the reaction products on the fluorescent DNA sequencer in a single work day. This scheme for automation of the fluorescent DNA sequencing method allows the fluorescent sequencer to be run at its full capacity every day and eliminates much of the labor required to obtain a high level of data output. Currently, we are able to perform and analyze 16 fluorescent-labeled reactions every day, with an average output of over 7000 bp per sequencer run.     

Improved initial yields in C-terminal sequence analysis by thiohydantoin chemistry using purified diphenylphosphoryl isothiocyanate: NMR evidence for a reaction intermediate in the coupling reaction

A thiohydantoin method for C-terminal sequence analysis of proteins on Zitex membranes involves derivatization of the free alpha-carboxyl group with diphenylphosphoryl isothiocyanate (DPPITC) plus treatment with pyridine to form a peptidylthiohydantoin derivative, cleavage of the thiohydantoin (TH) amino acid from the protein with potassium trimethylsilanolate, and identification of the released TH-amino acid by online reversed-phase HPLC. This automated chemistry, which was adapted to the Hewlett-Packard G 1009A sequencer, has been shown to identify two or three cycles on a wide variety of proteins, but suffers from low initial yields and instability of the DPPITC reagent. We report here an improved method for synthesis and purification of DPPITC. With this procedure the DPPITC reagent is a clear liquid that is stable at room temperature under vacuum for more than 9 months or for more than 24 months as a 1.0M solution in benzene at -20 degrees C. Using the purified reagent we were able to more than double the initial yield (from 30.7 to 72.4%) of TH-amino acid from a test protein and substantially decrease sequencer background. Examination of the reaction between DPPITC and the carboxylate of model N-terminally protected dipeptides with 31P NMR provides spectroscopic evidence for a postulated intermediate formed between the DPPITC and the peptide carboxylate. The reaction intermediate provides new insight into the coupling mechanism.     

Characterization of a new human apolipoprotein A-I Yame by direct sequencing of polymerase chain reaction-amplified DNA.

A new genetic variant apolipoprotein (apo), A-I Yame, was discovered during screening for apoA-I genetic variants with isoelectric focusing gel electrophoresis. To investigate the structural abnormality of apoA-I Yame, we amplified the apoA-I gene isolated from the proband's peripheral blood leukocytes with the polymerase chain reaction (PCR) method and directly sequenced these PCR fragments. ApoA-I Yame was found to have aspartic acid (GAC) substituted by tyrosine (TAC) at residue 13. We also identified this substitution by an automated DNA sequencer. This substitution was confirmed with amino acid sequencing of the isolated apoA-I Yame by Immobiline gel electrophoresis. This combined method, direct PCR from genomic DNA-derived individual peripheral blood leukocytes and subsequent direct sequencing, can be used to identify the entire sequence of apoA-I in a short period of time. Furthermore, with this method, it is possible to identify both alleles in heterozygous individuals.     

Chemical evidence for the separation of G gamma and A gamma chains by polyacrylamide gel electrophoresis in urea and triton X-100

Polyacrylamide gel electrophoresis in urea and Triton X-100 of a hemolysate from human fetal red blood cells produces four major protein bands: alpha, beta, and 2 gamma globin chains. We have verified that the latter two are the G gamma and A gamma globin chains which have respectively glycine or alanine at position 136. After incorporation of either [3H] alanine or [3H] glycine into newly synthesized globin each gamma chain was isolated by preparative electrophoresis. The chains were cleaved with cyanogen bromide at methionines 55 and 133, then subjected to automated sequencing, and the residues from each sequencer turn counted. Glycine incorporation was detected for the third turn (position 136) of the G gamma chain and alanine for the A gamma. Substantial metabolic conversion of [3H] glycine to serine and proline was also noted.